… specifically for cyanobacteria and/or chlorophyll-a?
I’ve nearly completed my first delta stage microscope (also my first open source project period) and I’m really excited because this has been a long process of learning & acquiring both skills and parts. I never went to college and I’ve more or less stumbled into being an (extremely) amateur biologist, so I’ve been carefully collecting material on fluorescence microscopy — though this is pretty challenging because i have no background or frame of reference for a lot of the academic material. This brings me to my dilemma:
How do I choose my excitation and emission filters so that they match the purpose of what I’m using the microscope for: observing local microalgae/cyanobacteria (really anything in the pondwater though)? By digging through the many open source fluorometer projects I was able to atleast narrow down the excitation filter i need for observing chlorophyll-a (#19 red from the roscolux booklet) but now I’m at a loss for the emission filter.
Does anyone have any experience they could quickly share?
i want to contribute something so let me add that when building the reflection module I found using a dremel way easier than the suggested oil glass cutter recommended in the instructions. the beam splitter is such a small piece of glass and the budget-friendly tool i sprung for wasn’t delicate/accurate enough. the dremel was much simpler in case anyone else is having problems with cutting the glass*
Thank you for everything.
Thanks for posting @spinoza, and thanks for the tip on the Dremel. I’ve never used one to cut glass, so that’s helpful to know. Our glass cutter isn’t overly expensive, and the same technique works pretty well with a £10 diamond scribe, but there’s very definitely a knack to it, that is not easy to convey other than in person! I smashed up ten or twenty microscope slides before I got the hang of it…
As for choosing your filters, I would start by looking up the excitation and emission spectra of the things you’re interested in. I guess if you’ve found fluorimeter projects, they may have posted spectra already?
A quick search found this paper on chlorophyll a (didn’t ask me to log in but I’m not sure if it’s open or not) so I think you can excite with blue (400-450nm) or red (~600nm) light, and the fluorescence looks to be near-infrared (>650nm). So, you could get a suitable long pass filter for emission, and a short pass for excitation - and your beamsplitter can either be 50:50, or you can use a 45 degree dichroic (more expensive but more efficient). I don’t know if you can search the Rosco website by wavelength, or if they deal with IR filters; I see they make one that blocks IR, but you specifically want to pass IR and block everything else! I dimly remember there’s some magic combination we used to use in the theatre when we wanted IR illumination (was it a particularly violent red + blue combination?) but that may not be officially documented and I don’t have a note of the filter numbers.
Oh, and I think you’ll want to make sure you remove the IR filter from the Pi camera!
Thank you very much for all the links.
I’m waiting on the last bit of components for my sangaboard v3 and then I’ll have all of the hardware stuff done. I’ll be sure to post pics of the build and share the results I’m able to get with various water samples.
Thank you again, it’s been a blast building this thing.
** UPDATE **
Found a really cool web app that has all the fluorescence information for all types of organisms/compounds. Super helpful if you know nothing (like myself).
https://www.aatbio.com/fluorescence-excitation-emission-spectrum-graph-viewer/C_Phycocyanin
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Thanks @spinoza that is a really handy tool!