Combine fluorescent and brightfield images taken of your cell culture using Photoshop. This tutorial was done using a PC3 cancer cell line that was stained with acridine orange for 5 minutes. The stain penetrated the membrane and binds with the DNA to produce “green” fluorescence (viewed using a FITC filter).
How this was done (for those text lovers out there):
- Take a bright field
- Turn off lights, add filter, refocus and take care to make sure second picture is in same position
- Open up both images in Photoshop
- Fluorescent image: Select all (Ctrl-A), Copy (Ctrl-C)
- Paste into brightfield image (will appear as new layer)
- With fluoro layer selected, go to Image>Adjustments>Channel Mixer
- Change Channel to whichever you want (red if you want to output red, green if you want green)
- Let’s say Green.
- Drag green slider up to ~110%
- Drag blue slider up somewhat
- Oversaturated green… then drag “Constant” down about 20-30%.
- Play with settings until you get desired colouring. Click OK.
- Change Blend mode on fluoro layer to “Color”. This gives best results for me. Alternatively Overlay and Screen work as well.
- Save as a PNG for a nice composite image.
Hope that helps some of you out there! Remember to stay cultured!