See also
This page was generated from examples/twocolor.ipynb.
Download the Jupyter Notebook for this section: twocolor.ipynb. View in nbviewer.
Two-color ImagingΒΆ
This is an example using scopyon (https://scopyon.readthedocs.io/).
[1]:
import scopyon
When you want to show debug messages, set the logging level.
[2]:
import logging
logging.basicConfig(level=logging.DEBUG)
Set the configuration first.
[3]:
config = scopyon.DefaultConfiguration()
config.default.detector.exposure_time = 33.0e-3 # second
A field of microscopic view could be calculated as follows:
[4]:
pixel_length = config.default.detector.pixel_length / config.default.magnification
L_2 = config.default.detector.image_size[0] * pixel_length * 0.5
Randomly generate positions of 250 molecules in two dimensions.
[5]:
import numpy.random
rng = numpy.random.RandomState(123)
N1 = 150
N2 = 50
N3 = 50
inputs = rng.uniform(-L_2, +L_2, size=(N1 + N2 + N3, 2))
Form an image where N1 and N2 molecules are labeled.
[6]:
img1 = scopyon.form_image(inputs[: N1 + N2], config=config, rng=rng)
DEBUG:scopyon.io: Type,Fluorophore
DEBUG:scopyon.io: Maker,"Invitrogen(Life Technologies)"
DEBUG:scopyon.io: ProductName,"Tetramethylrhodamine(TRITC)"
DEBUG:scopyon.io: Comment,"Tetramethylrhodamine goat anti-mouse IgG antibody/pH 8.0"
DEBUG:scopyon.io: Public,1
INFO:scopyon._epifm:--- Fluorophore: Tetramethylrhodamine(TRITC) PSF
INFO:scopyon._epifm: Wave Length = 5.780000000000062e-07 m
INFO:scopyon._epifm: Normalization = 13564.06122650865
INFO:scopyon._epifm: Fluorophore radius = 2e-08 m
INFO:scopyon._epifm: PSF Normalization Factor = 13564.06122650865
INFO:scopyon._epifm: Emission : Wave Length = 5.780000000000062e-07 m
INFO:scopyon._epifm:--- Shutter:
INFO:scopyon._epifm: Start-Time = 0.0 sec
INFO:scopyon._epifm: End-Time = None sec
INFO:scopyon._epifm:--- Light Source:LASER
INFO:scopyon._epifm: Wave Length = 5.32e-07 m
INFO:scopyon._epifm: Beam Flux Density = 400000.0 W/m2
INFO:scopyon._epifm: 1/e2 Radius = 2e-05 m
INFO:scopyon._epifm: Angle = 1.2566370614359172 radian
DEBUG:scopyon.io: Type,Dichroic
DEBUG:scopyon.io: Maker,"Semrock"
DEBUG:scopyon.io: ProductName,"FF562-Di03-25x36"
DEBUG:scopyon.io: Comment,""
DEBUG:scopyon.io: Public,1
INFO:scopyon._epifm:--- Dichroic Mirror:
INFO:scopyon._epifm:--- Magnification: x 241.0
INFO:scopyon._epifm:--- Detector: EMCCD
INFO:scopyon._epifm: Image Size = 512 x 512
INFO:scopyon._epifm: Pixel Size = 1.6e-05 m/pixel
INFO:scopyon._epifm: Focal Point = [0. 0. 0.]
INFO:scopyon._epifm: Exposure Time = 0.033 sec
INFO:scopyon._epifm: Quantum Efficiency = 92.0 %
INFO:scopyon._epifm: Readout Noise = 100 electron
INFO:scopyon._epifm: Dark Count = 0 electron/sec
INFO:scopyon._epifm: EM gain = x 300
INFO:scopyon._epifm:Focal Center: [0. 0. 0.]
INFO:scopyon._epifm:--- A/D Converter: 16-bit
INFO:scopyon._epifm: Fullwell = 800000 electron
INFO:scopyon._epifm: none-Fixed Pattern Noise: 0.0 count
INFO:scopyon._epifm:--- Excitation Filter:
INFO:scopyon._epifm:--- Emission Filter:
INFO:scopyon._epifm:--- Background:
INFO:scopyon._epifm: Mean = 0.01 photons
INFO:scopyon._epifm:--- Fluorescence:
INFO:scopyon._epifm: Quantum Yield = 0.61
INFO:scopyon._epifm: Abs. Coefficient = 83400 1/(M cm)
INFO:scopyon._epifm: Abs. Cross-section = 3.1888261070088875e-16 cm^2
INFO:scopyon._epifm:--- Photobleaching:
INFO:scopyon._epifm: Photobleaching half life = 2.5
INFO:scopyon._epifm:processes = 1.
