def start_aqcuisition(self):
global_pos = np.array(
((-5000, -5000), (-5000, 5000), (5000, -5000), (5000, 5000)))
global_pos_name = ['A', 'B', 'C', 'D']
delta = 200
local_pos = np.transpose(
np.reshape(
np.meshgrid(np.array((-delta, 0, delta)),
np.array((-delta, 0, delta))), (2, -1)))
local_pos_name = [str(i) for i in range(9)]
self.cam = CamActuator()
self.cam.initializeCamera()
# Offset variables are used to generate replicates for good statistics
offset_y = offset_x = delta
cnt = 0
for i in range(global_pos.shape[0]):
for j in range(local_pos.shape[0]):
x = global_pos[i, 0] + local_pos[j, 0] + offset_x
y = global_pos[i, 1] + local_pos[j, 1] + offset_y
stage_movement_thread = StagemovementAbsoluteThread(x, y)
stage_movement_thread.start()
time.sleep(2)
stage_movement_thread.quit()
stage_movement_thread.wait()
image = self.cam.SnapImage(0.04)
filename = global_pos_name[i] + local_pos_name[j]
self.save_image(filename, image)
cnt += 1
print(
str(cnt) + '/' +
str(len(local_pos_name) * len(global_pos_name)))
self.cam.Exit()
def run(self):
#Make sure registration can only start when camera is connected
try:
self.cam = CamActuator()
self.cam.initializeCamera()
except:
print(sys.exc_info())
self.backend.ui_widget.normalOutputWritten(
'Unable to connect Hamamatsu camera')
return
self.cam.setROI(0, 0, 2048, 2048)
if self.device_1 == 'galvos':
reference_coordinates = self.gather_reference_coordinates_galvos()
self.dict_transformations['camera-galvos'] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
elif self.device_1 == 'dmd':
reference_coordinates = self.gather_reference_coordinates_dmd()
for laser in self.laser_list:
self.dict_transformations['camera-dmd-'+laser] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
elif self.device_1 == 'stage':
reference_coordinates = self.gather_reference_coordinates_stage()
self.dict_transformations['camera-stage'] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
self.cam.Exit()
## Save transformation to file
with open('CoordinatesManager/Registration/transformation.txt',
'w') as json_file:
dict_transformations_list_format = {}
for key, value in self.dict_transformations.items():
dict_transformations_list_format[key] = value.tolist()
json.dump(dict_transformations_list_format, json_file)
self.sig_finished_registration.emit(self.dict_transformations)
class DMDRegistator:
def __init__(self, parent):
self.DMD = DMDActuator.DMDActuator()
self.cam = CamActuator()
self.cam.initializeCamera()
def registration(self, laser='640', points=6):
x_coords = np.linspace(0, 1024, 5)[1:-1]
y_coords = np.linspace(0, 768, 4)[1:-1]
dmd_coordinates = np.vstack((x_coords, y_coords))
camera_coordinates = np.zeros(dmd_coordinates.shape)
cnt = 0
for i in range(points):
for j in range(y_coords.shape[0]):
mask = create_registration_image(i, j)
self.DMD.send_data_to_DMD(mask)
self.DMD.start_projection()
image = self.cam.SnapImage(0.4)
camera_coordinates[cnt, :] = touchingCoordinateFinder(
image, method='curvefit')
cnt += 1
self.DMD.stop_projection()
self.DMD.free_memory()
self.DMD.disconnect_DMD()
transformation = findTransformationCurvefit(camera_coordinates,
dmd_coordinates,
kx=3,
ky=2)
return transformation
def create_registration_image(x, y, sigma=75):
array = np.zeros((1024, 768))
array[skd.draw.rectangle((x - sigma, y - sigma), (x, y))] = 255
array[skd.draw.rectangle((x + sigma, y + sigma), (x, y))] = 255
return array
class RegistrationThread(QThread):
sig_finished_registration = pyqtSignal(dict)
def __init__(self, parent, laser=None):
QThread.__init__(self)
self.flag_finished = [0, 0, 0]
self.backend = parent
self.dmd = self.backend.DMD
if not isinstance(laser, list):
self.laser_list = [laser]
else:
self.laser_list = laser
self.dict_transformators = {}
self.dict_transformations = {}
self.dtype_ref_co = np.dtype([('camera', int, (3, 2)),
('dmd', int, (3, 2)),
('galvos', int, (3, 2)),
('stage', int, (3, 2))])
self.reference_coordinates = {}
def set_device_to_register(self, device_1, device_2='camera'):
self.device_1 = device_1
self.device_2 = device_2
def run(self):
#Make sure registration can only start when camera is connected
try:
self.cam = CamActuator()
self.cam.initializeCamera()
except:
print(sys.exc_info())
self.backend.ui_widget.normalOutputWritten(
'Unable to connect Hamamatsu camera')
return
self.cam.setROI(0, 0, 2048, 2048)
if self.device_1 == 'galvos':
reference_coordinates = self.gather_reference_coordinates_galvos()
self.dict_transformations['camera-galvos'] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
elif self.device_1 == 'dmd':
reference_coordinates = self.gather_reference_coordinates_dmd()
for laser in self.laser_list:
self.dict_transformations['camera-dmd-'+laser] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
elif self.device_1 == 'stage':
reference_coordinates = self.gather_reference_coordinates_stage()
self.dict_transformations['camera-stage'] = findTransform(reference_coordinates[0], \
reference_coordinates[1])
self.cam.Exit()
## Save transformation to file
with open('CoordinatesManager/Registration/transformation.txt',
'w') as json_file:
dict_transformations_list_format = {}
for key, value in self.dict_transformations.items():
dict_transformations_list_format[key] = value.tolist()
json.dump(dict_transformations_list_format, json_file)
self.sig_finished_registration.emit(self.dict_transformations)
def gather_reference_coordinates_stage(self):
image = np.zeros((2048, 2048, 3))
stage_coordinates = np.array([[-2800, 100], [-2500, 400],
[-1900, -200]])
self.backend.loadMask(mask=np.ones((768, 1024)))
self.backend.startProjection()
for idx, pos in enumerate(stage_coordinates):
stage_movement_thread = StagemovementAbsoluteThread(pos[0], pos[1])
stage_movement_thread.start()
time.sleep(0.5)
stage_movement_thread.quit()
stage_movement_thread.wait()
image[:, :, idx] = self.cam.SnapImage(0.04)
camera_coordinates = find_subimage_location(image, save=True)
self.backend.stopProjection()
self.backend.freeMemory()
return np.array([camera_coordinates, stage_coordinates])
def gather_reference_coordinates_galvos(self):
galvothread = DAQmission()
readinchan = []
camera_coordinates = np.zeros((3, 2))
galvo_coordinates = np.array([[0, 3], [3, -3], [-3, -3]])
for i in range(3):
pos_x = galvo_coordinates[i, 0]
pos_y = galvo_coordinates[i, 1]
galvothread.sendSingleAnalog('galvosx', pos_x)
galvothread.sendSingleAnalog('galvosy', pos_y)
image = self.cam.SnapImage(0.04)
camera_coordinates[i, :] = gaussian_fitting(image)
del galvothread
return np.array([camera_coordinates, galvo_coordinates])
def gather_reference_coordinates_dmd(self):
galvo_coordinates = np.zeros((3, 2))
for laser in self.laser_list:
self.flag_finished = [0, 0, 0]
self.backend.ui_widget.sig_control_laser.emit(laser, 5)
self.registration_single_laser(laser)
self.backend.ui_widget.sig_control_laser.emit(laser, 0)
return np.array(
[self.camera_coordinates, self.dmd_coordinates, galvo_coordinates])
def registration_single_laser(self, laser):
date_time = datetime.datetime.now().timetuple()
image_id = ''
for i in range(5):
image_id += str(date_time[i]) + '_'
image_id += str(date_time[5]) + '_l' + laser
self.camera_coordinates = np.zeros((3, 2))
self.touchingCoordinateFinder = []
for i in range(3):
self.touchingCoordinateFinder.append(
touchingCoordinateFinder_Thread(i, method='curvefit'))
self.touchingCoordinateFinder[
i].sig_finished_coordinatefinder.connect(
self.touchingCoordinateFinder_finished)
for i in range(3):
self.loadFileName = './CoordinatesManager/Registration_Images/TouchingSquares/registration_mask_' + str(
i) + '.png'
# Transpose because mask in file is rotated by 90 degrees.
mask = np.transpose(plt.imread(self.loadFileName))
self.backend.loadMask(mask)
self.backend.startProjection()
time.sleep(0.5)
self.image = self.cam.SnapImage(0.0015)
time.sleep(0.5)
self.backend.stopProjection()
self.backend.freeMemory()
# Start touchingCoordinateFinder thread
self.touchingCoordinateFinder[i].put_image(self.image)
self.touchingCoordinateFinder[i].start()
self.dmd_coordinates = self.read_dmd_coordinates_from_file()
# Block till all touchingCoordinateFinder_Thread threads are finished
while np.prod(self.flag_finished) == 0:
time.sleep(0.1)
def read_dmd_coordinates_from_file(self):
file = open(
'./CoordinatesManager/Registration_Images/TouchingSquares/positions.txt',
'r')
self.dmd_coordinates = []
for ln in file.readlines():
self.dmd_coordinates.append(ln.strip().split(','))
file.close()
return np.asarray(self.dmd_coordinates).astype(int)
def touchingCoordinateFinder_finished(self, sig):
self.camera_coordinates[sig, :] = np.flip(
self.touchingCoordinateFinder[sig].coordinates)
self.flag_finished[sig] = 1
def __init__(self, parent):
self.DMD = DMDActuator.DMDActuator()
self.cam = CamActuator()
self.cam.initializeCamera()
class CameraThread(QThread):
snapsignal = pyqtSignal(np.ndarray)
livesignal = pyqtSignal(np.ndarray)
def __init__(self, camerahandle=None):
self.exposuretime = 0.02 # seconds
self.GUIframerate = 25 # frames per second
self.sleeptime = np.max([1/self.GUIframerate, self.exposuretime])
self.frame = np.random.rand(2048, 2048)
self.mutex = QMutex()
# Camera attributes
if camerahandle == None:
self.camera = CamActuator()
self.camera.initializeCamera()
else:
self.camera = camerahandle
self.camera.hcam.setPropertyValue("exposure_time", self.exposuretime)
# QThread attributes
super().__init__()
self.isrunning = False
self.moveToThread(self)
self.started.connect(self.live)
def stop(self):
self.isrunning = False
self.quit()
self.wait()
def snap(self):
time.sleep(self.sleeptime)
snap = np.random.rand(2048, 2048)
self.mutex.lock()
snap = copy(self.frame)
self.mutex.unlock()
self.snapsignal.emit(snap)
return snap
@pyqtSlot()
def live(self):
print('camera thread started')
self.camera.isLiving = True
self.camera.hcam.acquisition_mode = "run_till_abort"
# Wait a second for camera acquisition to start
self.camera.hcam.startAcquisition()
QThread.msleep(1000)
# Emit and get frames from the camera at a rate of 1/sleeptime
self.isrunning = True
while self.isrunning:
QThread.msleep(int(self.sleeptime *1000))
try:
[frames, dims] = self.camera.hcam.getFrames()
self.mutex.lock()
self.frame = np.resize(frames[-1].np_array, (dims[1], dims[0]))
self.livesignal.emit(self.frame)
self.mutex.unlock()
except:
pass
self.camera.hcam.stopAcquisition()
self.camera.isLiving = False
self.camera.Exit()
print('camera thread stopped')
def run(self):
"""
# ==========================================================================================================================================================
# Initialization
# ==========================================================================================================================================================
"""
#%%
"""
# =============================================================================
# connect the Objective motor
# =============================================================================
"""
print('----------------------Starting to connect the Objective motor-------------------------')
self.pi_device_instance = PIMotor()
print('Objective motor connected.')
self.errornum = 0
self.ObjCurrentPos = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
"""
# =============================================================================
# connect the Hmamatsu camera
# =============================================================================
"""
self._use_camera = False
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for waveform_and_cam_key in self.RoundQueueDict[key][1]:
if "CameraPackage_" in waveform_and_cam_key:
if len(self.RoundQueueDict[key][1][waveform_and_cam_key]) != 0:
self._use_camera = True
if self._use_camera == True:
print('Connecting camera...')
self.HamamatsuCam = CamActuator()
self.HamamatsuCam.initializeCamera()
else:
print('No camera involved.')
"""
# =============================================================================
# connect the Insight X3
# =============================================================================
"""
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.Laserinstance = InsightX3('COM11')
try:
querygap = 1.1
# self.Laserinstance.SetWatchdogTimer(0) # Disable the laser watchdog!
# Status_watchdog_thread = threading.Thread(target = self.Status_watchdog, args=[querygap], daemon=True)
# Status_watchdog_thread.start()
time.sleep(1)
#-------------Initialize laser--------------
self.watchdog_flag = False
time.sleep(0.5)
warmupstatus = 0
while int(warmupstatus) != 100:
try:
warmupstatus = self.Laserinstance.QueryWarmupTime()
time.sleep(0.6)
except:
time.sleep(0.6)
# if int(warmupstatus) == 100:
# self.warmupstatus = True
# print('Laser fully warmed up.')
# if 'Laser state:Ready' in self.Status_list:
# self.Laserinstance.Turn_On_PumpLaser()
# self.laserRun = True
# elif 'Laser state:RUN' in self.Status_list:
# self.laserRun = True
self.watchdog_flag = True
time.sleep(0.5)
except:
print('Laser not connected.')
# If turn on the laser shutter in the beginning
if 'Shutter_Open' in self.GeneralSettingDict['StartUpEvents']:
time.sleep(0.5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
#%%
"""
# ==========================================================================================================================================================
# Execution
# ==========================================================================================================================================================
"""
GridSequence = 0
TotalGridNumber = self.meshgridnumber**2
ScanningMaxCoord = int(np.amax(self.RoundCoordsDict['CoordsPackage_1'])) # Get the largest coordinate
for EachGrid in range(TotalGridNumber):
"""
# =============================================================================
# For each small repeat unit in the scanning meshgrid
# =============================================================================
"""
ScanningGridOffset_Row = int(GridSequence % self.meshgridnumber) * (ScanningMaxCoord + self.GeneralSettingDict['Scanning step']) # Offset coordinate row value for each well.
ScanningGridOffset_Col = int(GridSequence/self.meshgridnumber) * (ScanningMaxCoord + self.GeneralSettingDict['Scanning step']) # Offset coordinate colunm value for each well.
GridSequence += 1
time.sleep(0.5)
for EachRound in range(int(len(self.RoundQueueDict)/2-1)): # EachRound is the round sequence number starting from 0, while the actual number used in dictionary is 1.
print ('----------------------------------------------------------------------------')
print('Below is Grid {}, Round {}.'.format(EachGrid, EachRound+1)) # EachRound+1 is the corresponding round number when setting the dictionary starting from round 1.
"""
# =============================================================================
# Unpack the settings for each round
# =============================================================================
"""
# Initialize variables
CoordOrder = 0 # Counter for n th coordinates, for appending cell properties array.
CellPropertiesDict = {}
ND_filter1_Pos = None
ND_filter2_Pos = None
EM_filter_Pos = None
cp_end_index = -1
self.IndexLookUpCellPropertiesDict = {} #look up dictionary for each cell properties
#-------------Unpack the focus stack information.
ZStackinfor = self.GeneralSettingDict['FocusStackInfoDict']['RoundPackage_{}'.format(EachRound+1)]
self.ZStackNum = int(ZStackinfor[ZStackinfor.index('Focus')+5])
self.ZStackStep = float(ZStackinfor[ZStackinfor.index('Being')+5:len(ZStackinfor)])
#-------------Unpack infor for stage move.
CoordsNum = int(len(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)])/2) #Each pos has 2 coords
#-------------Unpack infor for filter event. In the list, the first one is for ND filter and the second one is for emission filter.
FilterEventIndexList = [i for i,x in enumerate(self.RoundQueueDict['FilterEvents']) if 'Round_{}'.format(EachRound+1) in x]
if len(FilterEventIndexList) > 0:
NDposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[0]]
NDnumber = NDposText[NDposText.index('ToPos_')+6:len(NDposText)]
EMposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[1]]
EMprotein = EMposText[EMposText.index('ToPos_')+6:len(EMposText)]
# "COM9" for filter 1 port, which has ND values from 0 to 3.
# "COM7" for filter 2 port, which has ND values from 0 to 0.5.
if NDnumber == '0':
ND_filter1_Pos = 0
ND_filter2_Pos = 0
elif NDnumber == '1':
ND_filter1_Pos = 1
ND_filter2_Pos = 0
elif NDnumber == '2':
ND_filter1_Pos = 2
ND_filter2_Pos = 0
elif NDnumber == '2.3':
ND_filter1_Pos = 2
ND_filter2_Pos = 2
elif NDnumber == '2.5':
ND_filter1_Pos = 2
ND_filter2_Pos = 3
elif NDnumber == '0.5':
ND_filter1_Pos = 0
ND_filter2_Pos = 3
elif NDnumber == '0.3':
ND_filter1_Pos = 0
ND_filter2_Pos = 2
if EMprotein == 'Arch':
EM_filter_Pos = 0
elif EMprotein == 'eGFP' or EMprotein == 'Citrine':
EM_filter_Pos = 1
#-------------Unpack infor for Insight X3. In the list, the first one is for shutter event and the second one is for wavelength event.
