DIY Fluorescence microscopy?

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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maguee
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DIY Fluorescence microscopy?

#1 Post by maguee » Mon Feb 21, 2022 2:59 pm

I have seen a few videos and tutorials on the internet about homemade Fluorescence microscopy.

Is there any info or experience in this forum?
Thank you!!

Plasmid
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Re: DIY Fluorescence microscopy?

#2 Post by Plasmid » Mon Feb 21, 2022 3:48 pm

Are you looking for info on how to assemble the hardware or on the dyes?

Alexander
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Re: DIY Fluorescence microscopy?

#3 Post by Alexander » Mon Feb 21, 2022 4:02 pm

In a first step differentiate between transmitted light and epi-fluorescence.

Transmitted light:
You need some excitation filter and an analyzer filter. The first under and the second over the specimen. Very much the same as polarization. The light source must deliver the needed wave length. This technology has two significant disadvantages. First all the light that passes the excitation filter goes up to the analyzer and the eye-peaces. The background of your picture will never be black. Second the excitation takes place on the wrong side of the specimen und the (much weaker) fluorescence light must pass the specimen which reduces contrast.
For UV excitation a 4 mm thick BG12 is best. As analyzer a K510 will work well. For blue excitation the filter combination could be K430 and K510 or K530.

Epi-fluorescence:
Mount the light source, preferable a LED on the stage table and illuminate the specimen from above and from the side. Again you need an analyzer filter. The mayor problem is the little working distance of stronger objectives. There is very little room to bring the excitation light onto the specimen.

Plasmid
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Re: DIY Fluorescence microscopy?

#4 Post by Plasmid » Mon Feb 21, 2022 7:14 pm

Alexander wrote:
Mon Feb 21, 2022 4:02 pm
In a first step differentiate between transmitted light and epi-fluorescence.

Transmitted light:
You need some excitation filter and an analyzer filter. The first under and the second over the specimen. Very much the same as polarization. "




I've have very knowledge on this technique, do you know of any dedicated condensers and or microscope platforms sold for this technique?.

Alexander
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Re: DIY Fluorescence microscopy?

#5 Post by Alexander » Mon Feb 21, 2022 7:28 pm

Transmitted light fluorescence is no longer used in professional labs. It survives as a hobbyists technology only. Any microscope that allows filters under and above the specimen will do.

maguee
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Re: DIY Fluorescence microscopy?

#6 Post by maguee » Mon Feb 21, 2022 10:17 pm

Alexander wrote:
Mon Feb 21, 2022 4:02 pm
In a first step differentiate between transmitted light and epi-fluorescence.

Transmitted light:
You need some excitation filter and an analyzer filter. The first under and the second over the specimen. Very much the same as polarization. The light source must deliver the needed wave length. This technology has two significant disadvantages. First all the light that passes the excitation filter goes up to the analyzer and the eye-peaces. The background of your picture will never be black. Second the excitation takes place on the wrong side of the specimen und the (much weaker) fluorescence light must pass the specimen which reduces contrast.
For UV excitation a 4 mm thick BG12 is best. As analyzer a K510 will work well. For blue excitation the filter combination could be K430 and K510 or K530.

Epi-fluorescence:
Mount the light source, preferable a LED on the stage table and illuminate the specimen from above and from the side. Again you need an analyzer filter. The mayor problem is the little working distance of stronger objectives. There is very little room to bring the excitation light onto the specimen.
Thank you alexander!! Very usefull info.

Hobbyst46
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Re: DIY Fluorescence microscopy?

#7 Post by Hobbyst46 » Mon Feb 21, 2022 10:56 pm

One reason that trans-fluorescence has been phased out is, as commented above, its inability to fully separate the excitation from the fluorescence - the major challenge of basic fluorescence inspection and measurements..
Trans-fluorescence has been practiced with darkfield condensers; those provide illumination at an angle, so less excitation light passes directly into the objective. Still, they are inferior than epi-fluorescence.

For safety reasons, I would avoid any ultra violet trans-illumination. Too risky for your eyesight. Filters do not sufficiently protect from the harmful UV.

maguee
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Re: DIY Fluorescence microscopy?

#8 Post by maguee » Tue Feb 22, 2022 8:36 am

Hobbyst46 wrote:
Mon Feb 21, 2022 10:56 pm
One reason that trans-fluorescence has been phased out is, as commented above, its inability to fully separate the excitation from the fluorescence - the major challenge of basic fluorescence inspection and measurements..
Trans-fluorescence has been practiced with darkfield condensers; those provide illumination at an angle, so less excitation light passes directly into the objective. Still, they are inferior than epi-fluorescence.

For safety reasons, I would avoid any ultra violet trans-illumination. Too risky for your eyesight. Filters do not sufficiently protect from the harmful UV.
Well pointed out. Even with high quality filters i dont see any reason to risk the eyesight. All the fluorescence microscopy videos i have seen are all about epi-fluorescence or trans fluorescence with a camera, so the eyes are never exposed.

Alexander
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Re: DIY Fluorescence microscopy?

#9 Post by Alexander » Tue Feb 22, 2022 9:18 am

For a hobbyist UV excitation is of limited use anyway. It is widely overrated by many. In most cases you are better off with blue excitation. Acridine orange as a stain is very versatile, cheap and easily available while stains for UV excitation tend to be very, very expensive and are rarely available to hobbyists.

I would experiment with a blue LED as excitation light and some green filter as an analyzer. The LED is easily mounted on the stage to provide epi-fluorescence.

apochronaut
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Re: DIY Fluorescence microscopy?

