Troubleshooting Fluorescence Microscopy
Jim Pawley upgraded the 39 Steps from a simple MacGuffin to a classic checklist for troubleshooting fluorescent microscopy images. Now 10 years old, it remains very relevant. It covers thirty-nine different parameters to check: from singlet state saturation to embedding medium. It’s a good place to start if you’re puzzled as to why your images don’t look as good as you expected.
Jim Pawley’s The 39 Steps (thanks to CW for the tip)
On a related note, if you’re in London, the stage production of The 39 Steps at the Criterion theater is a great comedy to check out.
BTW, here are the online microscopy and imaging references from the major microscope manufacturers. These can also help in troubleshooting.
Olympus Microscopy Resource Center
Nikon’s Microscopy U
Carl Zeiss MicroImaging Online Campus
Don’t forget catalogs as textbooks!
Pawley’s 39 Steps is less a checklist for troubleshooting fluorescence microscopy images and more a position for the impossibility of quantitative fluorescence imaging. He states explicitly that this list is not exhaustive and that other factors may pertain. The actual stated thesis of this paper is that without controlling for the other 38 factors, quantitative determination of the factor of interest (usually amount of fluorophore present) is impossible.