InsightX3EventIndexList = [i for i,x in enumerate(self.RoundQueueDict['InsightEvents']) if 'Round_{}'.format(EachRound+1) in x]
"""
# =============================================================================
# Execute Insight event at the beginning of each round
# =============================================================================
"""
if len(InsightX3EventIndexList) == 1:
print(InsightX3EventIndexList)
InsightText = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'Shutter_Open' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'Shutter_Close' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText[InsightText.index('To_')+3:len(InsightText)])
print(TargetWavelen)
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
self.watchdog_flag = True
time.sleep(0.5)
elif len(InsightX3EventIndexList) == 2:
InsightText_wl = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[1]]
InsightText_st = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'WavelengthTo' in InsightText_wl and 'Shutter_Open' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText_wl and 'Shutter_Close' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(1)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
time.sleep(2)
"""
# =============================================================================
# Execute filter event at the beginning of each round
# =============================================================================
"""
if ND_filter1_Pos != None and ND_filter2_Pos != None:
#Move filter 1
self.filter1 = ELL9Filter("COM9")
self.filter1.moveToPosition(ND_filter1_Pos)
time.sleep(1)
#Move filter 2
self.filter2 = ELL9Filter("COM7")
self.filter2.moveToPosition(ND_filter2_Pos)
time.sleep(1)
if EM_filter_Pos != None:
self.filter3 = ELL9Filter("COM15")
self.filter3.moveToPosition(EM_filter_Pos)
time.sleep(1)
self.currentCoordsSeq = 0
#%%
for EachCoord in range(CoordsNum):
#-----------------------------------------------At each stage coordinate:--------------------------------------------
self.error_massage = None
self.currentCoordsSeq += 1
"""
# =============================================================================
# Stage movement
# =============================================================================
"""
RowIndex = int(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord*2:EachCoord*2+2][0]) + ScanningGridOffset_Row
ColumnIndex = int(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord*2:EachCoord*2+2][1]) + ScanningGridOffset_Col
try:
self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
except:
self.error_massage = 'Fail_MoveStage'
self.errornum += 1
print('Stage move failed! Error number: {}'.format(int(self.errornum)))
print ('Round {}. Current index: {}.'.format(EachRound+1, [RowIndex,ColumnIndex]))
time.sleep(1)
"""
# =============================================================================
# Get the z stack objective positions ready
# =============================================================================
"""
#-------------------------------------------If focus correction applies----------------------------------------
if len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) > 0:
FocusPosArray = self.GeneralSettingDict['FocusCorrectionMatrixDict']['RoundPackage_{}_Grid_{}'.format(EachRound+1, EachGrid)]
# print(FocusPosArray)
FocusPosArray = FocusPosArray.flatten('F')
FocusPos_fromCorrection = FocusPosArray[EachCoord]
print('Target focus pos: '.format(FocusPos_fromCorrection))
# Without focus correction
if len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) == 0:
ZStacklinspaceStart = self.ObjCurrentPos['1'] - (math.floor(self.ZStackNum/2)-1)*self.ZStackStep
ZStacklinspaceEnd = self.ObjCurrentPos['1'] + (self.ZStackNum - math.floor(self.ZStackNum/2))*self.ZStackStep
# With focus correction
elif len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) > 0:
ZStacklinspaceStart = FocusPos_fromCorrection - (math.floor(self.ZStackNum/2)-1)*self.ZStackStep
ZStacklinspaceEnd = FocusPos_fromCorrection + (self.ZStackNum - math.floor(self.ZStackNum/2))*self.ZStackStep
ZStackPosList = np.linspace(ZStacklinspaceStart, ZStacklinspaceEnd, num = self.ZStackNum)
print(ZStackPosList)
"""
# =============================================================================
# Execute waveform packages
# =============================================================================
"""
self.WaveforpackageNum = int(len(self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]))
#Execute each individual waveform package
print('*******************************************Round {}. Current index: {}.**************************************************'.format(EachRound+1, [RowIndex,ColumnIndex]))
#------------------Move to Z stack focus-------------------
for EachZStackPos in range(self.ZStackNum):
print('--------------------------------------------Stack {}--------------------------------------------------'.format(EachZStackPos+1))
if self.ZStackNum > 1:
self.ZStackOrder = int(EachZStackPos +1) # Here the first one is 1, not starting from 0.
FocusPos = ZStackPosList[EachZStackPos]
print('Target focus pos: {}'.format(FocusPos))
pos = PIMotor.move(self.pi_device_instance.pidevice, FocusPos)
self.ObjCurrentPosInStack = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
print("Current position: {:.4f}".format(self.ObjCurrentPosInStack['1']))
time.sleep(0.3)
else:
self.ZStackOrder = 1
#------------For waveforms in each coordinate----------
for EachWaveform in range(self.WaveforpackageNum):
# Extract information
WaveformPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]['WaveformPackage_{}'.format(EachWaveform+1)]
CameraPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][1]["CameraPackage_{}".format(EachWaveform+1)]
WaveformPackageGalvoInfor = self.RoundQueueDict['GalvoInforPackage_{}'.format(EachRound+1)]['GalvoInfor_{}'.format(EachWaveform+1)]
self.readinchan = WaveformPackageToBeExecute[3]
self.RoundWaveformIndex = [EachRound+1, EachWaveform+1] # first is current round number, second is current waveform package number.
self.CurrentPosIndex = [RowIndex, ColumnIndex]
#----------------Camera operations-----------------
_camera_isUsed = False
if CameraPackageToBeExecute != {}: # if camera operations are configured
_camera_isUsed = True
CamSettigList = CameraPackageToBeExecute["Settings"]
self.HamamatsuCam.StartStreaming(BufferNumber = CameraPackageToBeExecute["Buffer_number"],
trigger_source = CamSettigList[CamSettigList.index("trigger_source")+1],
exposure_time = CamSettigList[CamSettigList.index("exposure_time")+1],
trigger_active = CamSettigList[CamSettigList.index("trigger_active")+1])
# Make sure that the camera is prepared before waveform execution.
# while self.HamamatsuCam.isStreaming == False:
# print('Waiting for camera...')
# time.sleep(0.5)
time.sleep(1)
print('Now start waveforms')
#----------------Waveforms operations--------------
if WaveformPackageGalvoInfor != 'NoGalvo': # Unpack the information of galvo scanning.
self.readinchan = WaveformPackageGalvoInfor[0]
self.repeatnum = WaveformPackageGalvoInfor[1]
self.PMT_data_index_array = WaveformPackageGalvoInfor[2]
self.averagenum = WaveformPackageGalvoInfor[3]
self.lenSample_1 = WaveformPackageGalvoInfor[4]
self.ScanArrayXnum = WaveformPackageGalvoInfor[5]
if self.clock_source == 'Dev1 as clock source':
self.adcollector = execute_analog_readin_optional_digital_thread()
self.adcollector.set_waves(WaveformPackageToBeExecute[0], WaveformPackageToBeExecute[1], WaveformPackageToBeExecute[2], WaveformPackageToBeExecute[3]) #[0] = sampling rate, [1] = analogcontainer_array, [2] = digitalcontainer_array, [3] = readinchan
self.adcollector.collected_data.connect(self.ProcessData)
self.adcollector.run()
time.sleep(0.2)
#self.ai_dev_scaling_coeff = self.adcollector.get_ai_dev_scaling_coeff()
elif self.clock_source == 'Cam as clock source' :
self.adcollector = execute_analog_and_readin_digital_optional_camtrig_thread()
self.adcollector.set_waves(WaveformPackageToBeExecute[0], WaveformPackageToBeExecute[1], WaveformPackageToBeExecute[2], WaveformPackageToBeExecute[3])
self.adcollector.collected_data.connect(self.ProcessData)
self.adcollector.run()
#------------------Camera saving-------------------
if _camera_isUsed == True:
self.HamamatsuCam.isSaving = True
tif_name = os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_Cam_'+'Zpos'+str(self.ZStackOrder)+'.tif')
self.HamamatsuCam.StopStreaming(saving_dir = tif_name)
# Make sure that the saving process is finished.
while self.HamamatsuCam.isSaving == True:
print('Camera saving...')
time.sleep(0.5)
time.sleep(1)
time.sleep(0.6) # Wait for receiving data to be done.
time.sleep(0.3)
print('*************************************************************************************************************************')
#%%
"""
# ==========================================================================================================================================================
# Finalizing
# ==========================================================================================================================================================
"""
# Switch off laser
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
self.Laserinstance.SaveVariables()
while True:
try:
self.Laserinstance.Turn_Off_PumpLaser()
break
except:
time.sleep(1)
# Disconnect camera
if self._use_camera == True:
self.HamamatsuCam.Exit()
# Disconnect focus motor
try:
PIMotor.CloseMotorConnection(self.pi_device_instance.pidevice)
print('Objective motor disconnected.')
except:
pass
class ScanningExecutionThread(QThread):
ScanningResult = pyqtSignal(np.ndarray, np.ndarray, object, object) #The signal for the measurement, we can connect to this signal
def __init__(self, RoundQueueDict, RoundCoordsDict, GeneralSettingDict, *args, **kwargs):
super().__init__(*args, **kwargs)
self.RoundQueueDict = RoundQueueDict
self.RoundCoordsDict = RoundCoordsDict
self.GeneralSettingDict = GeneralSettingDict
self.Status_list = None
self.ludlStage = LudlStage("COM12")
self.watchdog_flag = True
# self.PMTimageDict = {}
# for i in range(int(len(self.RoundQueueDict)/2-1)): # initial the nested PMTimageDict dictionary. -2 because default keys for insight and filter.
# self.PMTimageDict['RoundPackage_{}'.format(i+1)] = {}
self.clock_source = 'Dev1 as clock source' # Should be set by GUI.
self.scansavedirectory = self.GeneralSettingDict['savedirectory']
self.meshgridnumber = int(self.GeneralSettingDict['Meshgrid'])
def Try_until_Success(func):
""" This is the decorator to try to execute the function until succeed.
"""
def wrapper(*args, **kwargs):
success = None
failnumber = 0
while success is None:
if failnumber <8:
try:
returnValue = func(*args, **kwargs)
success = True
except:
failnumber += 1
print('Laser action failed, failnumber: {}'.format(failnumber))
time.sleep(0.2)
else:
print('Fail for 8 times, give up - -')
success = False
return returnValue
return wrapper
def run(self):
"""
# ==========================================================================================================================================================
# Initialization
# ==========================================================================================================================================================
"""
#%%
"""
# =============================================================================
# connect the Objective motor
# =============================================================================
"""
print('----------------------Starting to connect the Objective motor-------------------------')
self.pi_device_instance = PIMotor()
print('Objective motor connected.')
self.errornum = 0
self.ObjCurrentPos = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
"""
# =============================================================================
# connect the Hmamatsu camera
# =============================================================================
"""
self._use_camera = False
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for waveform_and_cam_key in self.RoundQueueDict[key][1]:
if "CameraPackage_" in waveform_and_cam_key:
if len(self.RoundQueueDict[key][1][waveform_and_cam_key]) != 0:
self._use_camera = True
if self._use_camera == True:
print('Connecting camera...')
self.HamamatsuCam = CamActuator()
self.HamamatsuCam.initializeCamera()
else:
print('No camera involved.')
"""
# =============================================================================
# connect the Insight X3
# =============================================================================
"""
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.Laserinstance = InsightX3('COM11')
try:
querygap = 1.1
# self.Laserinstance.SetWatchdogTimer(0) # Disable the laser watchdog!
# Status_watchdog_thread = threading.Thread(target = self.Status_watchdog, args=[querygap], daemon=True)
# Status_watchdog_thread.start()
time.sleep(1)
#-------------Initialize laser--------------
self.watchdog_flag = False
time.sleep(0.5)
warmupstatus = 0
while int(warmupstatus) != 100:
try:
warmupstatus = self.Laserinstance.QueryWarmupTime()
time.sleep(0.6)
except:
time.sleep(0.6)
# if int(warmupstatus) == 100:
# self.warmupstatus = True
# print('Laser fully warmed up.')
# if 'Laser state:Ready' in self.Status_list:
# self.Laserinstance.Turn_On_PumpLaser()
# self.laserRun = True
# elif 'Laser state:RUN' in self.Status_list:
# self.laserRun = True
self.watchdog_flag = True
time.sleep(0.5)
except:
print('Laser not connected.')
# If turn on the laser shutter in the beginning
if 'Shutter_Open' in self.GeneralSettingDict['StartUpEvents']:
time.sleep(0.5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
#%%
"""
# ==========================================================================================================================================================
# Execution
# ==========================================================================================================================================================
"""
GridSequence = 0
TotalGridNumber = self.meshgridnumber**2
ScanningMaxCoord = int(np.amax(self.RoundCoordsDict['CoordsPackage_1'])) # Get the largest coordinate
for EachGrid in range(TotalGridNumber):
"""
# =============================================================================
# For each small repeat unit in the scanning meshgrid
# =============================================================================
"""
ScanningGridOffset_Row = int(GridSequence % self.meshgridnumber) * (ScanningMaxCoord + self.GeneralSettingDict['Scanning step']) # Offset coordinate row value for each well.
ScanningGridOffset_Col = int(GridSequence/self.meshgridnumber) * (ScanningMaxCoord + self.GeneralSettingDict['Scanning step']) # Offset coordinate colunm value for each well.
GridSequence += 1
time.sleep(0.5)
for EachRound in range(int(len(self.RoundQueueDict)/2-1)): # EachRound is the round sequence number starting from 0, while the actual number used in dictionary is 1.
print ('----------------------------------------------------------------------------')
print('Below is Grid {}, Round {}.'.format(EachGrid, EachRound+1)) # EachRound+1 is the corresponding round number when setting the dictionary starting from round 1.
"""
# =============================================================================
# Unpack the settings for each round
# =============================================================================
"""
# Initialize variables
CoordOrder = 0 # Counter for n th coordinates, for appending cell properties array.
CellPropertiesDict = {}
ND_filter1_Pos = None
ND_filter2_Pos = None
EM_filter_Pos = None
cp_end_index = -1
self.IndexLookUpCellPropertiesDict = {} #look up dictionary for each cell properties
#-------------Unpack the focus stack information.
ZStackinfor = self.GeneralSettingDict['FocusStackInfoDict']['RoundPackage_{}'.format(EachRound+1)]
self.ZStackNum = int(ZStackinfor[ZStackinfor.index('Focus')+5])
self.ZStackStep = float(ZStackinfor[ZStackinfor.index('Being')+5:len(ZStackinfor)])
#-------------Unpack infor for stage move.
CoordsNum = int(len(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)])/2) #Each pos has 2 coords
#-------------Unpack infor for filter event. In the list, the first one is for ND filter and the second one is for emission filter.
FilterEventIndexList = [i for i,x in enumerate(self.RoundQueueDict['FilterEvents']) if 'Round_{}'.format(EachRound+1) in x]
if len(FilterEventIndexList) > 0:
NDposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[0]]
NDnumber = NDposText[NDposText.index('ToPos_')+6:len(NDposText)]
EMposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[1]]
EMprotein = EMposText[EMposText.index('ToPos_')+6:len(EMposText)]
# "COM9" for filter 1 port, which has ND values from 0 to 3.
# "COM7" for filter 2 port, which has ND values from 0 to 0.5.
if NDnumber == '0':
ND_filter1_Pos = 0
ND_filter2_Pos = 0
elif NDnumber == '1':
ND_filter1_Pos = 1
ND_filter2_Pos = 0
elif NDnumber == '2':
ND_filter1_Pos = 2
ND_filter2_Pos = 0
elif NDnumber == '2.3':
ND_filter1_Pos = 2
ND_filter2_Pos = 2
elif NDnumber == '2.5':
ND_filter1_Pos = 2
ND_filter2_Pos = 3
elif NDnumber == '0.5':
ND_filter1_Pos = 0
ND_filter2_Pos = 3
elif NDnumber == '0.3':
ND_filter1_Pos = 0
ND_filter2_Pos = 2
if EMprotein == 'Arch':
EM_filter_Pos = 0
elif EMprotein == 'eGFP' or EMprotein == 'Citrine':
EM_filter_Pos = 1
#-------------Unpack infor for Insight X3. In the list, the first one is for shutter event and the second one is for wavelength event.
