def snap_location(self,x="",y="",z="",offset="",path="",):
if offset:
self.move_XYstage(x,y)
self.use_afc(offset)
else:
self.move_XYstage(x,y,z)
self.mmc.snapImage()
self.img=self.mmc.getImage()
self.md=self.getFullSystemState()
if path:
FileOut.tif_writer(numpy_array=self.img,path=path,md_string=self.md)
##turn off afc so that you could do an MDA if you wanted
scope.mmc.enableContinuousFocus(False)
## Take your images
channels=[Illuminations.BF2]
if int(pos_label[3:])< 17:
channels=[Illuminations.BF2,Illuminations.dimGREEN,Illuminations.dimRED]
elif int(pos_label[3:])>17 and int(pos_label[3:])<25:
channels=[Illuminations.BF2,Illuminations.dimRED]
elif int(pos_label[3:])>25:
channels=[Illuminations.BF2,Illuminations.dimGREEN]
images,metadata=scope.complicated_multichannel_zstack(channels,
z_width=3,
z_increments=1,
channels_for_z=channels[:1])
file_name="Timelapse_%s_t%s" % (pos_label,i)
FileOut.mda_save2(out_dir=save_dir,prefix=file_name,
mda=images,md=metadata,channels=channels)
#for channel in channels:
# scope.set_illumination(channel)
# scope.mmc.snapImage()
# img=scope.mmc.getImage()
# md=scope.getFullSystemState()
# # save your images
# file_name="Timelapse_%s_%s_t%s" % (pos_label,channel.name,i)
# save_loc=os.path.join(save_dir,file_name)
# FileOut.tif_writer(numpy_array=img,path=save_loc,md_string=md)
# if pos_label=="Pos4":
# break ## this position will only take the first channel
# record location info
loc_data="%s;%s;%s;%s;%s;%s;%s;%s;%s;%s;%s;%s;%s;%s\n" % (pos_label,
i,
x,
scope.move_XYstage(location[0],location[1])
x1,y1,z1,o1=scope.get_x_y_z_offset()
scope.mmc.enableContinuousFocus(True)
scope.mmc.setAutoFocusOffset(Oinit)
while True:
if scope.mmc.isContinuousFocusLocked():
time.sleep(.3)
break
time.sleep(.3) ### added to see if it is reporting a lock before it is
x2,y2,z2,o2=scope.get_x_y_z_offset()
scope.mmc.enableContinuousFocus(False)
scope.mmc.snapImage()
img=scope.mmc.getImage()
file_name="Test%s_simple_wait_loc%s_t%s" % (test_count,loc_count,i)
save_loc=os.path.join(save_dir,file_name)
FileOut.tif_writer(numpy_array=img,path=save_loc)
locinfo[loc_count-1]+="%s\t%s\t%s\t%s\t%s\t%s\t%s\n" % (i,
x2,
y2,
z1,
z2,
o1,
o2)
print i
time.sleep(60)
loc_counter=0
for loc in locinfo:
loc_counter+=1
save_dir=r"C:\Users\Administrator\Desktop\MicroscopeControl\MicroscopeControl\Tests"
file_name="Test%s_position%s_info.tsv" % (test_count,loc_counter)
def multi_point_timelapse(
scope, save_dir, positions_file, illumination_pattern_object, n_timepoints, frequency, file_name_prefix="Timelapse"
):
"""
args:
scope -- initalized scope object -- see Microscopes.Microscope
"""
#####################################################
### Pre Imaging ###
#####################################################
#### Getting stage locations
positions = StagePositions.positionList_parser(open(positions_file).read())
pos_order = sorted(positions.keys(), key=lambda x: int(x[3:])) # numerical order
#### Getting the channels that will be taken
# channels=illumination_pattern_object.channels
# channels=["BF","qGFP","qmCherry"]
channels = ["qDIC", "qGFP", "qCherry"]
#### Set up the to save images in a structured set of directories
if os.path.exists(save_dir) is False:
os.mkdir(save_dir)
for pos_label in pos_order:
pos_dir = os.path.join(save_dir, pos_label)
if os.path.exists(pos_dir) is False:
os.mkdir(pos_dir)
for channel_name in channels:
channel_dir = os.path.join(pos_dir, channel_name)
if os.path.exists(channel_dir) is False:
