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Objective 5x
*Objective 5x
Zoom 4.0 x (except PBN as 3.0x)
*Zoom 4.0 x (except PBN as 3.0x)
Format 1280x1024
*Format 1280x1024
'''Need to confirm the calibration of the objective -> um/pixel; it seems to have changed'''
Live (play button icon)
*Live (play button icon)
Adjust AutoExposure/Analog Gain or enter set exposure time
*Adjust AutoExposure/Analog Gain or enter set exposure time
Acquire -> Capture (camera icon)
*Acquire -> Capture (camera icon)
captures image with flurophore channel (e.g. "FITC")
:captures image with flurophore channel (e.g. "FITC")
(have to restart Live after a capture to view live image again)
:(have to restart Live after a capture to view live image again)
Save As -> Tagged Image Format (TIFF)
*Save As -> Tagged Image Format (TIFF)
proprietary Nikon file format that preserves acquisition parameters
:proprietary Nikon file format that preserves acquisition parameters
Using TIFF files, Drag and drop one channel from one TIFF file onto second channel in second TIFF file to create a merged TIFF file.
*Using JPEG files, Drag and drop one channel from one JPEG file onto second channel in second JPEG file to create a merged JPEG file.
e.g., drag FITC on top of TRITC channel
:e.g., drag green (FITC) channel on top of red (TRITC) channel
Save merged image as TIFF file
*Save merged image as JPEG file
Save As JPEG
*Save As JPEG
Save merged image as JPEG file to convert from emission filter channels to RGB
:Save merged image as JPEG file to convert from emission filter channels to RGB
JPEG file of one fluorophore channel will have data in 2 or 3 RGB channels, so may need to delete unwanted RGB chanels (e.g. delete red channel from FITC JPEG, to leave only green channel.) Or, better still, convert fluorophore image to monochrome (i.e., so that luminance of flurophore channel is encoded as equivalent gray value from 0-255 in each RGB channel of the JPEG).