InsightX3EventIndexList = [i for i,x in enumerate(self.RoundQueueDict['InsightEvents']) if 'Round_{}'.format(EachRound+1) in x]
"""
# =============================================================================
# Execute Insight event at the beginning of each round
# =============================================================================
"""
if len(InsightX3EventIndexList) == 1:
print(InsightX3EventIndexList)
InsightText = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'Shutter_Open' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'Shutter_Close' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText[InsightText.index('To_')+3:len(InsightText)])
print(TargetWavelen)
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
self.watchdog_flag = True
time.sleep(0.5)
elif len(InsightX3EventIndexList) == 2:
InsightText_wl = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[1]]
InsightText_st = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'WavelengthTo' in InsightText_wl and 'Shutter_Open' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
while True:
try:
self.Laserinstance.Open_TunableBeamShutter()
break
except:
time.sleep(1)
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText_wl and 'Shutter_Close' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
while True:
try:
self.Laserinstance.SetWavelength(TargetWavelen)
break
except:
time.sleep(1)
time.sleep(5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(1)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
time.sleep(2)
"""
# =============================================================================
# Execute filter event at the beginning of each round
# =============================================================================
"""
if ND_filter1_Pos != None and ND_filter2_Pos != None:
#Move filter 1
self.filter1 = ELL9Filter("COM9")
self.filter1.moveToPosition(ND_filter1_Pos)
time.sleep(1)
#Move filter 2
self.filter2 = ELL9Filter("COM7")
self.filter2.moveToPosition(ND_filter2_Pos)
time.sleep(1)
if EM_filter_Pos != None:
self.filter3 = ELL9Filter("COM15")
self.filter3.moveToPosition(EM_filter_Pos)
time.sleep(1)
self.currentCoordsSeq = 0
#%%
for EachCoord in range(CoordsNum):
#-----------------------------------------------At each stage coordinate:--------------------------------------------
self.error_massage = None
self.currentCoordsSeq += 1
"""
# =============================================================================
# Stage movement
# =============================================================================
"""
RowIndex = int(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord*2:EachCoord*2+2][0]) + ScanningGridOffset_Row
ColumnIndex = int(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord*2:EachCoord*2+2][1]) + ScanningGridOffset_Col
try:
self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
except:
self.error_massage = 'Fail_MoveStage'
self.errornum += 1
print('Stage move failed! Error number: {}'.format(int(self.errornum)))
print ('Round {}. Current index: {}.'.format(EachRound+1, [RowIndex,ColumnIndex]))
time.sleep(1)
"""
# =============================================================================
# Get the z stack objective positions ready
# =============================================================================
"""
#-------------------------------------------If focus correction applies----------------------------------------
if len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) > 0:
FocusPosArray = self.GeneralSettingDict['FocusCorrectionMatrixDict']['RoundPackage_{}_Grid_{}'.format(EachRound+1, EachGrid)]
# print(FocusPosArray)
FocusPosArray = FocusPosArray.flatten('F')
FocusPos_fromCorrection = FocusPosArray[EachCoord]
print('Target focus pos: '.format(FocusPos_fromCorrection))
# Without focus correction
if len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) == 0:
ZStacklinspaceStart = self.ObjCurrentPos['1'] - (math.floor(self.ZStackNum/2)-1)*self.ZStackStep
ZStacklinspaceEnd = self.ObjCurrentPos['1'] + (self.ZStackNum - math.floor(self.ZStackNum/2))*self.ZStackStep
# With focus correction
elif len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) > 0:
ZStacklinspaceStart = FocusPos_fromCorrection - (math.floor(self.ZStackNum/2)-1)*self.ZStackStep
ZStacklinspaceEnd = FocusPos_fromCorrection + (self.ZStackNum - math.floor(self.ZStackNum/2))*self.ZStackStep
ZStackPosList = np.linspace(ZStacklinspaceStart, ZStacklinspaceEnd, num = self.ZStackNum)
print(ZStackPosList)
"""
# =============================================================================
# Execute waveform packages
# =============================================================================
"""
self.WaveforpackageNum = int(len(self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]))
#Execute each individual waveform package
print('*******************************************Round {}. Current index: {}.**************************************************'.format(EachRound+1, [RowIndex,ColumnIndex]))
#------------------Move to Z stack focus-------------------
for EachZStackPos in range(self.ZStackNum):
print('--------------------------------------------Stack {}--------------------------------------------------'.format(EachZStackPos+1))
if self.ZStackNum > 1:
self.ZStackOrder = int(EachZStackPos +1) # Here the first one is 1, not starting from 0.
FocusPos = ZStackPosList[EachZStackPos]
print('Target focus pos: {}'.format(FocusPos))
pos = PIMotor.move(self.pi_device_instance.pidevice, FocusPos)
self.ObjCurrentPosInStack = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
print("Current position: {:.4f}".format(self.ObjCurrentPosInStack['1']))
time.sleep(0.3)
else:
self.ZStackOrder = 1
#------------For waveforms in each coordinate----------
for EachWaveform in range(self.WaveforpackageNum):
# Extract information
WaveformPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]['WaveformPackage_{}'.format(EachWaveform+1)]
CameraPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][1]["CameraPackage_{}".format(EachWaveform+1)]
WaveformPackageGalvoInfor = self.RoundQueueDict['GalvoInforPackage_{}'.format(EachRound+1)]['GalvoInfor_{}'.format(EachWaveform+1)]
self.readinchan = WaveformPackageToBeExecute[3]
self.RoundWaveformIndex = [EachRound+1, EachWaveform+1] # first is current round number, second is current waveform package number.
self.CurrentPosIndex = [RowIndex, ColumnIndex]
#----------------Camera operations-----------------
_camera_isUsed = False
if CameraPackageToBeExecute != {}: # if camera operations are configured
_camera_isUsed = True
CamSettigList = CameraPackageToBeExecute["Settings"]
self.HamamatsuCam.StartStreaming(BufferNumber = CameraPackageToBeExecute["Buffer_number"],
trigger_source = CamSettigList[CamSettigList.index("trigger_source")+1],
exposure_time = CamSettigList[CamSettigList.index("exposure_time")+1],
trigger_active = CamSettigList[CamSettigList.index("trigger_active")+1])
# Make sure that the camera is prepared before waveform execution.
# while self.HamamatsuCam.isStreaming == False:
# print('Waiting for camera...')
# time.sleep(0.5)
time.sleep(1)
print('Now start waveforms')
#----------------Waveforms operations--------------
if WaveformPackageGalvoInfor != 'NoGalvo': # Unpack the information of galvo scanning.
self.readinchan = WaveformPackageGalvoInfor[0]
self.repeatnum = WaveformPackageGalvoInfor[1]
self.PMT_data_index_array = WaveformPackageGalvoInfor[2]
self.averagenum = WaveformPackageGalvoInfor[3]
self.lenSample_1 = WaveformPackageGalvoInfor[4]
self.ScanArrayXnum = WaveformPackageGalvoInfor[5]
if self.clock_source == 'Dev1 as clock source':
self.adcollector = execute_analog_readin_optional_digital_thread()
self.adcollector.set_waves(WaveformPackageToBeExecute[0], WaveformPackageToBeExecute[1], WaveformPackageToBeExecute[2], WaveformPackageToBeExecute[3]) #[0] = sampling rate, [1] = analogcontainer_array, [2] = digitalcontainer_array, [3] = readinchan
self.adcollector.collected_data.connect(self.ProcessData)
self.adcollector.run()
time.sleep(0.2)
#self.ai_dev_scaling_coeff = self.adcollector.get_ai_dev_scaling_coeff()
elif self.clock_source == 'Cam as clock source' :
self.adcollector = execute_analog_and_readin_digital_optional_camtrig_thread()
self.adcollector.set_waves(WaveformPackageToBeExecute[0], WaveformPackageToBeExecute[1], WaveformPackageToBeExecute[2], WaveformPackageToBeExecute[3])
self.adcollector.collected_data.connect(self.ProcessData)
self.adcollector.run()
#------------------Camera saving-------------------
if _camera_isUsed == True:
self.HamamatsuCam.isSaving = True
tif_name = os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_Cam_'+'Zpos'+str(self.ZStackOrder)+'.tif')
self.HamamatsuCam.StopStreaming(saving_dir = tif_name)
# Make sure that the saving process is finished.
while self.HamamatsuCam.isSaving == True:
print('Camera saving...')
time.sleep(0.5)
time.sleep(1)
time.sleep(0.6) # Wait for receiving data to be done.
time.sleep(0.3)
print('*************************************************************************************************************************')
#%%
"""
# ==========================================================================================================================================================
# Finalizing
# ==========================================================================================================================================================
"""
# Switch off laser
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.watchdog_flag = False
time.sleep(0.5)
while True:
try:
self.Laserinstance.Close_TunableBeamShutter()
break
except:
time.sleep(1)
time.sleep(0.5)
self.Laserinstance.SaveVariables()
while True:
try:
self.Laserinstance.Turn_Off_PumpLaser()
break
except:
time.sleep(1)
# Disconnect camera
if self._use_camera == True:
self.HamamatsuCam.Exit()
# Disconnect focus motor
try:
PIMotor.CloseMotorConnection(self.pi_device_instance.pidevice)
print('Objective motor disconnected.')
except:
pass
#%%
#--------------------------------------------------------------Reconstruct and save images from 1D recorded array.--------------------------------------------------------------------------------
def ProcessData(self, data_waveformreceived):
print('ZStackOrder is:'+str(self.ZStackOrder)+'numis_'+str(self.ZStackNum))
self.adcollector.save_as_binary(self.scansavedirectory)
self.channel_number = len(data_waveformreceived)
if self.channel_number == 1:
if 'Vp' in self.readinchan:
pass
elif 'Ip' in self.readinchan:
pass
elif 'PMT' in self.readinchan: # repeatnum, PMT_data_index_array, averagenum, ScanArrayXnum
self.data_collected_0 = data_waveformreceived[0]*-1
self.data_collected_0 = self.data_collected_0[0:len(self.data_collected_0)-1]
print(len(self.data_collected_0))
for imageSequence in range(self.repeatnum):
try:
self.PMT_image_reconstructed_array = self.data_collected_0[np.where(self.PMT_data_index_array == imageSequence+1)]
# if imageSequence == int(self.repeatnum)-1:
# self.PMT_image_reconstructed_array = self.PMT_image_reconstructed_array[0:len(self.PMT_image_reconstructed_array)-1] # Extra one sample at the end.
# print(self.PMT_image_reconstructed_array.shape)
Dataholder_average = np.mean(self.PMT_image_reconstructed_array.reshape(self.averagenum, -1), axis=0)
# print(Dataholder_average.shape)
Value_yPixels = int(self.lenSample_1/self.ScanArrayXnum)
self.PMT_image_reconstructed = np.reshape(Dataholder_average, (Value_yPixels, self.ScanArrayXnum))
self.PMT_image_reconstructed = self.PMT_image_reconstructed[:, 50:550] # Crop size based on: M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Xin\2019-12-30 2p beads area test 4um
# self.PMT_image_reconstructed = self.PMT_image_reconstructed[:, 70:326] # for 256*256 images
#---------------------------------------------For multiple images in one z pos, Stack the arrays into a 3d array--------------------------------------------------------------------------
if imageSequence == 0:
self.PMT_image_reconstructed_stack = self.PMT_image_reconstructed[np.newaxis, :, :] # Turns into 3d array
else:
self.PMT_image_reconstructed_stack = np.concatenate((self.PMT_image_reconstructed_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
print(self.PMT_image_reconstructed_stack.shape)
#---------------------------------------------Calculate the z max projection-----------------------------------------------------------------------
if self.repeatnum == 1: # Consider one repeat image situlation
if self.ZStackNum > 1:
if self.ZStackOrder == 1:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
else:
self.PMT_image_maxprojection_stack = np.concatenate((self.PMT_image_maxprojection_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
else:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
# Save the max projection image
if self.ZStackOrder == self.ZStackNum:
self.PMT_image_maxprojection = np.max(self.PMT_image_maxprojection_stack, axis=0)
LocalimgZprojection = Image.fromarray(self.PMT_image_maxprojection) #generate an image object
LocalimgZprojection.save(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zmax'+'.tif')) #save as tif
#
Localimg = Image.fromarray(self.PMT_image_reconstructed) #generate an image object
Localimg.save(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zpos'+str(self.ZStackOrder)+'.tif')) #save as tif
plt.figure()
plt.imshow(self.PMT_image_reconstructed, cmap = plt.cm.gray) # For reconstructed image we pull out the first layer, getting 2d img.
plt.show()
except:
print('No.{} image failed to generate.'.format(imageSequence))
elif self.channel_number == 2:
if 'PMT' not in self.readinchan:
pass
elif 'PMT' in self.readinchan:
self.data_collected_0 = data_waveformreceived[0]*-1
self.data_collected_0 = self.data_collected_0[0:len(self.data_collected_0)-1]
print(len(self.data_collected_0))
for imageSequence in range(self.repeatnum):
try:
self.PMT_image_reconstructed_array = self.data_collected_0[np.where(self.PMT_data_index_array == imageSequence+1)]
if imageSequence == int(self.repeatnum)-1:
self.PMT_image_reconstructed_array = self.PMT_image_reconstructed_array[0:len(self.PMT_image_reconstructed_array)-1] # Extra one sample at the end.
Dataholder_average = np.mean(self.PMT_image_reconstructed_array.reshape(self.averagenum, -1), axis=0)
Value_yPixels = int(self.lenSample_1/self.ScanArrayXnum)
self.PMT_image_reconstructed = np.reshape(Dataholder_average, (Value_yPixels, self.ScanArrayXnum))
self.PMT_image_reconstructed = self.PMT_image_reconstructed[:, 50:550]
# Stack the arrays into a 3d array
if imageSequence == 0:
self.PMT_image_reconstructed_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
else:
self.PMT_image_reconstructed_stack = np.concatenate((self.PMT_image_reconstructed_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
#---------------------------------------------Calculate the z max projection-----------------------------------------------------------------------
if self.repeatnum == 1: # Consider one repeat image situlation
if self.ZStackNum > 1:
if self.ZStackOrder == 1:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
else:
self.PMT_image_maxprojection_stack = np.concatenate((self.PMT_image_maxprojection_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
else:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
# Save the max projection image
if self.ZStackOrder == self.ZStackNum:
self.PMT_image_maxprojection = np.max(self.PMT_image_maxprojection_stack, axis=0)
LocalimgZprojection = Image.fromarray(self.PMT_image_maxprojection) #generate an image object
LocalimgZprojection.save(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zmax'+'.tif')) #save as tif
Localimg = Image.fromarray(self.PMT_image_reconstructed) #generate an image object
Localimg.save(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zpos'+str(self.ZStackOrder)+'.tif')) #save as tif
plt.figure()
plt.imshow(self.PMT_image_reconstructed, cmap = plt.cm.gray)
plt.show()
except:
print('No.{} image failed to generate.'.format(imageSequence))
print('ProcessData executed.')
#----------------------------------------------------------------WatchDog for laser----------------------------------------------------------------------------------
def Status_watchdog(self, querygap):
while True:
if self.watchdog_flag == True:
self.Status_list = self.Laserinstance.QueryStatus()
time.sleep(querygap)
else:
print('Watchdog stopped')
time.sleep(querygap)
class StageWidget(QWidget):
def __init__(self, parent=None, *args, **kwargs):
super().__init__(*args, **kwargs)
self.main_application = parent
self.set_image_saving_location(
os.getcwd() +
'/CoordinatesManager/Registration_Images/StageRegistration/')
self.init_gui()
self.ludlStage = LudlStage("COM12")
def init_gui(self):
layout = QGridLayout()
# self.setFixedSize(320,100)
self.box = roundQGroupBox()
self.box.setTitle("Stage")
box_layout = QGridLayout()
self.box.setLayout(box_layout)
self.setLayout(layout)
self.collect_data_button = QPushButton('Collect data')
self.collect_data_button.clicked.connect(self.start_aqcuisition)
box_layout.addWidget(self.collect_data_button)
layout.addWidget(self.box)
def set_image_saving_location(self, filepath):
self.image_file_path = filepath
def start_aqcuisition(self):
global_pos = np.array(
((-5000, -5000), (-5000, 5000), (5000, -5000), (5000, 5000)))
global_pos_name = ['A', 'B', 'C', 'D']
delta = 200
local_pos = np.transpose(
np.reshape(
np.meshgrid(np.array((-delta, 0, delta)),
np.array((-delta, 0, delta))), (2, -1)))
local_pos_name = [str(i) for i in range(9)]
self.cam = CamActuator()
self.cam.initializeCamera()
# Offset variables are used to generate replicates for good statistics
offset_y = offset_x = delta
cnt = 0
for i in range(global_pos.shape[0]):
for j in range(local_pos.shape[0]):
x = global_pos[i, 0] + local_pos[j, 0] + offset_x
y = global_pos[i, 1] + local_pos[j, 1] + offset_y
self.ludlStage.moveAbs(x=x, y=y)
# stage_movement_thread = StagemovementAbsoluteThread(x, y)
# stage_movement_thread.start()
# time.sleep(2)
# stage_movement_thread.quit()
# stage_movement_thread.wait()
image = self.cam.SnapImage(0.04)
filename = global_pos_name[i] + local_pos_name[j]
self.save_image(filename, image)
cnt += 1
print(
str(cnt) + '/' +
str(len(local_pos_name) * len(global_pos_name)))
self.cam.Exit()
def save_image(self, filename, image):
plt.imsave(self.image_file_path + filename + '.png', image)
class MainGUI(QWidget):
# signal_DMDmask is cictionary with laser specification as key and binary mask as content.
signal_DMDmask = pyqtSignal(dict)
signal_DMDcontour = pyqtSignal(list)
def __init__(self, *args, **kwargs):
super().__init__(*args, **kwargs)
os.chdir('./') # Set directory to current folder.