os.mkdir(channel_dir)
#### Set up the log file
pos_stats_file_path = os.path.join(save_dir, "Position_Stats.tsv")
fout = open(pos_stats_file_path, "w")
pos_record_values = [
"Pos",
"Time",
"x_set",
"y_set",
"z_set",
"afc_set",
"x_1",
"y_1",
"z_1",
"afc_1",
"x_2",
"y_2",
"z_2",
"afc_2",
"channels",
]
header = "\t".join(pos_record_values) + "\n"
fout.write(header)
fout.close()
#####################################################
### Imaging Loop ###
#####################################################
try:
### interate over timepoints
for t in range(0, n_timepoints):
print ["init", "init", "move", "foc ", "imag", "save", "stats"]
acquisition_start_time = time.time()
for pos_label in pos_order:
t1 = time.time()
## lookup where to go
pos = positions[pos_label]
x = pos.get_x()
y = pos.get_y()
z = pos.get_z()
afc = pos.get_afc_offset()
print "Locations settings: %s\t%s x\t%s y\t%s z\t%s offset" % (pos_label, x, y, z, afc)
## move scope to x,y
t2 = time.time()
scope.move_XYstage(x, y)
## see where the scope is
x1, y1, z1, afc1 = scope.get_stage_loc()
## focus the scope using afc offset
t3 = time.time()
scope.move_to_offset(afc, post_focus_delay=0.1, attempt_delay=0.1)
## see where the scope is now
x2, y2, z2, afc2 = scope.get_stage_loc()
## figure out the channels to image
# c_to_image=conditional_illuminations.get_channels(pos_label,t)
# c_stack_settings=conditional_illuminations.get_stack_settings(pos_label,t)
c_to_image = [Illuminations.BF2, Illuminations.dimGREEN, Illuminations.dimRED]
# c_to_image=["qDIC","qGFP","qCherry"]
c_stack_settings = {"qDIC": [5, 1]}
## take the images
t4 = time.time()
images, metadata = scope.multichannel_zstack(c_to_image, z_params=c_stack_settings, timed=True)
## save the images
t5 = time.time()
for c in range(0, len(c_to_image)):
stack = images[c]
md_stack = metadata[c]
try:
channel_name = c_to_image[c].name
except:
channel_name = c_to_image[c]
full_save_dir = os.path.join(save_dir, pos_label, channel_name)
full_file_name = "%s_%s_c%s%s_t%s" % (file_name_prefix, pos_label, c, channel_name, t)
FileOut.stack_writer(full_save_dir, full_file_name, stack, md_stack[len(md_stack) / 2])
t6 = time.time()
###log some stats
channels_aqcuired_string = "" # this is in progress
# for c in c_to_image:
# name=c.name
# intensity=c.SOLA_intensity
# length=c.exposure
# if name in c_stack_settings:
# z=c_stack_settings[name][0]/c_stack_settings[name][1]+1
# else: z=1
# channel_string=""
pos_record_stats = (
pos_label,
t,
x,
y,
z,
afc,
x1,
y1,
z1,
afc1,
x2,
y2,
z2,
afc2,
channels_aqcuired_string,
)
pos_record_stats = [str(i) for i in pos_record_stats]
pos_record_stats = "\t".join(pos_record_stats)
pos_record_stats += "\n"
fout = open(pos_stats_file_path, "a")
fout.write(pos_record_stats)
fout.close()
t7 = time.time()
times = [t1, t2, t3, t4, t5, t6, t7]
times = ["%.2f" % (i - times[0]) for i in times]
times_lab = ["Start", "Find Where to Go", "Move Stage", "Focused", "Imaged", "Saved", "Saved_Stats"]
for i in range(0, len(times)):
print times[i], times_lab[i]
##return the to first location
scope.move_to_offset(afc)
xnext = positions[pos_order[0]].get_x()
ynext = positions[pos_order[0]].get_y()
scope.move_XYstage(xnext, ynext)
scope.mmc.enableContinuousFocus(False)
### wait for the start of the next acquisition
acquisition_finish_time = time.time()
until_next = frequency - (acquisition_finish_time - acquisition_start_time)
verbose_sleeper(until_next)
print "Timelapse completed"
results = "Scan Completed"
except:
print "UH OH!"
