Geotrichum candidum

NileRed · Post by **NileRed** » Mon Apr 11, 2011 3:35 am

Hello,
Following 4 pictures are Geotrichum Candidum (grown on Malt Agar), this mold you can find on the surface of some stinky cheeses.
Fluorescence microscopy, dye – Calcofluor white (stain chitin, thus you can see only cell walls and septa), Objective - Carl Zeiss Jena 40x 0.95, 550 EOS, ISO 200.

Important that pictures were obtained using NO common expensive epi-fluorescent attachment.
I used just two interference filters (eBay) and as a light source – 10W UV LED (eBay), everything was not more than 150 USD, what is incomparable with cost of the common epi-fluorescent attachment.

Optic scheme was very simple: UV LED with cooler - collimator (metal, not plastic) - excitation filter (365 nm) - condenser-sample – objective - barrier filter (450LP)….see following picture.

How I have found recently, power of such LED was enough to excite even autofluorescence of xylem lignin which does not require (by determination) any dye addition.

fpelectronica · Post by **fpelectronica** » Mon Apr 11, 2011 5:16 am

Very interesting
Francisco

René · Post by **René** » Mon Apr 11, 2011 12:27 pm

Nice clean image, I'm surprised how much UV still gets through the condenser. But with 10 W pure UV, wow, you can bake everything! What's the exposure time by the way?

I've also seen simple setups in the normal visible light range with halogen lighting. With a darkfield condenser the result is even cleaner, as the barrier filter doesn't need to be so 'strong'. It's a good old way of doing this fluorescence work.

But also an epi setup can be tinkered for very little money, based on the old vertical illuminator with a coverslip at 45 degrees in the path. A little piece of dichroic mirror improves it a lot further, etc etc..

Good luck,
René

lauriek · Post by **lauriek** » Mon Apr 11, 2011 12:31 pm

Wow, lovely pictures and interesting technique, I will re-read this when I've had my dinner!

Cactusdave · Post by **Cactusdave** » Tue Apr 12, 2011 12:06 am

Very interesting technique. This LED light source could very easily be adapted for episcopic use. Any chance you could post more detail of your lamp mount, perhaps in the 'techniques' forum?

NileRed · Post by **NileRed** » Tue Apr 12, 2011 11:04 am

Thank you for your interest to my pictures and technique.

Definitely it was very simple idea, and how I know, use of LED as a light source in fluorescent microscopy is not something new, but my main goal was to find something very cheap and simple what is able to convert bright field microscope to so to say "fluorescence" one. Generaly my idea was to make fluorescence microscopy more available for hobbyists of different level.

Few comments: I usually use exposition time from 5 to 20 sec (often 10-15 sec), and ISO 100 or 200. All pictures shown here except second one were stacks (Zerene), contrast and brightness were increased for all of them.
Condenser here just slightly concentrates UV light on the sample, I think not so strong as objective in epi-fluorescent attachement and I have not seen strong bleaching for Calcofluor white and lignin or cutin with my UV LED.

Few words about cleanness: This is, unfortunately, not only function of objective, filter set and filters quality but also - which type of dye do you use and how do you use it, what type of mounting solution do you use and etc. 65 % Glycerine, what I used here, has his own fluorescence, because it contains some impurity. Water is better, but not for long shooting.

About filters: my personal feeling - The devil is hidden often in excitation filter. For example, having 25 W blue LED, blue interference excitation filter and green interference barrier filter I see using no sample - green background. In case of one cheap epifluorescence microscope with common epi attachment I saw the same - green background!

About dark field condenser method: Definitely, I have tried old method with dark field condenser using 25 W blue LED, I was not satisfied with result - too low intensity for not bright dyes or autofluorescence.

René · Post by **René** » Thu Apr 14, 2011 8:51 am

hmm, yes. At these exposure levels, bleaching is not very much of a problem. I'm not sure about the problem with excitation filters, the ones that come with fluorescence microscopy are quite efficient, at least 50% gets through. But for UV exciation they are not necessary. We use UV-leds routinely, in epi setup, home made. Couple of hundred $ all together for one setup, not more. LED used is a Nichia 365 or 385 nm, max 300mW output, but generally used below 100mW. I have also used the very cheap 20mA UV leds (not sure about the output, they weren't given in watts in any case). You might it back here on the forum. We use it for dinoflagelate staining with calcofuor, also DAPI for nuclear staining.

Good luck, René

g4lab · Post by **g4lab** » Sat Apr 16, 2011 10:38 am

I can furnish new Zeiss HBO 100 lamphouses with Leistung Electronics Jena
power supplies (also new and international for voltage and frequency) for $400, plus shipping. I also have some Zeiss filter sets available. (just the filters not the cube holders)

If interested contact me by PM.

For darkfield fluorescence you have to track down a quartz glass condensor which is a fairly rare thing to find.

NileRed · Post by **NileRed** » Mon Apr 18, 2011 3:56 am

Rene,
thank you for the comments. May I somehow see your "dinoflagelate staining with calcofuor, also DAPI for nuclear staining" pictures?

How I recently found, my bright field condenser emit some amount of green fluorescence under blue light exposure, so for blue light excitation with 25W blue LED I use no condenser and in short time I will post fern stem section autofluorescence using this setup.

With best wishes

g4lab,
thank you for the offer I will keep it in my mind.
With best wishes