self.setFont(QFont("Arial"))
# self.setMinimumSize(900, 1020)
self.setWindowTitle("Cell Selection")
self.layout = QGridLayout(self)
self.roi_list_freehandl_added = []
self.selected_ML_Index = []
self.selected_cells_infor_dict = {}
self.mask_color_multiplier = [1, 1, 0]
# =============================================================================
# Container for image display
# =============================================================================
graphContainer = StylishQT.roundQGroupBox()
graphContainerLayout = QGridLayout()
self.Imgviewtabs = QTabWidget()
MLmaskviewBox = QWidget()
MLmaskviewBoxLayout = QGridLayout()
self.Matdisplay_Figure = Figure()
self.Matdisplay_Canvas = FigureCanvas(self.Matdisplay_Figure)
self.Matdisplay_Canvas.setFixedWidth(500)
self.Matdisplay_Canvas.setFixedHeight(500)
self.Matdisplay_Canvas.mpl_connect('button_press_event', self._onclick)
self.Matdisplay_toolbar = NavigationToolbar(self.Matdisplay_Canvas,
self)
MLmaskviewBoxLayout.addWidget(self.Matdisplay_toolbar, 0, 0)
MLmaskviewBoxLayout.addWidget(self.Matdisplay_Canvas, 1, 0)
MLmaskviewBox.setLayout(MLmaskviewBoxLayout)
self.Imgviewtabs.addTab(MLmaskviewBox, "MaskRCNN")
# =============================================================================
# Mask editing tab
# =============================================================================
MLmaskEditBox = QWidget()
MLmaskEditBoxLayout = QGridLayout()
self.Mask_edit_view = DrawingWidget(self)
self.Mask_edit_view.enable_drawing(False) # Disable drawing first
# self.Mask_edit_view = pg.ImageView()
# self.Mask_edit_view.getView().setLimits(xMin = 0, xMax = 2048, yMin = 0, yMax = 2048, minXRange = 2048, minYRange = 2048, maxXRange = 2048, maxYRange = 2048)
self.Mask_edit_viewItem = self.Mask_edit_view.getImageItem()
# self.ROIitem = pg.PolyLineROI([[0,0], [80,0], [80,80], [0,80]], closed=True)
self.Mask_edit_view_getView = self.Mask_edit_view.getView()
# self.Mask_edit_view_getView.addItem(self.ROIitem)
self.Mask_edit_view.ui.roiBtn.hide()
self.Mask_edit_view.ui.menuBtn.hide()
self.Mask_edit_view.ui.normGroup.hide()
self.Mask_edit_view.ui.roiPlot.hide()
MLmaskEditBoxLayout.addWidget(self.Mask_edit_view, 0, 0)
MLmaskEditBox.setLayout(MLmaskEditBoxLayout)
self.Imgviewtabs.addTab(MLmaskEditBox, "Mask edit")
graphContainerLayout.addWidget(self.Imgviewtabs, 0, 0)
graphContainer.setLayout(graphContainerLayout)
# =============================================================================
# Operation container
# =============================================================================
operationContainer = StylishQT.roundQGroupBox()
operationContainerLayout = QGridLayout()
self.init_ML_button = QPushButton('Initialize ML', self)
operationContainerLayout.addWidget(self.init_ML_button, 0, 0)
self.init_ML_button.clicked.connect(self.init_ML)
#---------------------Load image from file-----------------------------
self.textbox_loadimg = QLineEdit(self)
operationContainerLayout.addWidget(self.textbox_loadimg, 1, 0)
self.button_import_img_browse = QPushButton('Browse', self)
operationContainerLayout.addWidget(self.button_import_img_browse, 1, 1)
self.button_import_img_browse.clicked.connect(self.get_img_file_tif)
self.run_ML_button = QPushButton('Analysis', self)
operationContainerLayout.addWidget(self.run_ML_button, 2, 0)
self.run_ML_button.clicked.connect(self.run_ML_onImg_and_display)
self.generate_MLmask_button = QPushButton('Mask', self)
operationContainerLayout.addWidget(self.generate_MLmask_button, 2, 1)
self.generate_MLmask_button.clicked.connect(self.generate_MLmask)
self.update_MLmask_button = QPushButton('Update mask', self)
operationContainerLayout.addWidget(self.update_MLmask_button, 3, 0)
self.update_MLmask_button.clicked.connect(self.update_mask)
self.enable_modify_MLmask_button = QPushButton('Enable free-hand',
self)
self.enable_modify_MLmask_button.setCheckable(True)
operationContainerLayout.addWidget(self.enable_modify_MLmask_button, 4,
0)
self.enable_modify_MLmask_button.clicked.connect(self.enable_free_hand)
# self.modify_MLmask_button = QPushButton('Add patch', self)
# operationContainerLayout.addWidget(self.modify_MLmask_button, 4, 1)
# self.modify_MLmask_button.clicked.connect(self.addedROIitem_to_Mask)
self.clear_roi_button = QPushButton('Clear ROIs', self)
operationContainerLayout.addWidget(self.clear_roi_button, 5, 0)
self.clear_roi_button.clicked.connect(self.clear_edit_roi)
# self.maskLaserComboBox = QComboBox()
# self.maskLaserComboBox.addItems(['640', '532', '488'])
# operationContainerLayout.addWidget(self.maskLaserComboBox, 6, 0)
#
# self.generate_transformed_mask_button = QPushButton('Transform mask', self)
# operationContainerLayout.addWidget(self.generate_transformed_mask_button, 6, 1)
# self.generate_transformed_mask_button.clicked.connect(self.generate_transformed_mask)
self.emit_transformed_mask_button = QPushButton('Emit mask', self)
operationContainerLayout.addWidget(self.emit_transformed_mask_button,
7, 1)
self.emit_transformed_mask_button.clicked.connect(
self.emit_mask_contour)
operationContainer.setLayout(operationContainerLayout)
# =============================================================================
# Mask para container
# =============================================================================
MaskparaContainer = StylishQT.roundQGroupBox()
MaskparaContainerContainerLayout = QGridLayout()
#----------------------------------------------------------------------
self.fillContourButton = QCheckBox()
self.invertMaskButton = QCheckBox()
self.thicknessSpinBox = QSpinBox()
self.thicknessSpinBox.setRange(1, 25)
MaskparaContainerContainerLayout.addWidget(QLabel('Fill contour:'), 0,
0)
MaskparaContainerContainerLayout.addWidget(self.fillContourButton, 0,
1)
MaskparaContainerContainerLayout.addWidget(QLabel('Invert mask:'), 1,
0)
MaskparaContainerContainerLayout.addWidget(self.invertMaskButton, 1, 1)
MaskparaContainerContainerLayout.addWidget(QLabel('Thickness:'), 2, 0)
MaskparaContainerContainerLayout.addWidget(self.thicknessSpinBox, 2, 1)
MaskparaContainer.setLayout(MaskparaContainerContainerLayout)
# =============================================================================
# Device operation container
# =============================================================================
deviceOperationContainer = StylishQT.roundQGroupBox()
deviceOperationContainerLayout = QGridLayout()
#----------------------------------------------------------------------
self.CamExposureBox = QDoubleSpinBox(self)
self.CamExposureBox.setDecimals(6)
self.CamExposureBox.setMinimum(0)
self.CamExposureBox.setMaximum(100)
self.CamExposureBox.setValue(0.001501)
self.CamExposureBox.setSingleStep(0.001)
deviceOperationContainerLayout.addWidget(self.CamExposureBox, 0, 1)
deviceOperationContainerLayout.addWidget(QLabel("Exposure time:"), 0,
0)
cam_snap_button = QPushButton('Cam snap', self)
deviceOperationContainerLayout.addWidget(cam_snap_button, 0, 2)
cam_snap_button.clicked.connect(self.cam_snap)
cam_snap_button = QPushButton('Cam snap', self)
deviceOperationContainerLayout.addWidget(cam_snap_button, 0, 2)
cam_snap_button.clicked.connect(self.cam_snap)
deviceOperationContainer.setLayout(deviceOperationContainerLayout)
self.layout.addWidget(graphContainer, 0, 0, 3, 1)
self.layout.addWidget(operationContainer, 0, 1)
self.layout.addWidget(MaskparaContainer, 1, 1)
self.layout.addWidget(deviceOperationContainer, 2, 1)
self.setLayout(self.layout)
#%%
# =============================================================================
# MaskRCNN detection part
# =============================================================================
# @run_in_thread
def init_ML(self):
# Initialize the detector instance and load the model.
self.ProcessML = ProcessImageML()
def get_img_file_tif(self):
self.img_tif_filePath, _ = QtWidgets.QFileDialog.getOpenFileName(
self, 'Single File',
'M:/tnw/ist/do/projects/Neurophotonics/Brinkslab/Data', "(*.tif)")
self.textbox_loadimg.setText(self.img_tif_filePath)
if self.img_tif_filePath != None:
self.Rawimage = imread(self.img_tif_filePath)
self.MLtargetedImg_raw = self.Rawimage.copy()
self.MLtargetedImg = self.convert_for_MaskRCNN(
self.MLtargetedImg_raw)
self.show_raw_image(self.MLtargetedImg)
self.addedROIitemMask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
self.MLmask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
def show_raw_image(self, image):
# display a single image
try:
self.Matdisplay_Figure.clear()
except:
pass
ax1 = self.Matdisplay_Figure.add_subplot(111)
ax1.set_xticks([])
ax1.set_yticks([])
ax1.imshow(image)
self.Matdisplay_Figure.tight_layout()
self.Matdisplay_Canvas.draw()
RGB_image = gray2rgb(image)
self.Mask_edit_viewItem.setImage(RGB_image)
def convert_for_MaskRCNN(self, input_img):
"""Convert the image size and bit-depth to make it suitable for MaskRCNN detection."""
if input_img.shape[0] > 1024 or input_img.shape[1] > 1024:
resized_img = resize(input_img, [1024, 1024],
preserve_range=True).astype(input_img.dtype)
minval = np.min(resized_img)
maxval = np.max(resized_img)
return ((resized_img - minval) / (maxval - minval) * 255).astype(
np.uint8)
def run_ML_onImg_and_display(self):
"""Run MaskRCNN on input image"""
self.Matdisplay_Figure.clear()
ax1 = self.Matdisplay_Figure.add_subplot(111)
# Depends on show_mask or not, the returned figure will be input raw image with mask or not.
self.MLresults, self.Matdisplay_Figure_axis, self.unmasked_fig = self.ProcessML.DetectionOnImage(
self.MLtargetedImg, axis=ax1, show_mask=False, show_bbox=False)
self.Mask = self.MLresults['masks']
self.Label = self.MLresults['class_ids']
self.Score = self.MLresults['scores']
self.Bbox = self.MLresults['rois']
self.SelectedCellIndex = 0
self.NumCells = int(len(self.Label))
self.selected_ML_Index = []
self.selected_cells_infor_dict = {}
self.Matdisplay_Figure_axis.imshow(self.unmasked_fig.astype(np.uint8))
self.Matdisplay_Figure.tight_layout()
self.Matdisplay_Canvas.draw()
#%%
# =============================================================================
# Configure click event to add clicked cell mask
# =============================================================================
def _onclick(self, event):
"""Highlights the cell selected in the figure by the user when clicked on"""
if self.NumCells > 0:
ShapeMask = np.shape(self.Mask)
# get coorinates at selected location in image coordinates
if event.xdata == None or event.ydata == None:
return
xcoor = min(max(int(event.xdata), 0), ShapeMask[1])
ycoor = min(max(int(event.ydata), 0), ShapeMask[0])
# search for the mask coresponding to the selected cell
for EachCell in range(self.NumCells):
if self.Mask[ycoor, xcoor, EachCell]:
self.SelectedCellIndex = EachCell
break
# highlight selected cell
if self.SelectedCellIndex not in self.selected_ML_Index:
# Get the selected cell's contour coordinates and mask patch
self.contour_verts, self.Cell_patch = self.get_cell_polygon(
self.Mask[:, :, self.SelectedCellIndex])
self.Matdisplay_Figure_axis.add_patch(self.Cell_patch)
self.Matdisplay_Canvas.draw()
self.selected_ML_Index.append(self.SelectedCellIndex)
self.selected_cells_infor_dict['cell{}_verts'.format(
str(self.SelectedCellIndex))] = self.contour_verts
else:
# If click on the same cell
self.Cell_patch.remove()
self.Matdisplay_Canvas.draw()
self.selected_ML_Index.remove(self.SelectedCellIndex)
self.selected_cells_infor_dict.pop('cell{}_verts'.format(
str(self.SelectedCellIndex)))
def get_cell_polygon(self, mask):
# Mask Polygon
# Pad to ensure proper polygons for masks that touch image edges.
padded_mask = np.zeros((mask.shape[0] + 2, mask.shape[1] + 2),
dtype=np.uint8)
padded_mask[1:-1, 1:-1] = mask
contours = find_contours(padded_mask, 0.5)
for verts in contours:
# Subtract the padding and flip (y, x) to (x, y)
verts = np.fliplr(verts) - 1
contour_polygon = mpatches.Polygon(
verts, facecolor=self.random_colors(1)[0])
return contours, contour_polygon
def random_colors(self, N, bright=True):
"""
Generate random colors.
To get visually distinct colors, generate them in HSV space then
convert to RGB.
"""
brightness = 1.0 if bright else 0.7
hsv = [(i / N, 1, brightness) for i in range(N)]
colors = list(map(lambda c: colorsys.hsv_to_rgb(*c), hsv))
random.shuffle(colors)
return colors
#%%
# =============================================================================
# For mask generation
# =============================================================================
def generate_MLmask(self):
""" Generate binary mask with all selected cells"""
self.MLmask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
if len(self.selected_ML_Index) > 0:
for selected_index in self.selected_ML_Index:
self.MLmask = np.add(self.MLmask, self.Mask[:, :,
selected_index])
self.intergrate_into_final_mask()
self.add_rois_of_selected()
else:
self.intergrate_into_final_mask()
# self.Mask_edit_viewItem.setImage(gray2rgb(self.MLtargetedImg))
def add_rois_of_selected(self):
"""
Using find_contours to get list of contour coordinates in the binary mask, and then generate polygon rois based on these coordinates.
"""
for selected_index in self.selected_ML_Index:
contours = self.selected_cells_infor_dict['cell{}_verts'.format(
str(selected_index))]
# contours = find_contours(self.Mask[:,:,selected_index], 0.5) # Find iso-valued contours in a 2D array for a given level value.
for n, contour in enumerate(contours):
contour_coord_array = contours[n]
#Swap columns
contour_coord_array[:,
0], contour_coord_array[:,
1] = contour_coord_array[:,
1], contour_coord_array[:, 0].copy(
)
#Down sample the coordinates otherwise it will be too dense.
contour_coord_array_del = np.delete(
contour_coord_array,
np.arange(2, contour_coord_array.shape[0] - 3, 2), 0)
self.selected_cells_infor_dict['cell{}_ROIitem'.format(str(selected_index))] = \
pg.PolyLineROI(positions=contour_coord_array_del, closed=True)
self.Mask_edit_view.getView().addItem(
self.selected_cells_infor_dict['cell{}_ROIitem'.format(
str(selected_index))])
def update_mask(self):
"""
Regenerate the masks for MaskRCNN and free-hand added (in case they are changed), and show in imageview.
!!!ISSUE: getLocalHandlePositions: moving handles changes the position read out, dragging roi as a whole doesn't.
"""
# Binary mask from ML detection
if len(self.selected_ML_Index) > 0:
# Delete items in dictionary that are not roi items
roi_dict = self.selected_cells_infor_dict.copy()
del_key_list = []
for key in roi_dict:
print(key)
if 'ROIitem' not in key:
del_key_list.append(key)
for key in del_key_list:
del roi_dict[key]
self.MLmask = ProcessImage.ROIitem2Mask(
roi_dict,
mask_resolution=(self.MLtargetedImg.shape[0],
self.MLtargetedImg.shape[1]))
# Binary mask of added rois
self.addedROIitemMask = ProcessImage.ROIitem2Mask(
self.roi_list_freehandl_added,
mask_resolution=(self.MLtargetedImg.shape[0],
self.MLtargetedImg.shape[1]))
self.intergrate_into_final_mask()
# if type(self.roi_list_freehandl_added) is list:
# for ROIitem in self.roi_list_freehandl_added:
#
# ROIitem.sigHoverEvent.connect(lambda: self.show_roi_detail(ROIitem))
#
# plt.figure()
# plt.imshow(self.addedROIitemMask)
# plt.show()
# =============================================================================
# For free-hand rois
# =============================================================================
def enable_free_hand(self):
if self.enable_modify_MLmask_button.isChecked():
self.Mask_edit_view.enable_drawing(True)
else:
self.Mask_edit_view.enable_drawing(False)
def add_freehand_roi(self, roi):
# For drawwidget
self.roi_list_freehandl_added.append(roi)
def clear_edit_roi(self):
"""
Clean up all the free-hand rois.
"""
for roi in self.roi_list_freehandl_added:
self.Mask_edit_view.getView().removeItem(roi)
self.roi_list_freehandl_added = []
if len(self.selected_cells_infor_dict) > 0:
# Remove all selected masks
for roiItemkey in self.selected_cells_infor_dict:
if 'ROIitem' in roiItemkey:
self.Mask_edit_view.getView().removeItem(
self.selected_cells_infor_dict[roiItemkey])
self.selected_cells_infor_dict = {}
self.MLmask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
self.intergrate_into_final_mask()
def intergrate_into_final_mask(self):
# Binary mask of added rois
self.addedROIitemMask = ProcessImage.ROIitem2Mask(
self.roi_list_freehandl_added,
mask_resolution=(self.MLtargetedImg.shape[0],
self.MLtargetedImg.shape[1]))
#Display the RGB mask, ML mask plus free-hand added.
self.Mask_edit_viewItem.setImage(gray2rgb(self.addedROIitemMask) * self.mask_color_multiplier + \
gray2rgb(self.MLmask) * self.mask_color_multiplier + gray2rgb(self.MLtargetedImg))
self.final_mask = self.MLmask + self.addedROIitemMask
# In case the input image is 2048*2048, and it is resized to fit in MaskRCNN, need to convert back to original size for DMD tranformation.
if self.final_mask.shape[0] != self.Rawimage.shape[
0] or self.final_mask.shape[1] != self.Rawimage.shape[1]:
self.final_mask = resize(
self.final_mask,
[self.Rawimage.shape[0], self.Rawimage.shape[1]],
preserve_range=True).astype(self.final_mask.dtype)
# self.final_mask = np.where(self.final_mask <= 1, self.final_mask, int(1))
plt.figure()
plt.imshow(self.final_mask)
plt.show()
# =============================================================================
# For DMD transformation and mask generation
# =============================================================================
def generate_transformed_mask(self):
self.read_transformations_from_file()
# self.transform_to_DMD_mask(laser = self.maskLaserComboBox.currentText(), dict_transformations = self.dict_transformations)
target_laser = self.maskLaserComboBox.currentText()
self.final_DMD_mask = self.finalmask_to_DMD_mask(
laser=target_laser, dict_transformations=self.dict_transformations)
plt.figure()
plt.imshow(self.final_DMD_mask)
plt.show()
def emit_mask_contour(self):
"""Use find_contours to get a list of (n,2)-ndarrays consisting of n (row, column) coordinates along the contour,
and then feed the list of signal:[list_of_rois, flag_fill_contour, contour_thickness, flag_invert_mode] to the
receive_mask_coordinates function in DMDWidget.
"""
contours = find_contours(self.final_mask, 0.5)
sig = [
contours,
self.fillContourButton.isChecked(),
self.thicknessSpinBox.value(),
self.invertMaskButton.isChecked()
]
self.signal_DMDcontour.emit(sig)
def emit_mask(self):
target_laser = self.maskLaserComboBox.currentText()
final_DMD_mask_dict = {}
final_DMD_mask_dict['camera-dmd-' + target_laser] = self.final_DMD_mask
self.signal_DMDmask.emit(final_DMD_mask_dict)
def read_transformations_from_file(self):
try:
with open(
r'M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\People\Xin Meng\Code\Python_test\DMDManager\Registration\transformation.txt',
'r') as json_file:
self.dict_transformations = json.load(json_file)
except:
print(
'No transformation could be loaded from previous registration run.'
