Fluorescent Double-Labeling

• Objective 5x
• Zoom 4.0 x (except PBN as 3.0x)
• Format 1280x1024

Need to confirm the calibration of the objective -> um/pixel; it seems to have changed

• Live (play button icon)

• Adjust AutoExposure/Analog Gain or enter set exposure time

• Acquire -> Capture (camera icon)

captures image with flurophore channel (e.g. "FITC")

(have to restart Live after a capture to view live image again

• Save As -> Tagged Image Format (TIFF)

proprietary Nikon file format that preserves acquisition parameters

• Save As -> JPEG

Save single fluorophore channel 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).

• Open JPEG files for each fluorophore/

(e.g., FITC JPEG and TRITC JPEG)

• 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 green (FITC) channel on top of red (TRITC) channel

• Save merged image as JPEG file