tb = StringIO.StringIO()
exc_type, exc_value, exc_traceback = sys.exc_info()
traceback.print_exception(exc_type, exc_value, exc_traceback, file=tb)
results = (
"There was some sort of error, "
"your scan most likely did not finish."
"The error was \n\n %s" % tb.getvalue()
)
return results
def plateScan(scope,
positions,
scan_illumination,
mda_channels,
prefix="",
blacklist=[],
well_limit=15,
well_count_dict={},
data_dir="",
visible=True,
stack_height=5,
slice_height=.5,
minAreaCOI=100,
maxAreaCOI=1000,
thresholdCOI=1000):
"""
Write the doc string!!
"""
if prefix=="":
prefix=time.asctime().replace(" ","_").replace(":","_")
if data_dir is not "":
if os.path.isdir(data_dir) is False:
os.mkdir(data_dir)
data_dir=os.path.join(data_dir,"Scan_%s" % prefix)
if os.path.isdir(data_dir) is False:
os.mkdir(data_dir)
order=-1
## set up the
rows=string.ascii_uppercase[1:7]
columns=range(2,12)
sites=range(0,225)
well_count_dict={}
for row in rows:
## flip the order that the columns are gone through
order=order*-1
for column in columns[::order]:
well="%s%s" % (row,column)
if well not in well_count_dict: well_count_dict[well]=0
if well not in blacklist:
for site in sites:
try:
#if scope.mmc.isContinuousFocusLocked() is False:
# ## try waiting a few seconds
# time.sleep(10)
# if scope.mmc.isContinuousFocusLocked() is False:
# ## tried waiting, that didnt work, lets exit
# print "Lost focus on %s site %s" % (well, site)
# return well_count_dict
x,y=positions["%s%s-Site_%s" % (row,column,site)].get_xy()
scope.mmc.setXYPosition("XYStage",x,y)
print ">Looking at %s%s-Site_%s" % (row,column,site)
site_name="%s%s-Site_%s" % (row,column,site)
site_imaged,mda,md = plateScanSite(scope,
scan_illumination,
mda_channels,
x,
y,
name=site_name,
visible=visible,
stack_height=stack_height,
slice_height=slice_height,
minAreaCOI=minAreaCOI,
maxAreaCOI=maxAreaCOI,
thresholdCOI=thresholdCOI)
well_count_dict[well]+=site_imaged
if mda:
FileOut.mda_save2(out_dir=data_dir,
prefix="%s%s-Site_%s" % (row,
column,
site),
mda=mda,
md=md,
channels=mda_channels)
del mda
del md
del site_imaged
if well_count_dict[well] >= well_limit:
count = well_count_dict[well]
print "I have found %s cells/stacks in %s" %(count,
well)
break
if scope.mmc.isContinuousFocusLocked() is False:
## try waiting a few seconds
time.sleep(10)
if scope.mmc.isContinuousFocusLocked() is False:
## tried waiting, that didnt work, lets exit
msg="Lost focus on %s site %s" % (well, site)
print msg
return str(well_count_dict)+"\n\n"+msg
except:
tb=StringIO.StringIO()
exc_type, exc_value, exc_traceback = sys.exc_info()
traceback.print_exception(exc_type,
exc_value,
exc_traceback,
file=tb)
message=str(well_count_dict)+"\n\n"+tb.getvalue()
return message
return well_count_dict
def legacy():
"""
This is the code that I had originall wrote to do this.
This code was really messy, but worked, so I am leaving it here.
It has been rewritten in the above functions, but could be useful to look
at.