)
return
# def transform_to_DMD_mask(self, laser, dict_transformations, flag_fill_contour = True, contour_thickness = 1, flag_invert_mode = False, mask_resolution = (1024, 768)):
# """
# Get roi vertices from all roi items and perform the transformation, and then create the mask for DMD.
# """
#
# #list of roi vertices each being (n,2) numpy array for added rois
# if len(self.roi_list_freehandl_added) > 0:
# self.addedROIitem_vertices = ProcessImage.ROIitem2Vertices(self.roi_list_freehandl_added)
# #addedROIitem_vertices needs to be seperated to be inidividual (n,2) np.array
# self.ROIitems_mask_transformed = ProcessImage.vertices_to_DMD_mask(self.addedROIitem_vertices, laser, dict_transformations, flag_fill_contour = True, contour_thickness = 1,\
# flag_invert_mode = False, mask_resolution = (1024, 768))
#
# #Dictionary with (n,2) numpy array for clicked cells
# if len(self.selected_cells_infor_dict) > 0:
# #Convert dictionary to np.array
# for roiItemkey in self.selected_cells_infor_dict:
# #Each one is 'contours' from find_contour
# if '_verts' in roiItemkey:
# self.selected_cells_infor_dict[roiItemkey]
#
# self.MLitems_mask_transformed = ProcessImage.vertices_to_DMD_mask(self.selected_cells_infor_dict, laser, dict_transformations, flag_fill_contour = True, contour_thickness = 1,\
# flag_invert_mode = False, mask_resolution = (1024, 768))
#
# if len(self.roi_list_freehandl_added) > 0:
# self.final_DMD_mask = self.ROIitems_mask_transformed + self.MLitems_mask_transformed
# self.final_DMD_mask[self.final_DMD_mask>1] = 1
# else:
# self.final_DMD_mask = self.MLitems_mask_transformed
#
# return self.final_DMD_mask
def finalmask_to_DMD_mask(self,
laser,
dict_transformations,
flag_fill_contour=True,
contour_thickness=1,
flag_invert_mode=False,
mask_resolution=(1024, 768)):
"""
Same goal as transform_to_DMD_mask, with input being the final binary mask and using find_contour to get all vertices and perform transformation,
and then coordinates to mask.
"""
self.final_DMD_mask = ProcessImage.binarymask_to_DMD_mask(self.final_mask, laser, dict_transformations, flag_fill_contour = True, \
contour_thickness = 1, flag_invert_mode = False, mask_resolution = (1024, 768))
return self.final_DMD_mask
def closeEvent(self, event):
QtWidgets.QApplication.quit()
event.accept()
# def apply_mask(self, image, mask, color, alpha=0.5):
# """Apply the given mask to the image.
# """
# for c in range(3):
# image[:, :, c] = np.where(mask == 1,
# image[:, :, c] *
# (1 - alpha) + alpha * color[c] * 255,
# image[:, :, c])
# return image
#%%
# @run_in_thread
def cam_snap(self):
"""Get a image from camera"""
self.cam = CamActuator()
self.cam.initializeCamera()
exposure_time = self.CamExposureBox.value()
self.Rawimage = self.cam.SnapImage(exposure_time)
self.cam.Exit()
print('Snap finished')
self.MLtargetedImg_raw = self.Rawimage.copy()
self.MLtargetedImg = self.convert_for_MaskRCNN(self.MLtargetedImg_raw)
self.show_raw_image(self.MLtargetedImg)
self.addedROIitemMask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
self.MLmask = np.zeros(
(self.MLtargetedImg.shape[0], self.MLtargetedImg.shape[1]))
def run(self):
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Connect devices.
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
"""
# =============================================================================
# connect the Objective motor
# =============================================================================
"""
print('----------------------Starting to connect the Objective motor-------------------------')
self.pi_device_instance = PIMotor()
print('Objective motor connected.')
self.errornum = 0
self.init_focus_position = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)['1']
print("init_focus_position : {}".format(self.init_focus_position))
"""
# =============================================================================
# connect the Hmamatsu camera
# =============================================================================
"""
self._use_camera = False
# Check if camera is used.
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for waveform_and_cam_key in self.RoundQueueDict[key][1]:
if "CameraPackage_" in waveform_and_cam_key:
if len(self.RoundQueueDict[key][1][waveform_and_cam_key]) != 0:
self._use_camera = True
if self._use_camera:
print('Connecting camera...')
self.HamamatsuCam = CamActuator()
self.HamamatsuCam.initializeCamera()
else:
print('No camera involved.')
"""
# =============================================================================
# connect the Insight X3
# =============================================================================
"""
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.Laserinstance = InsightX3('COM11')
try:
# querygap = 1.1
# self.Laserinstance.SetWatchdogTimer(0) # Disable the laser watchdog!
# Status_watchdog_thread = threading.Thread(target = self.Status_watchdog, args=[querygap], daemon=True)
# Status_watchdog_thread.start()
# time.sleep(1)
#-------------Initialize laser--------------
self.watchdog_flag = False
time.sleep(0.5)
warmupstatus = 0
while int(warmupstatus) != 100:
warmupstatus = self.Laserinstance.QueryWarmupTime()
time.sleep(0.6)
self.watchdog_flag = True
time.sleep(0.5)
except:
print('Laser not connected.')
# If turn on the laser shutter in the beginning
if 'Shutter_Open' in self.GeneralSettingDict['StartUpEvents']:
time.sleep(0.5)
self.Laserinstance.Open_TunableBeamShutter()
time.sleep(0.5)
"""
# =============================================================================
# Initialize ML
# =============================================================================
"""
self._use_ML = False
# Check if machine learning segmentation is used.
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for photocycle_key in self.RoundQueueDict[key][2]:
if "PhotocyclePackage_" in photocycle_key:
if len(self.RoundQueueDict[key][2][photocycle_key]) != 0:
self._use_ML = True
if self._use_ML:
from ImageAnalysis.ImageProcessing_MaskRCNN import ProcessImageML
self.Predictor = ProcessImageML()
print("ML loaded.")
#%%
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Execution
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
GridSequence = 0
TotalGridNumber = self.meshgridnumber**2
for EachGrid in range(TotalGridNumber):
"""
#------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH GRID ::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------
"""
self.Grid_index = EachGrid
"""
# =============================================================================
# For each small repeat unit in the scanning meshgrid
# =============================================================================
"""
time.sleep(0.5)
for EachRound in range(int(len(self.RoundQueueDict)/2-1)): # EachRound is the round sequence number starting from 0, while the actual number used in dictionary is 1.
"""
#-------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH ROUND ::::::::::::::::::::::::::::::::
#-------------------------------------------------------------------------------
"""
print ('----------------------------------------------------------------------------')
print('Below is Grid {}, Round {}.'.format(EachGrid, EachRound+1)) # EachRound+1 is the corresponding round number when setting the dictionary starting from round 1.
"""
# =============================================================================
# Execute Insight event at the beginning of each round
# =============================================================================
"""
self.laser_init(EachRound)
"""
# =============================================================================
# Execute filter event at the beginning of each round
# =============================================================================
"""
self.filters_init(EachRound)
"""
# =============================================================================
# Generate focus position list at the beginning of each round
# =============================================================================
"""
if EachRound == 0:
ZStackinfor = self.GeneralSettingDict['FocusStackInfoDict']['RoundPackage_{}'.format(EachRound+1)]
ZStackNum = int(ZStackinfor[ZStackinfor.index('Focus')+5])
ZStackStep = float(ZStackinfor[ZStackinfor.index('Being')+5:len(ZStackinfor)])
# Generate position list.
ZStacklinspaceStart = self.init_focus_position - (math.floor(ZStackNum/2)) * ZStackStep
ZStacklinspaceEnd = self.init_focus_position + (ZStackNum - math.floor(ZStackNum/2)-1) * ZStackStep
self.ZStackPosList = np.linspace(ZStacklinspaceStart, ZStacklinspaceEnd, num = ZStackNum)
self.currentCoordsSeq = 0
#%%
#-------------Unpack infor for stage move.
CoordsNum = len(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)])
for EachCoord in range(CoordsNum):
"""
#------------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH COORDINATE ::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------------
"""
self.error_massage = None
self.currentCoordsSeq += 1
"""
# =============================================================================
# Stage movement
# =============================================================================
"""
self.coord_array = self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord]
# Offset coordinate row value for each well.
ScanningGridOffset_Row = int(EachGrid % self.meshgridnumber) * (self.GeneralSettingDict['StageGridOffset'])
# Offset coordinate colunm value for each well.
ScanningGridOffset_Col = int(EachGrid/self.meshgridnumber) * (self.GeneralSettingDict['StageGridOffset'])
RowIndex = self.coord_array['row'] + ScanningGridOffset_Row
ColumnIndex = self.coord_array['col'] + ScanningGridOffset_Col
try:
# for _ in range(2): # Repeat twice
# self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
# time.sleep(1)
self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
time.sleep(1.2)
except:
self.error_massage = 'Fail_MoveStage'
self.errornum += 1
print('Stage move failed! Error number: {}'.format(int(self.errornum)))
time.sleep(0.2)
"""
# =============================================================================
# Focus position
# Unpack the focus stack information, conduct auto-focusing if set.
# =============================================================================
"""
# Here also generate the ZStackPosList.
self.ZStackNum = self.unpack_focus_stack(EachGrid, EachRound, EachCoord)
self.stack_focus_degree_list = []
print('*******************************************Round {}. Current index: {}.**************************************************'.format\
(EachRound+1, [RowIndex,ColumnIndex]))
#------------------Move to Z stack focus-------------------
for EachZStackPos in range(self.ZStackNum):
"""
#------------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH ZSTACK ::::::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------------
"""
print('--------------------------------------------Stack {}--------------------------------------------------'.format(EachZStackPos+1))
if self.ZStackNum > 1:
self.ZStackOrder = int(EachZStackPos +1) # Here the first one is 1, not starting from 0.
self.FocusPos = self.ZStackPosList[EachZStackPos]# + self.wavelength_offset
# Add the focus degree of previous image to the list.
# For stack of 3 only.
if EachZStackPos > 0:
try:
self.stack_focus_degree_list.append(self.FocusDegree_img_reconstructed)
except:
# FocusDegree_img_reconstructed is not generated with camera imaging.
pass
print('stack_focus_degree_list is {}'.format(self.stack_focus_degree_list))
#-----Suppose now it's the 3rd stack position-----
if len(self.stack_focus_degree_list) == 2:
if self.stack_focus_degree_list[-1] < self.stack_focus_degree_list[-2]:
self.FocusPos = self.ZStackPosList[0] - (self.ZStackPosList[1] - self.ZStackPosList[0])/2
print('Focus degree decreasing, run the other direction.')
#-------------------------------------------------
print('Target focus pos: {}'.format(self.FocusPos))
self.pi_device_instance.move(self.FocusPos)
# self.auto_focus_positionInStack = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
# print("Current position: {:.4f}".format(self.auto_focus_positionInStack['1']))
time.sleep(0.3)
else:
self.ZStackOrder = 1
self.FocusPos = self.init_focus_position
"""
# =============================================================================
# Execute waveform packages
# =============================================================================
"""
self.Waveform_sequence_Num = int(len(self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]))
#------------For waveforms in each coordinate----------
for EachWaveform in range(self.Waveform_sequence_Num):
"""
# =============================================================================
# For photo-cycle
# =============================================================================
"""
# Get the photo cycle information
PhotocyclePackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][2] \
["PhotocyclePackage_{}".format(EachWaveform+1)]
# See if in this waveform sequence photo cycle is involved.
# PhotocyclePackageToBeExecute is {} if not configured.
if len(PhotocyclePackageToBeExecute) > 0:
# Load the previous acquired camera image
self.cam_tif_name = r"M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Octoscope\2020-8-13 Screening Archon1 library V5 and 6\V6\Round2_Coords181_R19800C0_PMT_0Zmax.tif"
previous_cam_img = imread(self.cam_tif_name)
img_width = previous_cam_img.shape[1]
img_height = previous_cam_img.shape[0]
# Get the segmentation of full image.
fig, ax = plt.subplots()
MLresults = self.Predictor.DetectionOnImage(previous_cam_img, axis = ax)
ROI_number = len(MLresults["scores"])
print('roi number: {}'.format(ROI_number))
for each_ROI in range(ROI_number):
# if MLresults['class_ids'][each_ROI] == 3:
ROIlist = MLresults['rois'][each_ROI]
print(ROIlist)
# np array's column([1]) is the width of image, and is the row in stage coordinates.
ROI_center_width = int(ROIlist[1] + (ROIlist[3] - ROIlist[1])/2)
print('ROI_center_width '.format(ROIlist))
ROI_center_height = int(ROIlist[0] + (ROIlist[2] + ROIlist[0])/2)
print('ROI_center_height '.format(ROIlist))
cam_stage_transform_factor = 1.135
stage_move_col = (int((img_width)/2) - ROI_center_width) * cam_stage_transform_factor
print(stage_move_col)
stage_move_row = (int((img_height)/2) - ROI_center_height) * cam_stage_transform_factor
print(stage_move_row)
# Move cell of interest to the center of field of view
self.ludlStage.moveRel(xRel = stage_move_row, yRel= stage_move_col)
time.sleep(1)
# Move the cell back
self.ludlStage.moveRel(xRel = -1*stage_move_row, yRel= -1*stage_move_col)
time.sleep(1)
#--------------------------------------------------
"""
# =============================================================================
# Execute pre-set operations at EACH COORDINATE.
# =============================================================================
"""
self.inidividual_coordinate_operation(EachRound, EachWaveform, RowIndex, ColumnIndex)
time.sleep(0.6) # Wait for receiving data to be done.
time.sleep(0.5)
print('*************************************************************************************************************************')
#%%
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Disconnect devices.
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
# Switch off laser
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.watchdog_flag = False
time.sleep(0.5)
self.Laserinstance.Close_TunableBeamShutter()
time.sleep(0.5)
self.Laserinstance.SaveVariables()
self.Laserinstance.Turn_Off_PumpLaser()
# Disconnect camera
if self._use_camera == True:
self.HamamatsuCam.Exit()
# Disconnect focus motor
try:
self.pi_device_instance.CloseMotorConnection()
print('Objective motor disconnected.')
except:
pass
class ScanningExecutionThread(QThread):
ScanningResult = pyqtSignal(np.ndarray, np.ndarray, object, object) #The signal for the measurement, we can connect to this signal
#%%
def __init__(self, RoundQueueDict, RoundCoordsDict, GeneralSettingDict, *args, **kwargs):
super().__init__(*args, **kwargs)
self.RoundQueueDict = RoundQueueDict
self.RoundCoordsDict = RoundCoordsDict
self.GeneralSettingDict = GeneralSettingDict
self.Status_list = None
self.ludlStage = LudlStage("COM12")
self.watchdog_flag = True
self.clock_source = 'DAQ' # Should be set by GUI.
self.scansavedirectory = self.GeneralSettingDict['savedirectory']
self.meshgridnumber = int(self.GeneralSettingDict['Meshgrid'])
self.wavelength_offset = 0 # An offset of 0.002 mm is observed between 900 and 1280 nm.
#%%
def run(self):
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Connect devices.
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
"""
# =============================================================================
# connect the Objective motor
# =============================================================================
"""
print('----------------------Starting to connect the Objective motor-------------------------')
self.pi_device_instance = PIMotor()
print('Objective motor connected.')
self.errornum = 0
self.init_focus_position = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)['1']
print("init_focus_position : {}".format(self.init_focus_position))
"""
# =============================================================================
# connect the Hmamatsu camera
# =============================================================================
"""
self._use_camera = False
# Check if camera is used.
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for waveform_and_cam_key in self.RoundQueueDict[key][1]:
if "CameraPackage_" in waveform_and_cam_key:
if len(self.RoundQueueDict[key][1][waveform_and_cam_key]) != 0:
self._use_camera = True
if self._use_camera:
print('Connecting camera...')
self.HamamatsuCam = CamActuator()
self.HamamatsuCam.initializeCamera()
else:
print('No camera involved.')
"""
# =============================================================================
# connect the Insight X3
# =============================================================================
"""
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.Laserinstance = InsightX3('COM11')
try:
# querygap = 1.1
# self.Laserinstance.SetWatchdogTimer(0) # Disable the laser watchdog!
# Status_watchdog_thread = threading.Thread(target = self.Status_watchdog, args=[querygap], daemon=True)
# Status_watchdog_thread.start()
# time.sleep(1)
#-------------Initialize laser--------------
self.watchdog_flag = False
time.sleep(0.5)
warmupstatus = 0
while int(warmupstatus) != 100:
warmupstatus = self.Laserinstance.QueryWarmupTime()
time.sleep(0.6)
self.watchdog_flag = True
time.sleep(0.5)
except:
print('Laser not connected.')
# If turn on the laser shutter in the beginning
if 'Shutter_Open' in self.GeneralSettingDict['StartUpEvents']:
time.sleep(0.5)
self.Laserinstance.Open_TunableBeamShutter()
time.sleep(0.5)
"""
# =============================================================================
# Initialize ML
# =============================================================================
"""
self._use_ML = False
# Check if machine learning segmentation is used.
for key in self.RoundQueueDict:
if "RoundPackage_" in key:
for photocycle_key in self.RoundQueueDict[key][2]:
if "PhotocyclePackage_" in photocycle_key:
if len(self.RoundQueueDict[key][2][photocycle_key]) != 0:
self._use_ML = True
if self._use_ML:
from ImageAnalysis.ImageProcessing_MaskRCNN import ProcessImageML
self.Predictor = ProcessImageML()
print("ML loaded.")
