def __init__(self, cycle, fc):
self.maestro = MaestroFunctions()
self.cycle_name = cycle
self.flowcell = fc
self.aexpose = Autoexpose()
self.TDI_flag = 0
self.num_arrays = 1
self.auto_exposure = 0
"""
is this being called to do darkfield imaging or fluorescence imaging?
darkfield imaging is specified by cycle name starting w/ 'WL';
assume all other cycle names specify fluorescence imaging
for darkfield imaging, numeric suffix on cycle name tells whether
the run is for one flowcell or two
"""
if self.cycle_name.find("WL") is not -1:
self.fluor_imaging = 0
if self.cycle_name.find("1") is not -1: # this is a single-fc run
self.dual_fc = 0
else:
self.dual_fc = 1
# WL2 specifies dual-flowcell run
self.flowcell = self.flowcell + 2
"""
specifies how many images to average for each WL image; we use 5
because the time required to acquire & average 5 is approx equal to
the time required to segment the image
"""
self.WL_num_to_avg = 1
# specifies the integration time (in msec) for brightfield imaging
self.WL_integration_time = 35
self.WL_gain = 70
# construct cycle name for WL imaging
today = datetime.date.today()
fn = "%02d%02d%04d" % (today.month, today.day, today.year)
self.cycle_name = "%c%c%c%c%c%c" % (fn[0], fn[1], fn[2], fn[3], fn[6], fn[7])
# end if
else:
self.fluor_imaging = 1
self.cycle_name = cycle
# end else
self.flowcell = fc
# these are used for both manual exposures and autoexposure;
# the order of these values corresponds to the fluor order
# in array fluors[]
self.integration_times = [35, 35, 35, 35, 35]
self.manual_gains = [45, 120, 70, 50]
# fluors = ['cy5', 'fam', 'cy3', 'txred']
# this is run as a thread, so invoke Thread's constructor
threading.Thread.__init__(self)
class Imager(threading.Thread):
global WL_num_to_avg
global WL_integration_time
global fluor_imaging
global cycle_name
global flowcell
global dual_fc
global integration_times
global manual_gains
global aexpose
global num_arrays
global num_imgs
global TDI_flag
global auto_exposure
def __init__(self, cycle, fc):
self.maestro = MaestroFunctions()
self.cycle_name = cycle
self.flowcell = fc
self.aexpose = Autoexpose()
self.TDI_flag = 0
self.num_arrays = 1
self.auto_exposure = 0
"""
is this being called to do darkfield imaging or fluorescence imaging?
darkfield imaging is specified by cycle name starting w/ 'WL';
assume all other cycle names specify fluorescence imaging
for darkfield imaging, numeric suffix on cycle name tells whether
the run is for one flowcell or two
"""
if self.cycle_name.find("WL") is not -1:
self.fluor_imaging = 0
if self.cycle_name.find("1") is not -1: # this is a single-fc run
self.dual_fc = 0
else:
self.dual_fc = 1
# WL2 specifies dual-flowcell run
self.flowcell = self.flowcell + 2
"""
specifies how many images to average for each WL image; we use 5
because the time required to acquire & average 5 is approx equal to
the time required to segment the image
"""
self.WL_num_to_avg = 1
# specifies the integration time (in msec) for brightfield imaging
self.WL_integration_time = 35
self.WL_gain = 70
# construct cycle name for WL imaging
today = datetime.date.today()
fn = "%02d%02d%04d" % (today.month, today.day, today.year)
self.cycle_name = "%c%c%c%c%c%c" % (fn[0], fn[1], fn[2], fn[3], fn[6], fn[7])
# end if
else:
self.fluor_imaging = 1
self.cycle_name = cycle
# end else
self.flowcell = fc
# these are used for both manual exposures and autoexposure;
# the order of these values corresponds to the fluor order
# in array fluors[]
self.integration_times = [35, 35, 35, 35, 35]
self.manual_gains = [45, 120, 70, 50]
# fluors = ['cy5', 'fam', 'cy3', 'txred']
# this is run as a thread, so invoke Thread's constructor
threading.Thread.__init__(self)
def run(self):
# are we doing fluorescent imaging (assume 4 colors) or
# darkfield imaging?
if self.fluor_imaging == 1:
if 0:
self.aexpose = self.aexpose.autoe()
self.manual_gains = self.aexpose
print "using gains found with autoexposgain.py"
else:
print "using gains entered by user"
# this is the order of fluors in the integration and gain arrays;
# it is also the order used by autoexposure
fluors = ["cy5", "fam", "cy3", "txred"]
"""
this is based on the nonamer synthesis, and is the sequence of
the probe strand therefore, the sequence of the template
(bead-bound) strand is the complement
"""
fluor_to_base = {"cy5": "T", "cy3": "A", "txred": "C", "fam": "G", "none": "N"}
# order to do the fluorescence imaging; fam is the most photolabile,
# so we do it first
imaging_order = ["fam", "cy3", "txred", "cy5", "none"]
# now do 4 colors of imaging; each time, perform the stage alignment
# first to minimize image alignment drift
i = 0
while i < 4:
# perform stage alignment
cmd = (os.environ["POLONATOR_PATH"] + "/bin/Polonator-stagealign %d") % (self.flowcell)
print cmd
os.system(cmd)
print "STARTING CHECK ........................"