#10 Post by apochronaut » Tue Feb 22, 2022 3:36 pm

Diascopic fluorescence is not entirely out of the picture. It is still used with a DF condenser for certain applications. For those in North America who have easier access to ebay.com, occasionally older fluorescent systems show up for sale and often quite cheaply. The AO model 20 was designed in an era when diascopic fluorescence was still much practised but epi fluorescence was up and coming. It can do both. It has a 2 gang filter carousel pack under the illuminator window capable of carrying 6 glass filters. The epi fluor units carry a 200 watt bulb and the entire unit dovetails in under the head. The microscope needs two power supplies to do epi fluor.
The transmitted system will need a DF condenser and iris equipped objectives. The condensers are still around for sometimes 50.00. The 50X .80 planachro with iris are still common and often very cheap (75.00 or less). The 100X 1.25 planachro with iris are less common now but still around. The last one I saw was $150.00. The entire epi systems go for in the neighbourhood of 150.00.

Some of the better microscope brokers/dealers that handle Chinese microscopes have stepped up to offering tier 2 Chinese microscopes aimed more towards professionals. LW Scientific is one such. Fisher another. There are quite a few but I will focus on LW because they are quite in evidence and offer epi fluorescent scopes. One of them in the past was the B200 , which now 10 or so years forward are showing up at bargain prices, used. This not an Amscope and in fact the very same microscope is still available factory direct as a 20 watt halogen model or as a 100 watt research model, with an either 20mm or 23mm f.o.v., 4 or 5 objectives. They can be either 160mm or infinity, with the infinity system having the same corrections as the older AO 34mm system, although in this case they are 45mm parfocal planachros. Prior, formerly (c.2005) sold a Pol version of the 20 watt model at in the neighbourhood of $2500.00 then, and as of a year ago, the 23mm f.o.v. trinocular, 5 objective 100 watt version was $2600.00, factory direct, f.o.b. This is not a shabby microscope and LW once offered an epi fluorescent infinity corrected version. Occasionally they now show up on ebay. One recently for $79.00. It only had one fouled objective, which cleaned up nicely. These get overlooked mainly because most microscope buyers are searching known brands and when they do encounter a stencil brand, they can't tell the difference between a good Chinese microscope and a poor one. Trinocular heads are still available for them: even the 23mm ones from the factory( about 350.00 f.o.b.). I mention this because trinocular versions are not common in the aftermarket.
For someone looking to get into fluorescence , the above 2 microscopes are good cheaper options.

maguee
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Re: DIY Fluorescence microscopy?

#11 Post by maguee » Tue Feb 22, 2022 10:07 pm

Alexander wrote:
Tue Feb 22, 2022 9:18 am
For a hobbyist UV excitation is of limited use anyway. It is widely overrated by many. In most cases you are better off with blue excitation. Acridine orange as a stain is very versatile, cheap and easily available while stains for UV excitation tend to be very, very expensive and are rarely available to hobbyists.

I would experiment with a blue LED as excitation light and some green filter as an analyzer. The LED is easily mounted on the stage to provide epi-fluorescence.
Thanks Alexander. In fact i was thinking about blue led, but i have just check the price of acridine orange and its probably one of the cheapest florescence stains but still a bit more expensive than i thought. I will continue in BF DF PC and polarization with non professional waveplates unless i can try fluorescence microscopy in a cheaper way.

crb5
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Re: DIY Fluorescence microscopy?

#12 Post by crb5 » Sat Feb 26, 2022 12:13 am

You can use a cheap LED flashlight (torch) to illuminate a sample for fluorescence detection with low power objective lenses (up to about 10x which have enough working distance to shine on to the sample without casting a shadow). A laser pointer can be used for higher power objectives (up to about 40x) - make sure there is a blocking emission filter in the microscope and/or record the image via a camera rather than by eye.

https://www.youtube.com/watch?v=w1tG2wj_TAU

We have used such set ups to look at microplastics using stereo and compound microscopes.

https://www.researchgate.net/publicatio ... _Detection

Greg Howald
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Re: DIY Fluorescence microscopy?

#13 Post by Greg Howald » Sat Feb 26, 2022 3:50 am

I Appreciate this post as my brother is interested in the subject matter and I can share with him. The forum again proves very informative.

BramHuntingNematodes
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Re: DIY Fluorescence microscopy?

#14 Post by BramHuntingNematodes » Sat Feb 26, 2022 4:58 am

I wanted to see if a very simple setup could work and I have just observed some fluorescence. I took a hand section of some moss and shone a 405nm laser on it. This laser arrived free with eyeglasses ordered online, I guess as a free prize or something. A 25A wratten filter was placed in front of the camera, and it did seem to work! I was not able to discern much useful detail, but the operating principle seems to have been proved.
1942 Bausch and Lomb Series T Dynoptic, Custom Illumination

maguee
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Re: DIY Fluorescence microscopy?

#15 Post by maguee » Sat Nov 18, 2023 7:37 am

Is there any way to repair a dicroic mirror?

maguee
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Re: DIY Fluorescence microscopy?

#16 Post by maguee » Wed Nov 22, 2023 3:33 pm

Well, i have a working fluorescence microscope. An old model without the Mercury lamp. I just put a blue/Green led torch in the tube, select a position for the built-in filters and... It just works.
It was a cheap opportunity i couldnt let go. I have to do some minor manteinance.


But... Fluorescent dyes are very expensive and i am more interested in autoflorescence. Any ideas about what examples should i look for? I have seen ukmicroscopy posts about autoflorescence, but i would like to know more about this.

Hobbyst46
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Re: DIY Fluorescence microscopy?

#17 Post by Hobbyst46 » Wed Nov 22, 2023 4:28 pm

maguee wrote:
Sat Nov 18, 2023 7:37 am
Is there any way to repair a dicroic mirror?
If the coating is peeling off or has changed color, the mirror cannot be repaired.

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