#%%
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Execution
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
GridSequence = 0
TotalGridNumber = self.meshgridnumber**2
for EachGrid in range(TotalGridNumber):
"""
#------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH GRID ::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------
"""
self.Grid_index = EachGrid
"""
# =============================================================================
# For each small repeat unit in the scanning meshgrid
# =============================================================================
"""
time.sleep(0.5)
for EachRound in range(int(len(self.RoundQueueDict)/2-1)): # EachRound is the round sequence number starting from 0, while the actual number used in dictionary is 1.
"""
#-------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH ROUND ::::::::::::::::::::::::::::::::
#-------------------------------------------------------------------------------
"""
print ('----------------------------------------------------------------------------')
print('Below is Grid {}, Round {}.'.format(EachGrid, EachRound+1)) # EachRound+1 is the corresponding round number when setting the dictionary starting from round 1.
"""
# =============================================================================
# Execute Insight event at the beginning of each round
# =============================================================================
"""
self.laser_init(EachRound)
"""
# =============================================================================
# Execute filter event at the beginning of each round
# =============================================================================
"""
self.filters_init(EachRound)
"""
# =============================================================================
# Generate focus position list at the beginning of each round
# =============================================================================
"""
if EachRound == 0:
ZStackinfor = self.GeneralSettingDict['FocusStackInfoDict']['RoundPackage_{}'.format(EachRound+1)]
ZStackNum = int(ZStackinfor[ZStackinfor.index('Focus')+5])
ZStackStep = float(ZStackinfor[ZStackinfor.index('Being')+5:len(ZStackinfor)])
# Generate position list.
ZStacklinspaceStart = self.init_focus_position - (math.floor(ZStackNum/2)) * ZStackStep
ZStacklinspaceEnd = self.init_focus_position + (ZStackNum - math.floor(ZStackNum/2)-1) * ZStackStep
self.ZStackPosList = np.linspace(ZStacklinspaceStart, ZStacklinspaceEnd, num = ZStackNum)
self.currentCoordsSeq = 0
#%%
#-------------Unpack infor for stage move.
CoordsNum = len(self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)])
for EachCoord in range(CoordsNum):
"""
#------------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH COORDINATE ::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------------
"""
self.error_massage = None
self.currentCoordsSeq += 1
"""
# =============================================================================
# Stage movement
# =============================================================================
"""
self.coord_array = self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord]
# Offset coordinate row value for each well.
ScanningGridOffset_Row = int(EachGrid % self.meshgridnumber) * (self.GeneralSettingDict['StageGridOffset'])
# Offset coordinate colunm value for each well.
ScanningGridOffset_Col = int(EachGrid/self.meshgridnumber) * (self.GeneralSettingDict['StageGridOffset'])
RowIndex = self.coord_array['row'] + ScanningGridOffset_Row
ColumnIndex = self.coord_array['col'] + ScanningGridOffset_Col
try:
# for _ in range(2): # Repeat twice
# self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
# time.sleep(1)
self.ludlStage.moveAbs(RowIndex,ColumnIndex) # Row/Column indexs of np.array are opposite of stage row-col indexs.
time.sleep(1.2)
except:
self.error_massage = 'Fail_MoveStage'
self.errornum += 1
print('Stage move failed! Error number: {}'.format(int(self.errornum)))
time.sleep(0.2)
"""
# =============================================================================
# Focus position
# Unpack the focus stack information, conduct auto-focusing if set.
# =============================================================================
"""
# Here also generate the ZStackPosList.
self.ZStackNum = self.unpack_focus_stack(EachGrid, EachRound, EachCoord)
self.stack_focus_degree_list = []
print('*******************************************Round {}. Current index: {}.**************************************************'.format\
(EachRound+1, [RowIndex,ColumnIndex]))
#------------------Move to Z stack focus-------------------
for EachZStackPos in range(self.ZStackNum):
"""
#------------------------------------------------------------------------------------
#:::::::::::::::::::::::::::::::: AT EACH ZSTACK ::::::::::::::::::::::::::::::::::::
#------------------------------------------------------------------------------------
"""
print('--------------------------------------------Stack {}--------------------------------------------------'.format(EachZStackPos+1))
if self.ZStackNum > 1:
self.ZStackOrder = int(EachZStackPos +1) # Here the first one is 1, not starting from 0.
self.FocusPos = self.ZStackPosList[EachZStackPos]# + self.wavelength_offset
# Add the focus degree of previous image to the list.
# For stack of 3 only.
if EachZStackPos > 0:
try:
self.stack_focus_degree_list.append(self.FocusDegree_img_reconstructed)
except:
# FocusDegree_img_reconstructed is not generated with camera imaging.
pass
print('stack_focus_degree_list is {}'.format(self.stack_focus_degree_list))
#-----Suppose now it's the 3rd stack position-----
if len(self.stack_focus_degree_list) == 2:
if self.stack_focus_degree_list[-1] < self.stack_focus_degree_list[-2]:
self.FocusPos = self.ZStackPosList[0] - (self.ZStackPosList[1] - self.ZStackPosList[0])/2
print('Focus degree decreasing, run the other direction.')
#-------------------------------------------------
print('Target focus pos: {}'.format(self.FocusPos))
self.pi_device_instance.move(self.FocusPos)
# self.auto_focus_positionInStack = self.pi_device_instance.pidevice.qPOS(self.pi_device_instance.pidevice.axes)
# print("Current position: {:.4f}".format(self.auto_focus_positionInStack['1']))
time.sleep(0.3)
else:
self.ZStackOrder = 1
self.FocusPos = self.init_focus_position
"""
# =============================================================================
# Execute waveform packages
# =============================================================================
"""
self.Waveform_sequence_Num = int(len(self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]))
#------------For waveforms in each coordinate----------
for EachWaveform in range(self.Waveform_sequence_Num):
"""
# =============================================================================
# For photo-cycle
# =============================================================================
"""
# Get the photo cycle information
PhotocyclePackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][2] \
["PhotocyclePackage_{}".format(EachWaveform+1)]
# See if in this waveform sequence photo cycle is involved.
# PhotocyclePackageToBeExecute is {} if not configured.
if len(PhotocyclePackageToBeExecute) > 0:
# Load the previous acquired camera image
self.cam_tif_name = r"M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Octoscope\2020-8-13 Screening Archon1 library V5 and 6\V6\Round2_Coords181_R19800C0_PMT_0Zmax.tif"
previous_cam_img = imread(self.cam_tif_name)
img_width = previous_cam_img.shape[1]
img_height = previous_cam_img.shape[0]
# Get the segmentation of full image.
fig, ax = plt.subplots()
MLresults = self.Predictor.DetectionOnImage(previous_cam_img, axis = ax)
ROI_number = len(MLresults["scores"])
print('roi number: {}'.format(ROI_number))
for each_ROI in range(ROI_number):
# if MLresults['class_ids'][each_ROI] == 3:
ROIlist = MLresults['rois'][each_ROI]
print(ROIlist)
# np array's column([1]) is the width of image, and is the row in stage coordinates.
ROI_center_width = int(ROIlist[1] + (ROIlist[3] - ROIlist[1])/2)
print('ROI_center_width '.format(ROIlist))
ROI_center_height = int(ROIlist[0] + (ROIlist[2] + ROIlist[0])/2)
print('ROI_center_height '.format(ROIlist))
cam_stage_transform_factor = 1.135
stage_move_col = (int((img_width)/2) - ROI_center_width) * cam_stage_transform_factor
print(stage_move_col)
stage_move_row = (int((img_height)/2) - ROI_center_height) * cam_stage_transform_factor
print(stage_move_row)
# Move cell of interest to the center of field of view
self.ludlStage.moveRel(xRel = stage_move_row, yRel= stage_move_col)
time.sleep(1)
# Move the cell back
self.ludlStage.moveRel(xRel = -1*stage_move_row, yRel= -1*stage_move_col)
time.sleep(1)
#--------------------------------------------------
"""
# =============================================================================
# Execute pre-set operations at EACH COORDINATE.
# =============================================================================
"""
self.inidividual_coordinate_operation(EachRound, EachWaveform, RowIndex, ColumnIndex)
time.sleep(0.6) # Wait for receiving data to be done.
time.sleep(0.5)
print('*************************************************************************************************************************')
#%%
"""~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
# Disconnect devices.
# ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~"""
# Switch off laser
if len(self.RoundQueueDict['InsightEvents']) != 0:
self.watchdog_flag = False
time.sleep(0.5)
self.Laserinstance.Close_TunableBeamShutter()
time.sleep(0.5)
self.Laserinstance.SaveVariables()
self.Laserinstance.Turn_Off_PumpLaser()
# Disconnect camera
if self._use_camera == True:
self.HamamatsuCam.Exit()
# Disconnect focus motor
try:
self.pi_device_instance.CloseMotorConnection()
print('Objective motor disconnected.')
except:
pass
#%%
def laser_init(self, EachRound):
"""
Execute Insight event at the beginning of each round
Parameters
----------
EachRound : int
Round index.
Returns
-------
None.
"""
#-Unpack infor for Insight X3. In the list, the first one is for shutter event and the second one is for wavelength event.
InsightX3EventIndexList = [i for i,x in enumerate(self.RoundQueueDict['InsightEvents']) if 'Round_{}'.format(EachRound+1) in x]
if len(InsightX3EventIndexList) == 1:
print(InsightX3EventIndexList)
InsightText = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'Shutter_Open' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
self.Laserinstance.Open_TunableBeamShutter()
time.sleep(0.5)
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'Shutter_Close' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
self.Laserinstance.Close_TunableBeamShutter()
time.sleep(0.5)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText[InsightText.index('To_')+3:len(InsightText)])
if TargetWavelen == 1280:
self.wavelength_offset = -0.002 # give an offset if wavelength goes to 1280.
elif TargetWavelen == 900:
self.wavelength_offset = 0
self.Laserinstance.SetWavelength(TargetWavelen)
time.sleep(5)
self.watchdog_flag = True
time.sleep(0.5)
elif len(InsightX3EventIndexList) == 2:
InsightText_wl = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[1]]
InsightText_st = self.RoundQueueDict['InsightEvents'][InsightX3EventIndexList[0]]
if 'WavelengthTo' in InsightText_wl and 'Shutter_Open' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
self.Laserinstance.SetWavelength(TargetWavelen)
if TargetWavelen == 1280:
self.wavelength_offset = -0.002 # give an offset if wavelength goes to 1280.
elif TargetWavelen == 900:
self.wavelength_offset = 0
time.sleep(5)
self.Laserinstance.Open_TunableBeamShutter()
print('Laser shutter open.')
self.watchdog_flag = True
time.sleep(0.5)
elif 'WavelengthTo' in InsightText_wl and 'Shutter_Close' in InsightText_st:
self.watchdog_flag = False
time.sleep(0.5)
TargetWavelen = int(InsightText_wl[InsightText_wl.index('To_')+3:len(InsightText_wl)])
self.Laserinstance.SetWavelength(TargetWavelen)
if TargetWavelen == 1280:
self.wavelength_offset = -0.002 # give an offset if wavelength goes to 1280.
elif TargetWavelen == 900:
self.wavelength_offset = 0
time.sleep(5)
self.Laserinstance.Close_TunableBeamShutter()
time.sleep(1)
print('Laser shutter closed.')
self.watchdog_flag = True
time.sleep(0.5)
time.sleep(2)
def filters_init(self, EachRound):
"""
Execute filter event at the beginning of each round.
Parameters
----------
EachRound : int
Round index.
Returns
-------
None.
"""
#-Unpack infor for filter event. In the list, the first one is for ND filter and the second one is for emission filter.
FilterEventIndexList = [i for i,x in enumerate(self.RoundQueueDict['FilterEvents']) if 'Round_{}'.format(EachRound+1) in x]
if len(FilterEventIndexList) > 0:
NDposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[0]]
NDnumber = NDposText[NDposText.index('ToPos_')+6:len(NDposText)]
EMposText = self.RoundQueueDict['FilterEvents'][FilterEventIndexList[1]]
EMprotein = EMposText[EMposText.index('ToPos_')+6:len(EMposText)]
# "COM9" for filter 1 port, which has ND values from 0 to 3.
# "COM7" for filter 2 port, which has ND values from 0 to 0.5.
if NDnumber == '0':
ND_filter1_Pos = 0
ND_filter2_Pos = 0
elif NDnumber == '1':
ND_filter1_Pos = 1
ND_filter2_Pos = 0
elif NDnumber == '2':
ND_filter1_Pos = 2
ND_filter2_Pos = 0
elif NDnumber == '2.3':
ND_filter1_Pos = 2
ND_filter2_Pos = 2
elif NDnumber == '2.5':
ND_filter1_Pos = 2
ND_filter2_Pos = 3
elif NDnumber == '0.5':
ND_filter1_Pos = 0
ND_filter2_Pos = 3
elif NDnumber == '0.3':
ND_filter1_Pos = 0
ND_filter2_Pos = 2
if EMprotein == 'Arch':
EM_filter_Pos = 0
elif EMprotein == 'eGFP' or EMprotein == 'Citrine':
EM_filter_Pos = 1
# Execution
if ND_filter1_Pos != None and ND_filter2_Pos != None:
#Move filter 1
self.filter1 = ELL9Filter("COM9")
self.filter1.moveToPosition(ND_filter1_Pos)
time.sleep(1)
#Move filter 2
self.filter2 = ELL9Filter("COM7")
self.filter2.moveToPosition(ND_filter2_Pos)
time.sleep(1)
if EM_filter_Pos != None:
self.filter3 = ELL9Filter("COM15")
self.filter3.moveToPosition(EM_filter_Pos)
time.sleep(1)
def unpack_focus_stack(self, EachGrid, EachRound, EachCoord):
"""
Unpack the focus stack information.
Determine focus position either from pre-set numbers of by auto-focusing.
Parameters
----------
EachGrid : int
Current grid index.
EachRound : int
Current round index.
EachCoord : int
Current coordinate index.
Returns
-------
ZStackNum : int
Number of focus positions in stack.
ZStackPosList : list
List of focus stack positions for objective to go to.
"""
ZStackinfor = self.GeneralSettingDict['FocusStackInfoDict']['RoundPackage_{}'.format(EachRound+1)]
ZStackNum = int(ZStackinfor[ZStackinfor.index('Focus')+5])
ZStackStep = float(ZStackinfor[ZStackinfor.index('Being')+5:len(ZStackinfor)])
# If manual focus correction applies, unpact the target focus infor.
if len(self.GeneralSettingDict['FocusCorrectionMatrixDict']) > 0:
FocusPosArray = self.GeneralSettingDict['FocusCorrectionMatrixDict']['RoundPackage_{}_Grid_{}'.format(EachRound+1, EachGrid)]
FocusPosArray = FocusPosArray.flatten('F')
FocusPos_fromCorrection = FocusPosArray[EachCoord]
ZStacklinspaceStart = FocusPos_fromCorrection - (math.floor(ZStackNum/2))* ZStackStep
ZStacklinspaceEnd = FocusPos_fromCorrection + (ZStackNum - math.floor(ZStackNum/2)-1) * ZStackStep
# With auto-focus correction
else:
# If go for auto-focus at this coordinate
auto_focus_flag = self.coord_array['auto_focus_flag']
# auto_focus_flag = False
print('focus_position {}'.format(self.coord_array['focus_position']))
#-----------------------Auto focus---------------------------------
if auto_focus_flag == "yes":
if self.coord_array['focus_position'] == -1.:
instance_FocusFinder = FocusFinder(motor_handle = self.pi_device_instance)
print("--------------Start auto-focusing-----------------")
# instance_FocusFinder.total_step_number = 7
# instance_FocusFinder.init_step_size = 0.013
# self.auto_focus_position = instance_FocusFinder.gaussian_fit()
self.auto_focus_position = instance_FocusFinder.bisection()
relative_move_coords = [[1550,0],[0,1550],[1550,1550]]
trial_num = 0
while self.auto_focus_position == False: # If there's no cell in FOV
if trial_num <= 2:
print('No cells found. move to next pos.')
# Move to next position in real scanning coordinates.
self.ludlStage.moveRel(relative_move_coords[trial_num][0],relative_move_coords[trial_num][1])
time.sleep(1)
print('Now stage pos is {}'.format(self.ludlStage.getPos()))
self.auto_focus_position = instance_FocusFinder.bisection()
# Move back
self.ludlStage.moveRel(-1*relative_move_coords[trial_num][0],-1*relative_move_coords[trial_num][1])
trial_num += 1
else:
print('No cells in neighbouring area.')
self.auto_focus_position = self.ZStackPosList[int(len(self.ZStackPosList)/2)]
break
print("--------------End of auto-focusing----------------")
time.sleep(1)
# Record the position, try to write it in the NEXT round dict.
try:
self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound + 2)][EachCoord]['focus_position'] = self.auto_focus_position
except:# If it's already the last round, skip.
pass
# Generate position list.
ZStacklinspaceStart = self.auto_focus_position - (math.floor(ZStackNum/2)) * ZStackStep
ZStacklinspaceEnd = self.auto_focus_position + (ZStackNum - math.floor(ZStackNum/2)-1) * ZStackStep
else: # If there's already position from last round, move to it.
self.previous_auto_focus_position = self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound+1)][EachCoord]['focus_position']
try:
# Record the position, try to write it in the NEXT round dict.
self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound + 2)][EachCoord]['focus_position'] = self.previous_auto_focus_position
except:
pass
# Generate position list.
ZStacklinspaceStart = self.previous_auto_focus_position - (math.floor(ZStackNum/2)) * ZStackStep
ZStacklinspaceEnd = self.previous_auto_focus_position + (ZStackNum - math.floor(ZStackNum/2)-1) * ZStackStep
# self.previous_auto_focus_position = self.auto_focus_position
# print("=====================Move to last recorded position=================")
# self.pi_device_instance.move(self.previous_auto_focus_position)
# time.sleep(0.2)
# instance_FocusFinder = FocusFinder(motor_handle = self.pi_device_instance)
# print("--------------Start auto-focusing-----------------")
# self.auto_focus_position = instance_FocusFinder.bisection()
# try:
# if self.auto_focus_position[0] == False: # If there's no cell in FOV
# # Skip? mid_position = [False, self.current_pos]
# self.auto_focus_position = self.auto_focus_position[1]
# except:
# pass
# print("--------------End of auto-focusing----------------")
# time.sleep(1)
# # Record the position, try to write it in the NEXT round dict.
# try:
# self.RoundCoordsDict['CoordsPackage_{}'.format(EachRound + 2)][EachCoord]['focus_position'] = self.auto_focus_position
# except:# If it's already the last round, skip.
# pass
# Generate the position list, for none-auto-focus coordinates they will use the same list variable.
self.ZStackPosList = np.linspace(ZStacklinspaceStart, ZStacklinspaceEnd, num = ZStackNum)
print('ZStackPos is : {}'.format(self.ZStackPosList))
#------------------------------------------------------------------
# If not auto focus, use the same list variable self.ZStackPosList.
else:
pass
return ZStackNum
def inidividual_coordinate_operation(self, EachRound, EachWaveform, RowIndex, ColumnIndex):
"""
Execute pre-set operations at each coordinate.
