Fluorescence Composite

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):
Image cells

  1. Take a bright field
  2. Turn off lights, add filter, refocus and take care to make sure second picture is in same position

Photo editing

  1. Open up both images in Photoshop
  2. Fluorescent image: Select all (Ctrl-A), Copy (Ctrl-C)
  3. Paste into brightfield image (will appear as new layer)
  4. With fluoro layer selected, go to Image>Adjustments>Channel Mixer
  5. Change Channel to whichever you want (red if you want to output red, green if you want green)
  6. Let’s say Green.
  7. Drag green slider up to ~110%
  8. Drag blue slider up somewhat
  9. Oversaturated green… then drag “Constant” down about 20-30%.
  10. Play with settings until you get desired colouring. Click OK.
  11. Change Blend mode on fluoro layer to “Color”. This gives best results for me. Alternatively Overlay and Screen work as well.
  12. Save as a PNG for a nice composite image.

Hope that helps some of you out there! Remember to stay cultured!