class Servo:
def __init__(self, *args, **kwargs):
super().__init__(*args, **kwargs)
self.sampling_rate = 10000
self.PWM_frequency = 50
self.mission = DAQmission()
def power_on(self):
self.mission.sendSingleDigital(channel='servo_power_1', value=True)
# time.sleep(1.5)
def power_off(self):
self.mission.sendSingleDigital(channel='servo_power_1', value=False)
def rotate(self, degree):
# Convert degree to duty cycle in PWM.
if degree >= 0 and degree <= 360:
dutycycle = 0.02 #round((degree/180) * 0.05 + 0.05, 6)
PWM_wave = self.blockWave(self.sampling_rate,
self.PWM_frequency,
dutycycle,
repeats=50)
PWM_wave = np.where(PWM_wave == 0, False, True)
# plt.figure()
# plt.plot(PWM_wave)
# plt.show()
PWM_wave_organized = np.array(
[('servo_modulation_1', PWM_wave),
('servo_power_1', np.ones(len(PWM_wave), dtype=bool))],
dtype=[('Sepcification', 'U20'),
('Waveform', bool, (len(PWM_wave), ))])
self.mission.runWaveforms(clock_source = "DAQ", sampling_rate = self.sampling_rate, analog_signals = {},\
digital_signals = PWM_wave_organized, readin_channels = {})
else:
print('Rotation degree out of range!')
def blockWave(self,
sampleRate,
frequency,
dutycycle,
repeats,
voltMin=0,
voltMax=5):
"""
Generates a one period blockwave.
sampleRate samplerate set on the DAQ (int)
frequency frequency you want for the block wave (int)
voltMin minimum value of the blockwave (float)
voltMax maximum value of the blockwave (float)
dutycycle duty cycle of the wave (wavelength at voltMax) (float)
"""
wavelength = int(sampleRate /
frequency) #Wavelength in number of samples
#The high values
high = np.ones(math.ceil(wavelength * dutycycle)) * voltMax
#Low values
low = np.ones(math.floor(wavelength * (1 - dutycycle))) * voltMin
#Adding them
single_period = np.append(high, low)
"""
Repeats the wave a set number of times and returns a new repeated wave.
"""
extendedWave = np.array([])
for i in range(repeats):
extendedWave = np.append(extendedWave, single_period)
return extendedWave
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)