Parameters
----------
EachRound : int
Current round index.
EachWaveform : int
Current waveform package index.
RowIndex : int
Current sample stage row index.
ColumnIndex : int
Current sample stage row index.
Returns
-------
None.
"""
# Extract information
WaveformPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][0]['WaveformPackage_{}'.format(EachWaveform+1)]
CameraPackageToBeExecute = self.RoundQueueDict['RoundPackage_{}'.format(EachRound+1)][1]["CameraPackage_{}".format(EachWaveform+1)]
WaveformPackageGalvoInfor = self.RoundQueueDict['GalvoInforPackage_{}'.format(EachRound+1)]['GalvoInfor_{}'.format(EachWaveform+1)]
self.readinchan = WaveformPackageToBeExecute[3]
self.RoundWaveformIndex = [EachRound+1, EachWaveform+1] # first is current round number, second is current waveform package number.
self.CurrentPosIndex = [RowIndex, ColumnIndex]
#----------------Camera operations-----------------
_camera_isUsed = False
if CameraPackageToBeExecute != {}: # if camera operations are configured
_camera_isUsed = True
CamSettigList = CameraPackageToBeExecute["Settings"]
self.HamamatsuCam.StartStreaming(BufferNumber = CameraPackageToBeExecute["Buffer_number"],
trigger_source = CamSettigList[CamSettigList.index("trigger_source")+1],
exposure_time = CamSettigList[CamSettigList.index("exposure_time")+1],
trigger_active = CamSettigList[CamSettigList.index("trigger_active")+1],
subarray_hsize = CamSettigList[CamSettigList.index("subarray_hsize")+1],
subarray_vsize = CamSettigList[CamSettigList.index("subarray_vsize")+1],
subarray_hpos = CamSettigList[CamSettigList.index("subarray_hpos")+1],
subarray_vpos = CamSettigList[CamSettigList.index("subarray_vpos")+1])
# HamamatsuCam starts another thread to pull out frames from buffer.
# Make sure that the camera is prepared before waveform execution.
# while self.HamamatsuCam.isStreaming == False:
# print('Waiting for camera...')
# time.sleep(0.5)
time.sleep(1)
print('Now start waveforms')
#----------------Waveforms operations--------------
if WaveformPackageGalvoInfor != 'NoGalvo': # Unpack the information of galvo scanning.
self.readinchan = WaveformPackageGalvoInfor[0]
self.repeatnum = WaveformPackageGalvoInfor[1]
self.PMT_data_index_array = WaveformPackageGalvoInfor[2]
self.averagenum = WaveformPackageGalvoInfor[3]
self.lenSample_1 = WaveformPackageGalvoInfor[4]
self.ScanArrayXnum = WaveformPackageGalvoInfor[5]
self.adcollector = DAQmission()
# self.adcollector.collected_data.connect(self.ProcessData)
self.adcollector.runWaveforms(clock_source = self.clock_source, sampling_rate = WaveformPackageToBeExecute[0],
analog_signals = WaveformPackageToBeExecute[1], digital_signals = WaveformPackageToBeExecute[2],
readin_channels = WaveformPackageToBeExecute[3])
self.adcollector.save_as_binary(self.scansavedirectory)
self.recorded_raw_data = self.adcollector.get_raw_data()
self.Process_raw_data()
#------------------Camera saving-------------------
if _camera_isUsed == True:
self.HamamatsuCam.isSaving = True
img_text = "_Cam_"+str(self.RoundWaveformIndex[1])+"_Zpos" + str(self.ZStackOrder)
self.cam_tif_name = self.generate_tif_name(extra_text = img_text)
self.HamamatsuCam.StopStreaming(saving_dir = self.cam_tif_name)
# Make sure that the saving process is finished.
while self.HamamatsuCam.isSaving == True:
print('Camera saving...')
time.sleep(0.5)
time.sleep(1)
time.sleep(0.5)
def Process_raw_data(self):
self.channel_number = len(self.recorded_raw_data)
self.data_collected_0 = self.recorded_raw_data[0][0:len(self.recorded_raw_data[0])-1]*-1
print(len(self.data_collected_0))
if self.channel_number == 1:
if 'Vp' in self.readinchan:
pass
elif 'Ip' in self.readinchan:
pass
elif 'PMT' in self.readinchan: # repeatnum, PMT_data_index_array, averagenum, ScanArrayXnum
# Reconstruct the image from np array and save it.
self.PMT_image_processing()
elif self.channel_number == 2:
if 'PMT' not in self.readinchan:
pass
elif 'PMT' in self.readinchan:
self.PMT_image_processing()
print('ProcessData executed.')
# def ProcessData(self, data_waveformreceived):
# print('ZStackOrder is:'+str(self.ZStackOrder)+'numis_'+str(self.ZStackNum))
# self.adcollector.save_as_binary(self.scansavedirectory)
# self.channel_number = len(data_waveformreceived)
# self.data_collected_0 = data_waveformreceived[0]*-1
# self.data_collected_0 = self.data_collected_0[0:len(self.data_collected_0)-1]
# print(len(self.data_collected_0))
# if self.channel_number == 1:
# if 'Vp' in self.readinchan:
# pass
# elif 'Ip' in self.readinchan:
# pass
# elif 'PMT' in self.readinchan: # repeatnum, PMT_data_index_array, averagenum, ScanArrayXnum
# # Reconstruct the image from np array and save it.
# self.PMT_image_processing()
# elif self.channel_number == 2:
# if 'PMT' not in self.readinchan:
# pass
# elif 'PMT' in self.readinchan:
# self.PMT_image_processing()
# print('ProcessData executed.')
def PMT_image_processing(self):
"""
Reconstruct the image from np array and save it.
Returns
-------
None.
"""
for imageSequence in range(self.repeatnum):
try:
self.PMT_image_reconstructed_array = self.data_collected_0[np.where(self.PMT_data_index_array == imageSequence+1)]
Dataholder_average = np.mean(self.PMT_image_reconstructed_array.reshape(self.averagenum, -1), axis=0)
Value_yPixels = int(self.lenSample_1/self.ScanArrayXnum)
self.PMT_image_reconstructed = np.reshape(Dataholder_average, (Value_yPixels, self.ScanArrayXnum))
self.PMT_image_reconstructed = self.PMT_image_reconstructed[:, 50:550] # Crop size based on: M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Xin\2019-12-30 2p beads area test 4um
# self.PMT_image_reconstructed = self.PMT_image_reconstructed[:, 70:326] # for 256*256 images
# Evaluate the focus degree of re-constructed image.
self.FocusDegree_img_reconstructed = ProcessImage.local_entropy(self.PMT_image_reconstructed.astype('float32'))
print('FocusDegree_img_reconstructed is {}'.format(self.FocusDegree_img_reconstructed))
# Save the individual file.
with skimtiff.TiffWriter(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0]) + '_Grid' + str(self.Grid_index) +'_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zpos'+str(self.ZStackOrder)+'.tif'), imagej = True) as tif:
tif.save(self.PMT_image_reconstructed.astype('float32'), compress=0, metadata = {"FocusPos: " : str(self.FocusPos)})
plt.figure()
plt.imshow(self.PMT_image_reconstructed, cmap = plt.cm.gray) # For reconstructed image we pull out the first layer, getting 2d img.
plt.show()
#---------------------------------------------For multiple images in one z pos, Stack the arrays into a 3d array--------------------------------------------------------------------------
# if imageSequence == 0:
# self.PMT_image_reconstructed_stack = self.PMT_image_reconstructed[np.newaxis, :, :] # Turns into 3d array
# else:
# self.PMT_image_reconstructed_stack = np.concatenate((self.PMT_image_reconstructed_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
# print(self.PMT_image_reconstructed_stack.shape)
#---------------------------------------------Calculate the z max projection-----------------------------------------------------------------------
if self.repeatnum == 1: # Consider one repeat image situlation
if self.ZStackNum > 1:
if self.ZStackOrder == 1:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
else:
self.PMT_image_maxprojection_stack = np.concatenate((self.PMT_image_maxprojection_stack, self.PMT_image_reconstructed[np.newaxis, :, :]), axis=0)
else:
self.PMT_image_maxprojection_stack = self.PMT_image_reconstructed[np.newaxis, :, :]
# Save the max projection image
if self.ZStackOrder == self.ZStackNum:
self.PMT_image_maxprojection = np.max(self.PMT_image_maxprojection_stack, axis=0)
# Save the zmax file.
with skimtiff.TiffWriter(os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+ '_Grid' + str(self.Grid_index) + '_Coords'+str(self.currentCoordsSeq)+'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+'_PMT_'+str(imageSequence)+'Zmax'+'.tif'), imagej = True) as tif:
tif.save(self.PMT_image_maxprojection.astype('float32'), compress=0, metadata = {"FocusPos: " : str(self.FocusPos)})
except:
print('No.{} image failed to generate.'.format(imageSequence))
def generate_tif_name(self, extra_text = "_"):
tif_name = os.path.join(self.scansavedirectory, 'Round'+str(self.RoundWaveformIndex[0])+'_Coords'+str(self.currentCoordsSeq)+ \
'_R'+str(self.CurrentPosIndex[0])+'C'+str(self.CurrentPosIndex[1])+ extra_text +'.tif')
return tif_name
#----------------------------------------------------------------WatchDog for laser----------------------------------------------------------------------------------
def Status_watchdog(self, querygap):
while True:
if self.watchdog_flag == True:
self.Status_list = self.Laserinstance.QueryStatus()
time.sleep(querygap)
else:
print('Watchdog stopped')
time.sleep(querygap)
class CoordinatesWidgetUI(QWidget):
sig_cast_mask_coordinates_to_dmd = pyqtSignal(list)
sig_cast_mask_coordinates_to_galvo = pyqtSignal(list)
sig_start_registration = pyqtSignal()
sig_finished_registration = pyqtSignal()
sig_cast_camera_image = pyqtSignal(np.ndarray)
def __init__(self, parent=None, *args, **kwargs):
super().__init__(*args, **kwargs)
self.main_application = parent
self.init_gui()
def closeEvent(self, event):
try:
self.DMD
except:
pass
else:
self.DMD.disconnect_DMD()
QtWidgets.QApplication.quit()
event.accept()
def init_gui(self):
self.setWindowTitle("Coordinate control")
self.layout = QGridLayout()
self.setMinimumSize(1250, 1000)
self.setLayout(self.layout)
self.image_mask_stack = QTabWidget()
self.selection_view = DrawingWidget(self)
self.selection_view.enable_drawing(True)
self.selection_view.getView().setLimits(xMin=0,
xMax=2048,
yMin=0,
yMax=2048,
minXRange=2048,
minYRange=2048,
maxXRange=2048,
maxYRange=2048)
self.selection_view.ui.roiBtn.hide()
self.selection_view.ui.menuBtn.hide()
self.selection_view.ui.normGroup.hide()
self.selection_view.ui.roiPlot.hide()
# self.selection_view.setImage(plt.imread('CoordinatesManager/Registration_Images/StageRegistration/Distance200_Offset0/A1.png'))
self.mask_view = SquareImageView()
self.mask_view.getView().setLimits(xMin=0,
xMax=2048,
yMin=0,
yMax=2048,
minXRange=2048,
minYRange=2048,
maxXRange=2048,
maxYRange=2048)
self.mask_view.ui.roiBtn.hide()
self.mask_view.ui.menuBtn.hide()
self.mask_view.ui.normGroup.hide()
self.mask_view.ui.roiPlot.hide()
self.mask_view.ui.histogram.hide()
self.image_mask_stack.addTab(self.selection_view, 'Select')
self.image_mask_stack.addTab(self.mask_view, 'Mask')
self.layout.addWidget(self.image_mask_stack, 0, 0, 5, 1)
# ---------------------- Mask generation Container --------------
self.maskGeneratorContainer = roundQGroupBox()
self.maskGeneratorContainer.setFixedSize(320, 220)
self.maskGeneratorContainer.setTitle("Mask generator")
self.maskGeneratorContainerLayout = QGridLayout()
self.maskGeneratorLayout = QGridLayout()
self.maskGeneratorContainer.setLayout(
self.maskGeneratorContainerLayout)
self.loadMaskFromFileButton = QPushButton('Open mask')
self.loadMaskFromFileButton.clicked.connect(self.load_mask_from_file)
self.addRoiButton = QPushButton("Add ROI")
self.createMaskButton = QPushButton("Create mask")
self.removeSelectionButton = QPushButton("Remove ROIs")
self.addRoiButton.clicked.connect(self.add_polygon_roi)
self.createMaskButton.clicked.connect(self.create_mask)
self.removeSelectionButton.clicked.connect(self.remove_selection)
self.maskGeneratorContainerLayout.addWidget(self.addRoiButton, 1, 0)
self.maskGeneratorContainerLayout.addWidget(self.createMaskButton, 2,
0)
self.maskGeneratorContainerLayout.addWidget(self.removeSelectionButton,
1, 1)
self.selectionOptionsContainer = roundQGroupBox()
self.selectionOptionsContainer.setTitle('Options')
self.selectionOptionsLayout = QGridLayout()
self.fillContourButton = QCheckBox()
self.invertMaskButton = QCheckBox()
self.thicknessSpinBox = QSpinBox()
self.thicknessSpinBox.setRange(1, 25)
self.selectionOptionsLayout.addWidget(QLabel('Fill contour:'), 0, 0)
self.selectionOptionsLayout.addWidget(self.fillContourButton, 0, 1)
self.selectionOptionsLayout.addWidget(QLabel('Invert mask:'), 1, 0)
self.selectionOptionsLayout.addWidget(self.invertMaskButton, 1, 1)
self.selectionOptionsLayout.addWidget(QLabel('Thickness:'), 2, 0)
self.selectionOptionsLayout.addWidget(self.thicknessSpinBox, 2, 1)
self.selectionOptionsContainer.setLayout(self.selectionOptionsLayout)
self.snapFovButton = QPushButton('Image FOV')
self.snapFovButton.clicked.connect(self.snap_fov)
self.maskGeneratorContainerLayout.addWidget(self.snapFovButton, 0, 0,
1, 1)
self.maskGeneratorContainerLayout.addWidget(
self.loadMaskFromFileButton, 0, 1, 1, 1)
self.maskGeneratorContainerLayout.addWidget(
self.selectionOptionsContainer, 2, 1, 2, 1)
self.layout.addWidget(self.maskGeneratorContainer, 0, 1)
self.DMDWidget = DMDWidget.DMDWidget()
self.layout.addWidget(self.DMDWidget, 1, 1)
self.DMDWidget.sig_request_mask_coordinates.connect(
lambda: self.cast_mask_coordinates('dmd'))
self.sig_cast_mask_coordinates_to_dmd.connect(
self.DMDWidget.receive_mask_coordinates)
self.DMDWidget.sig_start_registration.connect(
lambda: self.sig_start_registration.emit())
self.DMDWidget.sig_finished_registration.connect(
lambda: self.sig_finished_registration.emit())
self.GalvoWidget = GalvoWidget.GalvoWidget()
self.layout.addWidget(self.GalvoWidget, 2, 1)
self.GalvoWidget.sig_request_mask_coordinates.connect(
lambda: self.cast_mask_coordinates('galvo'))
self.sig_cast_mask_coordinates_to_galvo.connect(
self.GalvoWidget.receive_mask_coordinates)
self.GalvoWidget.sig_start_registration.connect(
lambda: self.sig_start_registration.emit())
self.GalvoWidget.sig_finished_registration.connect(
lambda: self.sig_finished_registration.emit())
self.ManualRegistrationWidget = ManualRegistration.ManualRegistrationWidget(
self)
self.ManualRegistrationWidget.sig_request_camera_image.connect(
self.cast_camera_image)
self.sig_cast_camera_image.connect(
self.ManualRegistrationWidget.receive_camera_image)
self.layout.addWidget(self.ManualRegistrationWidget, 3, 1)
self.StageRegistrationWidget = StageRegistrationWidget.StageWidget(
self)
self.layout.addWidget(self.StageRegistrationWidget, 4, 1)
def cast_transformation_to_DMD(self, transformation, laser):
self.DMDWidget.transform[laser] = transformation
self.DMDWidget.save_transformation()
def cast_transformation_to_galvos(self, sig):
transformation = sig
self.GalvoWidget.transform = transformation
self.GalvoWidget.save_transformation()
def cast_camera_image(self):
image = self.selection_view.image
if type(image) == np.ndarray:
self.sig_cast_camera_image.emit(image)
def snap_fov(self):
self.DMDWidget.interupt_projection()
self.DMDWidget.project_full_white()
self.cam = CamActuator()
self.cam.initializeCamera()
image = self.cam.SnapImage(0.04)
self.cam.Exit()
self.selection_view.setImage(image)
def cast_mask_coordinates(self, receiver):
list_of_rois = self.get_list_of_rois()
sig = [
list_of_rois,
self.fillContourButton.isChecked(),
self.thicknessSpinBox.value(),
self.invertMaskButton.isChecked()
]
if receiver == 'dmd':
self.sig_cast_mask_coordinates_to_dmd.emit(sig)
else:
self.sig_cast_mask_coordinates_to_galvo.emit(sig)
def get_list_of_rois(self):
view = self.selection_view
list_of_rois = []
for roi in view.roilist:
roi_handle_positions = roi.getLocalHandlePositions()
roi_origin = roi.pos()
for idx, pos in enumerate(roi_handle_positions):
roi_handle_positions[idx] = pos[1]
num_vertices = len(roi_handle_positions)
vertices = np.zeros([num_vertices, 2])
for idx, vertex in enumerate(roi_handle_positions):
vertices[idx, :] = np.array(
[vertex.x() + roi_origin.x(),
vertex.y() + roi_origin.y()])
list_of_rois.append(vertices)
return list_of_rois
def create_mask(self):
flag_fill_contour = self.fillContourButton.isChecked()
flag_invert_mode = self.invertMaskButton.isChecked()
contour_thickness = self.thicknessSpinBox.value()
list_of_rois = self.get_list_of_rois()
self.mask = ProcessImage.CreateBinaryMaskFromRoiCoordinates(list_of_rois, \
fill_contour = flag_fill_contour, \
contour_thickness = contour_thickness, \
invert_mask = flag_invert_mode)
self.mask_view.setImage(self.mask)
def remove_selection(self):
self.selection_view.clear_rois()
def set_camera_image(self, sig):
self.selection_view.setImage(sig)
def add_polygon_roi(self):
view = self.selection_view
x = (view.getView().viewRect().x()) * 0.3
y = (view.getView().viewRect().y()) * 0.3
a = (view.getView().viewRect().width() + x) * 0.3
b = (view.getView().viewRect().height() + y) * 0.3
c = (view.getView().viewRect().width() + x) * 0.7
d = (view.getView().viewRect().height() + y) * 0.7
polygon_roi = pg.PolyLineROI([[a, b], [c, b], [c, d], [a, d]],
pen=view.pen,
closed=True,
movable=True,
removable=True)
view.getView().addItem(polygon_roi)
view.append_to_roilist(polygon_roi)
def load_mask_from_file(self):
"""
Open a file manager to browse through files, load image file
"""
self.loadFileName, _ = QtWidgets.QFileDialog.getOpenFileName(
self, 'Select file', './CoordinateManager/Images/',
"(*.png, *.tiff, *.jpg)")
try:
image = plt.imread(self.loadFileName)
self.mask = image
self.mask_view.setImage(self.mask)
except:
print('fail to load file.')