INFO:scopyon._epifm:time: 0.0 sec (0)
INFO:scopyon._epifm:center (width, height): 0.0 0.0
INFO:scopyon._epifm:scaling [m/pixel]: 6.639004149377593e-08
INFO:scopyon._epifm: 000-th file in 000-th frame: 0.0 + 0.033 sec
INFO:scopyon._epifm:Total camera_pixel = 2.190565e+04
INFO:scopyon._epifm:200 optoinfos are registered.
INFO:scopyon._epifm:Apply detector effects.
Generate another image where N2 and N3 molecules are labeled.
[7]:
img2 = scopyon.form_image(inputs[150: ], config=config, rng=rng)
DEBUG:scopyon.io: Type,Fluorophore
DEBUG:scopyon.io: Maker,"Invitrogen(Life Technologies)"
DEBUG:scopyon.io: ProductName,"Tetramethylrhodamine(TRITC)"
DEBUG:scopyon.io: Comment,"Tetramethylrhodamine goat anti-mouse IgG antibody/pH 8.0"
DEBUG:scopyon.io: Public,1
INFO:scopyon._epifm:--- Fluorophore: Tetramethylrhodamine(TRITC) PSF
INFO:scopyon._epifm: Wave Length = 5.780000000000062e-07 m
INFO:scopyon._epifm: Normalization = 13564.06122650865
INFO:scopyon._epifm: Fluorophore radius = 2e-08 m
INFO:scopyon._epifm: PSF Normalization Factor = 13564.06122650865
INFO:scopyon._epifm: Emission : Wave Length = 5.780000000000062e-07 m
INFO:scopyon._epifm:--- Shutter:
INFO:scopyon._epifm: Start-Time = 0.0 sec
INFO:scopyon._epifm: End-Time = None sec
INFO:scopyon._epifm:--- Light Source:LASER
INFO:scopyon._epifm: Wave Length = 5.32e-07 m
INFO:scopyon._epifm: Beam Flux Density = 400000.0 W/m2
INFO:scopyon._epifm: 1/e2 Radius = 2e-05 m
INFO:scopyon._epifm: Angle = 1.2566370614359172 radian
DEBUG:scopyon.io: Type,Dichroic
DEBUG:scopyon.io: Maker,"Semrock"
DEBUG:scopyon.io: ProductName,"FF562-Di03-25x36"
DEBUG:scopyon.io: Comment,""
DEBUG:scopyon.io: Public,1
INFO:scopyon._epifm:--- Dichroic Mirror:
INFO:scopyon._epifm:--- Magnification: x 241.0
INFO:scopyon._epifm:--- Detector: EMCCD
INFO:scopyon._epifm: Image Size = 512 x 512
INFO:scopyon._epifm: Pixel Size = 1.6e-05 m/pixel
INFO:scopyon._epifm: Focal Point = [0. 0. 0.]
INFO:scopyon._epifm: Exposure Time = 0.033 sec
INFO:scopyon._epifm: Quantum Efficiency = 92.0 %
INFO:scopyon._epifm: Readout Noise = 100 electron
INFO:scopyon._epifm: Dark Count = 0 electron/sec
INFO:scopyon._epifm: EM gain = x 300
INFO:scopyon._epifm:Focal Center: [0. 0. 0.]
INFO:scopyon._epifm:--- A/D Converter: 16-bit
INFO:scopyon._epifm: Fullwell = 800000 electron
INFO:scopyon._epifm: none-Fixed Pattern Noise: 0.0 count
INFO:scopyon._epifm:--- Excitation Filter:
INFO:scopyon._epifm:--- Emission Filter:
INFO:scopyon._epifm:--- Background:
INFO:scopyon._epifm: Mean = 0.01 photons
INFO:scopyon._epifm:--- Fluorescence:
INFO:scopyon._epifm: Quantum Yield = 0.61
INFO:scopyon._epifm: Abs. Coefficient = 83400 1/(M cm)
INFO:scopyon._epifm: Abs. Cross-section = 3.1888261070088875e-16 cm^2
INFO:scopyon._epifm:--- Photobleaching:
INFO:scopyon._epifm: Photobleaching half life = 2.5
INFO:scopyon._epifm:processes = 1.
INFO:scopyon._epifm:time: 0.0 sec (0)
INFO:scopyon._epifm:center (width, height): 0.0 0.0
INFO:scopyon._epifm:scaling [m/pixel]: 6.639004149377593e-08
INFO:scopyon._epifm: 000-th file in 000-th frame: 0.0 + 0.033 sec
INFO:scopyon._epifm:Total camera_pixel = 1.087719e+04
INFO:scopyon._epifm:100 optoinfos are registered.
INFO:scopyon._epifm:Apply detector effects.
Make a two-color image by merging two images.
[8]:
img = scopyon.Image.RGB(red=img1, green=img2)
[9]:
img