"""
## initialize the scope
scope=DGLab_Leica2.DGLab_Leica2()
## set up the
rows=string.ascii_uppercase[1:7]
columns=range(2,12)
sites=range(0,225)
pos_string=open("PositionList_96well_15x15sites.pos").read()
positions=StagePositions.positionList_parser(pos_string)
scope.mmc.enableContinuousFocus(True)
scope.set_illumination(Illuminations.Cy5)
#start_time=time.asctime().replace(" ","_").replace(":","_")
start_time="Thu_Aug_13_14_38_51_2015"
done_list={}
well_counter={}
## if this is a continuation of an old scan, this will add on to the
## existing log file.
if os.path.exists("Scan_%s.txt" % start_time):
completed_fin=open("Scan_%s.txt" % start_time,"r")
for line in completed_fin:
site=line.split(";")[0]
try:
if "No cells of interest" not in line.split(";")[1]:
well=site.split("-")
if well not in well_counter:
well_counter[well]=0
well_counter+=1
except:
pass
completed_fin.close()
status_out=open("Scan_%s.txt" % start_time,"r")
status_string=status_out.read()
status_out.close()
status_out=open("Scan_%s.txt" % start_time,"w")
status_out.write(status_string)
else:
status_out=open("Scan_%s.txt" % start_time,"w")
data_dir="Scan_%s" % start_time
if os.path.isdir(data_dir) is False:
os.mkdir(data_dir)
order=-1
for row in rows:
## flip the order that the columns are gone through
order=order*-1
for column in columns[::order]:
well="%s%s" % (row,column)
if well not in well_counter:well_counter[well]=0
if well_counter[well]<15:
## reset the well count for this new well.
well_count=well_counter[well]
## iterate through the site in this well.
for site in sites: ## Test case
if "%s%s-Site_%s" % (row,column,site) not in done_list.keys():
done_list["%s%s-Site_%s" % (row,column,site)]=True
print ">Looking at %s%s-Site_%s" % (row,column,site)
x,y=positions["%s%s-Site_%s" % (row,column,site)].get_xy()
## take an image to use for a trigger
scope.set_illumination(Illuminations.qCy5)
scope.mmc.setXYPosition("XYStage",x,y)
scope.mmc.snapImage()
img=scope.mmc.getImage()
## show image
plt.imshow(img)
plt.draw()
## analyze this image
cois=Threshold.coi_finder(img,
threshold=1000,
min_area=100,
max_area=1000)
if cois:
do_stack=True
progress_line=">%s%s-Site_%s;cois=%s\n" % (row,column,site,cois)
else:
do_stack=False
progress_line=">%s%s-Site_%s;No cells of interest\n" % (row,column,site)
status_out.write(progress_line)
## run the stack if there was a COI found
if do_stack:
print ">Stack at %s%s-Site_%s" % (row,column,site)
well_count+=1
well_counter[well]+=1
channels=[Illuminations.DIC,
Illuminations.GFP,
Illuminations.qCy5,
Illuminations.DAPI]
scope.mmc.enableContinuousFocus(False)
mda,md=scope.multi_channel_acquisition(channels)
FileOut.mda_save(out_dir=data_dir,
prefix="%s%s-Site_%s" % (row,column,site),
mda=mda,
md=md,
channels=channels)
print "Stack taken at %s%s-Site_%s" % (row,column,site)
## turn AFC back on
scope.mmc.enableContinuousFocus(True)
## move onto the next well if we have acquired enough
if well_count > 15:
break
## break out of the well if you can't focus
if scope.mmc.isContinuousFocusLocked() is False:
time.sleep(5)
if scope.mmc.isContinuousFocusLocked() is False:
print "Couldnt focus 3"
break
## break out of the column if you still cant focus
if scope.mmc.isContinuousFocusLocked() is False:
time.sleep(5)
if scope.mmc.isContinuousFocusLocked() is False:
print "Couldnt focus 2"
break
## break out of the row if you STILL cant focus.
if scope.mmc.isContinuousFocusLocked() is False:
time.sleep(5)
if scope.mmc.isContinuousFocusLocked() is False:
print "Couldnt focus 1"
break
status_out.close()