def __init__(self, source_of_image = "PMT", init_search_range = 0.010, total_step_number = 5, imaging_conditions = {'edge_volt':5}, \
motor_handle = None, camera_handle = None, twophoton_handle = None, *args, **kwargs):
"""
Parameters
----------
source_of_image : string, optional
The input source of image. The default is PMT.
init_search_range : int, optional
The step size when first doing coarse searching. The default is 0.010.
total_step_number : int, optional
Number of steps in total to find optimal focus. The default is 5.
imaging_conditions : list
Parameters for imaging.
For PMT, it specifies the scanning voltage.
For camera, it specifies the AOTF voltage and exposure time.
motor_handle : TYPE, optional
Handle to control PI motor. The default is None.
twophoton_handle : TYPE, optional
Handle to control Insight X3. The default is None.
Returns
-------
None.
"""
super().__init__(*args, **kwargs)
# The step size when first doing coarse searching.
self.init_search_range = init_search_range
# Number of steps in total to find optimal focus.
self.total_step_number = total_step_number
# Parameters for imaging.
self.imaging_conditions = imaging_conditions
if motor_handle == None:
# Connect the objective if the handle is not provided.
self.pi_device_instance = PIMotor()
else:
self.pi_device_instance = motor_handle
# Current position of the focus.
self.current_pos = self.pi_device_instance.GetCurrentPos()
# Number of steps already tried.
self.steps_taken = 0
# The focus degree of previous position.
self.previous_degree_of_focus = 0
# Number of going backwards.
self.turning_point = 0
# The input source of image.
self.source_of_image = source_of_image
if source_of_image == "PMT":
self.galvo = RasterScan(Daq_sample_rate = 500000, edge_volt = self.imaging_conditions['edge_volt'])
elif source_of_image == "Camera":
if camera_handle == None:
# If no camera instance fed in, initialize camera.
self.HamamatsuCam_ins = CamActuator()
self.HamamatsuCam_ins.initializeCamera()
else:
self.HamamatsuCam_ins = camera_handle
class FocusFinder():
def __init__(self, source_of_image = "PMT", init_search_range = 0.010, total_step_number = 5, imaging_conditions = {'edge_volt':5}, \
motor_handle = None, camera_handle = None, twophoton_handle = None, *args, **kwargs):
"""
Parameters
----------
source_of_image : string, optional
The input source of image. The default is PMT.
init_search_range : int, optional
The step size when first doing coarse searching. The default is 0.010.
total_step_number : int, optional
Number of steps in total to find optimal focus. The default is 5.
imaging_conditions : list
Parameters for imaging.
For PMT, it specifies the scanning voltage.
For camera, it specifies the AOTF voltage and exposure time.
motor_handle : TYPE, optional
Handle to control PI motor. The default is None.
twophoton_handle : TYPE, optional
Handle to control Insight X3. The default is None.
Returns
-------
None.
"""
super().__init__(*args, **kwargs)
# The step size when first doing coarse searching.
self.init_search_range = init_search_range
# Number of steps in total to find optimal focus.
self.total_step_number = total_step_number
# Parameters for imaging.
self.imaging_conditions = imaging_conditions
if motor_handle == None:
# Connect the objective if the handle is not provided.
self.pi_device_instance = PIMotor()
else:
self.pi_device_instance = motor_handle
# Current position of the focus.
self.current_pos = self.pi_device_instance.GetCurrentPos()
# Number of steps already tried.
self.steps_taken = 0
# The focus degree of previous position.
self.previous_degree_of_focus = 0
# Number of going backwards.
self.turning_point = 0
# The input source of image.
self.source_of_image = source_of_image
if source_of_image == "PMT":
self.galvo = RasterScan(Daq_sample_rate = 500000, edge_volt = self.imaging_conditions['edge_volt'])
elif source_of_image == "Camera":
if camera_handle == None:
# If no camera instance fed in, initialize camera.
self.HamamatsuCam_ins = CamActuator()
self.HamamatsuCam_ins.initializeCamera()
else:
self.HamamatsuCam_ins = camera_handle
def gaussian_fit(self, move_to_focus = True):
# The upper edge.
upper_position = self.current_pos + self.init_search_range
# The lower edge.
lower_position = self.current_pos - self.init_search_range
# Generate the sampling positions.
sample_positions = np.linspace(lower_position, upper_position, self.total_step_number)
degree_of_focus_list = []
for each_pos in sample_positions:
# Go through each position and write down the focus degree.
degree_of_focus = self.evaluate_focus(round(each_pos, 6))
degree_of_focus_list.append(degree_of_focus)
print(degree_of_focus_list)
try:
interpolated_fitted_curve = ProcessImage.gaussian_fit(degree_of_focus_list)
# Generate the inpterpolated new focus position axis.
x_axis_new = np.linspace(lower_position, upper_position, len(interpolated_fitted_curve))
# Generate a dictionary and find the position where has the highest focus degree.
max_focus_pos = dict(zip(interpolated_fitted_curve, x_axis_new))[np.amax(interpolated_fitted_curve)]
if True: # Plot the fitting.
plt.plot(sample_positions, np.asarray(degree_of_focus_list),'b+:',label='data')
plt.plot(x_axis_new, interpolated_fitted_curve,'ro:',label='fit')
plt.legend()
plt.title('Fig. Fit for focus degree')
plt.xlabel('Position')
plt.ylabel('Focus degree')
plt.show()
max_focus_pos = round(max_focus_pos, 6)
print(max_focus_pos)
self.pi_device_instance.move(max_focus_pos)
# max_focus_pos_focus_degree = self.evaluate_focus(round(max_focus_pos, 6))
except:
print("Fitting failed. Find max in the list.")
max_focus_pos = sample_positions[degree_of_focus_list.index(max(degree_of_focus_list))]
print(max_focus_pos)
if move_to_focus == True:
self.pi_device_instance.move(max_focus_pos)
return max_focus_pos
def bisection(self):
"""
Bisection way of finding focus.
Returns
-------
mid_position : float
DESCRIPTION.
"""
# The upper edge in which we run bisection.
upper_position = self.current_pos + self.init_search_range
# The lower edge in which we run bisection.
lower_position = self.current_pos - self.init_search_range
for step_index in range(1, self.total_step_number + 1):
# In each step of bisection finding.
# In the first round, get degree of focus at three positions.
if step_index == 1:
# Get degree of focus in the mid.
mid_position = (upper_position + lower_position)/2
degree_of_focus_mid = self.evaluate_focus(mid_position)
print("mid focus degree: {}".format(round(degree_of_focus_mid, 5)))
# Break the loop if focus degree is below threshold which means
# that there's no cell in image.
if not ProcessImage.if_theres_cell(self.galvo_image.astype('float32')):
print('no cell')
mid_position = False
break
# Move to top and evaluate.
degree_of_focus_up = self.evaluate_focus(obj_position = upper_position)
print("top focus degree: {}".format(round(degree_of_focus_up, 5)))
# Move to bottom and evaluate.
degree_of_focus_low = self.evaluate_focus(obj_position = lower_position)
print("bot focus degree: {}".format(round(degree_of_focus_low, 5)))
# Sorting dicitonary of degrees in ascending.
biesection_range_dic = {"top":[upper_position, degree_of_focus_up],
"bot":[lower_position, degree_of_focus_low]}
# In the next rounds, only need to go to center and update boundaries.
elif step_index > 1:
# The upper edge in which we run bisection.
upper_position = biesection_range_dic["top"][0]
# The lower edge in which we run bisection.
lower_position = biesection_range_dic["bot"][0]
# Get degree of focus in the mid.
mid_position = (upper_position + lower_position)/2
degree_of_focus_mid = self.evaluate_focus(mid_position)
print("Current focus degree: {}".format(round(degree_of_focus_mid, 5)))
# If sits in upper half, make the middle values new bottom.
if biesection_range_dic["top"][1] > biesection_range_dic["bot"][1]:
biesection_range_dic["bot"] = [mid_position, degree_of_focus_mid]
else:
biesection_range_dic["top"] = [mid_position, degree_of_focus_mid]
print("The upper pos: {}; The lower: {}".format(biesection_range_dic["top"][0], biesection_range_dic["bot"][0]))
return mid_position
def evaluate_focus(self, obj_position = None):
"""
Evaluate the focus degree of certain objective position.
Parameters
----------
obj_position : float, optional
The target objective position. The default is None.
Returns
-------
degree_of_focus : float
Degree of focus.
"""
if obj_position != None:
self.pi_device_instance.move(obj_position)
# Get the image.
if self.source_of_image == "PMT":
self.galvo_image = self.galvo.run()
plt.figure()
plt.imshow(self.galvo_image)
plt.show()
if False:
with skimtiff.TiffWriter(os.path.join(r'M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Xin\2020-11-17 gaussian fit auto-focus cells\trial_11', str(obj_position).replace(".", "_")+ '.tif')) as tif:
tif.save(self.galvo_image.astype('float32'), compress=0)
degree_of_focus = ProcessImage.local_entropy(self.galvo_image.astype('float32'))
elif self.source_of_image == "Camera":
# First configure the AOTF.
self.AOTF_runner = DAQmission()
# Find the AOTF channel key
for key in self.imaging_conditions:
if 'AO' in key:
# like '488AO'
AOTF_channel_key = key
# Set the AOTF first.
self.AOTF_runner.sendSingleDigital('blankingall', True)
self.AOTF_runner.sendSingleAnalog(AOTF_channel_key, self.imaging_conditions[AOTF_channel_key])
# Snap an image from camera
self.camera_image = self.HamamatsuCam_ins.SnapImage(self.imaging_conditions['exposure_time'])
time.sleep(0.5)
# Set back AOTF
self.AOTF_runner.sendSingleDigital('blankingall', False)
self.AOTF_runner.sendSingleAnalog(AOTF_channel_key, 0)
plt.figure()
plt.imshow(self.camera_image)
plt.show()
if False:
with skimtiff.TiffWriter(os.path.join(r'M:\tnw\ist\do\projects\Neurophotonics\Brinkslab\Data\Xin\2021-03-06 Camera AF\beads', str(obj_position).replace(".", "_")+ '.tif')) as tif:
tif.save(self.camera_image.astype('float32'), compress=0)
degree_of_focus = ProcessImage.variance_of_laplacian(self.camera_image.astype('float32'))
time.sleep(0.2)
return degree_of_focus
class DMDRegistator:
def __init__(self, DMD, *args, **kwargs):
self.DMD = DMD
self.cam = CamActuator()
self.cam.initializeCamera()
def registration(self,
laser='640',
grid_points_x=2,
grid_points_y=3,
registration_pattern='circles'):
x_coords = np.linspace(0, 768, grid_points_x + 2)[1:-1]
y_coords = np.linspace(0, 1024, grid_points_y + 2)[1:-1]
x_mesh, y_mesh = np.meshgrid(x_coords, y_coords)
x_coords = np.ravel(x_mesh)
y_coords = np.ravel(y_mesh)
dmd_coordinates = np.stack((x_coords, y_coords), axis=1)
camera_coordinates = np.zeros(dmd_coordinates.shape)
for i in range(dmd_coordinates.shape[0]):
x = int(dmd_coordinates[i, 0])
y = int(dmd_coordinates[i, 1])
if registration_pattern == 'squares':
mask = DMDRegistator.create_registration_image_touching_squares(
x, y)
else:
mask = DMDRegistator.create_registration_image_circle(x, y)
self.DMD.send_data_to_DMD(mask)
self.DMD.start_projection()
image = self.cam.SnapImage(0.01)
plt.imsave(
os.getcwd() +
'/CoordinatesManager/Registration_Images/TouchingSquares/image_'
+ str(i) + '.png', image)
camera_coordinates[
i, :] = readRegistrationImages.touchingCoordinateFinder(
image, method='curvefit')
self.DMD.stop_projection()
print('DMD coordinates:')
print(dmd_coordinates)
print('Found camera coordinates:')
print(camera_coordinates)
self.DMD.free_memory()
self.cam.Exit()
transformation = CoordinateTransformations.polynomial2DFit(
camera_coordinates, dmd_coordinates, order=1)
print('Transformation found for x:')
print(transformation[:, :, 0])
print('Transformation found for y:')
print(transformation[:, :, 1])
return transformation
def create_registration_image_touching_squares(x, y, sigma=75):
array = np.zeros((768, 1024))
array[skimage.draw.rectangle((x - sigma, y - sigma), (x, y))] = 255
array[skimage.draw.rectangle((x + sigma, y + sigma), (x, y))] = 255
return array
def create_registration_image_circle(x, y, sigma=75):
array = np.zeros((768, 1024))
array[skimage.draw.circle(x, y, sigma)] = 255
return array
def __init__(self, DMD, *args, **kwargs):
self.DMD = DMD
self.cam = CamActuator()
self.cam.initializeCamera()
class GalvoRegistrator:
def __init__(self, *args, **kwargs):
self.cam = CamActuator()
self.cam.initializeCamera()
def registration(self, grid_points_x=3, grid_points_y=3):
"""
By default, generate 9 galvo voltage coordinates from (-5,-5) to (5,5),
take the camera images of these points, return a function matrix that
transforms camera_coordinates into galvo_coordinates using polynomial transform.
Parameters
----------
grid_points_x : TYPE, optional
DESCRIPTION. The default is 3.
grid_points_y : TYPE, optional
DESCRIPTION. The default is 3.
Returns
-------
transformation : TYPE
DESCRIPTION.
"""
galvothread = DAQmission()
readinchan = []
x_coords = np.linspace(-10, 10, grid_points_x + 2)[1:-1]
y_coords = np.linspace(-10, 10, grid_points_y + 2)[1:-1]
xy_mesh = np.reshape(np.meshgrid(x_coords, y_coords), (2, -1),
order='F').transpose()
galvo_coordinates = xy_mesh
camera_coordinates = np.zeros((galvo_coordinates.shape))
for i in range(galvo_coordinates.shape[0]):
galvothread.sendSingleAnalog('galvosx', galvo_coordinates[i, 0])
galvothread.sendSingleAnalog('galvosy', galvo_coordinates[i, 1])
time.sleep(1)
image = self.cam.SnapImage(0.06)
plt.imsave(
os.getcwd() +
'/CoordinatesManager/Registration_Images/2P/image_' + str(i) +
'.png', image)
camera_coordinates[i, :] = readRegistrationImages.gaussian_fitting(
image)
print('Galvo Coordinate')
print(galvo_coordinates)
print('Camera coordinates')
print(camera_coordinates)
del galvothread
self.cam.Exit()
transformation_cam2galvo = CoordinateTransformations.polynomial2DFit(
camera_coordinates, galvo_coordinates, order=1)
transformation_galvo2cam = CoordinateTransformations.polynomial2DFit(
galvo_coordinates, camera_coordinates, order=1)
print('Transformation found for x:')
print(transformation_cam2galvo[:, :, 0])
print('Transformation found for y:')
print(transformation_cam2galvo[:, :, 1])
print('galvo2cam found for x:')
print(transformation_galvo2cam[:, :, 0])
print('galvo2cam found for y:')
print(transformation_galvo2cam[:, :, 1])
return transformation_cam2galvo