if self.auto_exposure == 1:
j = 60
curr_name_new = "%s" % (imaging_order[i])
cmd = os.environ["POLONATOR_PATH"] + "/bin/PolonatorUtils gotostagealignpos 0 0"
print cmd
os.system(cmd)
cmd = os.environ["POLONATOR_PATH"] + "/bin/PolonatorUtils snap1 %s %f %d %d" % (
curr_name_new,
0.035,
j,
j + 150,
)
print cmd
os.system(cmd)
# end if
# turn the darkfield illuminator off if it was left on
self.maestro.darkfield_off()
print "STARTING SETUP IMAGE........................"
# setup controller for current color
self.maestro.setup_imaging(
imaging_order[i],
self.integration_times[fluors.index(imaging_order[i])] - 5,
1,
self.num_arrays,
self.flowcell,
0,
self.TDI_flag,
)
# start Polonator-acq which receives images from camera
# and sends to processing pipeline
#
# construct current image-set name (cycle name plus base identity)
curr_name = "%s_%s" % (self.cycle_name, fluor_to_base[imaging_order[i]])
# this must be called w/ sudo so that the thread has a high
# enough priority to ensure images aren't missed often
tcmd = (os.environ["POLONATOR_PATH"] + "/bin/Polonator-acquirer %s %d %d %d %d") % (
curr_name,
self.integration_times[fluors.index(imaging_order[i])] - 5,
self.manual_gains[fluors.index(imaging_order[i])],
self.flowcell,
self.num_arrays,
)
print tcmd
cmd = os.environ["POLONATOR_PATH"] + "/bin/Polonator-acquirer"
print "STARTING ACQUIRE........................"
# spawn thread to receive the images and wait for it to return;
# it will exit w/ value of 10 if it did not acquire all images -- in
# this case, repeat the current color
if (
os.spawnlp(
os.P_WAIT,
cmd,
cmd,
curr_name,
str(self.integration_times[fluors.index(imaging_order[i])]),
str(self.manual_gains[fluors.index(imaging_order[i])]),
str(self.flowcell),
str(self.num_arrays),
)
!= 10
):
# move on to next color if all images were received
i += 1
else:
print "Polonator-acquirer exited with error"
# we're doing darkfield imaging; assume we've just started the run and
# tell Polonator-stagealign to zero out the offsetlog and collect a new
# base image
else:
# generate the 'base' image for the stage alignment tool
# if this is a dual-flowcell run, we must convert the flowcell == 2 or 3
# to flowcell == 0 or 1
if self.dual_fc == 1:
cmd = (os.environ["POLONATOR_PATH"] + "/bin/Polonator-stagealign %d 1") % (self.flowcell - 2)
else:
cmd = (os.environ["POLONATOR_PATH"] + "/bin/Polonator-stagealign %d 1") % (self.flowcell)
print cmd
os.system(cmd)
# we are doing darkfield imaging
self.maestro.darkfield_on()
self.maestro.setup_imaging(
"none",
self.WL_integration_time - 5,
self.WL_num_to_avg,
self.num_arrays,
self.flowcell,
1,
self.TDI_flag,
)
"""
start Polonator-acq
during brightfield imaging (the start of the run), we need to tell
the processing computer whether it should expect 1 or 2 flowcells'
worth of data; we do this by changing the flowcell number from
0 for a single-flowcell run to either 2 or 3 for a dual flowcell
run (fc 0 or 1, respectively)
the imaging software (Polonator-acquirer) understands this
convention and converts the 2 or a 3 to a 0 or a 1 after sending
the proper flag to the processing pipeline
"""
cmd = (os.environ["POLONATOR_PATH"] + "/bin/Polonator-acquirer %s %d %d %d %d %d") % (
self.cycle_name,
self.WL_integration_time - 5,
self.WL_gain,
self.flowcell,
self.num_arrays,
self.WL_num_to_avg,
)
print cmd
os.system(cmd)
self.maestro.darkfield_off()
return 1