Stereo Microscope : Fluorescence Adapter

[Hobbyst46]

Michael,

Here is a crazy idea to try WITHOUT a stereo microscope - but an ordinary microscope. With a long WD objective. Say a 6,3X (mine) or 4X.
Make a thin-walled sleeve (plastic, cardboard, metal), of an inner diameter just slightly wider than the external diameter of the objective barrel.

Close the bottom opening of the sleeve with a small barrier filter. Say, a 8-10mm circle cut from an orange photography filter. Have the objective enclosed within the sleeve. The filter is thin enough to allow focusing of the objective on a slide, as usual.

Find a narrow beam 5mm, 20-50mA super-bright blue LED (450nm). Solder to stiff wire leads than can position the LED to shine sideways, diagonally, from above onto the slide on the stage beneath the filter+objective. It will be sufficient to uniformly illuminate the 3mm FOV without auxiliary lenses. No dimmers, use continuous maximal current from the power supply.

Sprinkle tiny fragments or cuttings of a green leaf or algae on the slide. Turn off all other room lights.

If I see red fluorescent spots through the eyepieces, and can photograph them, the principle works. Photography is a challenge though, since consumer cameras do not perform well with such weak signals.
How about that?

[MichaelG.]

That sounds perfectly logical as a 'proof of concept'

MichaelG.

[MichaelG.]

Edit: We did not discuss the necessity of a dimmer. Perhaps the Nightsea goose-neck is equipped with a dimmer. I am sure we can do without it. With fluorescence, usually, the brighter the excitation the better, with two exceptions: (a) a too intense light beam can damage the (live) organism, and (b) a too light beam can cause "photo bleaching" - a chemical change to the fluorescing molecule. These two factors, IMO, are not expected with the stereo microscope, since the illumination is not really focused (since we do not see any lenses between the light head and the specimen).
Cheers.

More 'points well made' thank you.

Both dimming and pulsing are available as options ... Text and video here:
https://www.emsdiasum.com/microscopy/pr ... htsea.aspx

MichaelG.

[Hobbyst46]

Pulsing is indeed routinely used to cope with photobleaching and photodamage in research studies, but it requires synchronization of the fluorescence detector (or camera) with the light source. But, again, I doubt that such measures are really justified when doing low-magnification microscopy. Especially for amatuer, non-quantitative jobs.

[Hobbyst46]

Pulsing is indeed routinely used to cope with photobleaching and photodamage in research studies, but it requires synchronization of the fluorescence detector (or camera) with the light source. Again, I doubt that such measures are justified for low-magnification, especially amateur, non-quantitative microscopy.

Having inspected again the barrel diameter (20mm) and working distance (4.9mm) of the 6.3X objective, I give up the "external excitation illumination". Too small space for a filter and too shallow angle (nearly horizontal) for a LED beam. My next low magnification is a 2.5X objective. 18mm barrel diameter and 8.7mm WD - yet the magnification is so low...

[Hobbyst46]

That sounds perfectly logical as a 'proof of concept'

I assembled a simple illumination setup for a biological, not stereo microscope. It is shown in the drawing. The objective is a 2.5x0.08, WD of about 9mm. I attached the barrier filter to the objective barrel by means of an adhesive tape, wound around the barrel and adjacent filter. I wrapped them on the outside with a tight-fitting cylinder made from black paper, to admit light passage only through the bottom surface of the filter and not its peripheral surface.

The filter was an orange absorption circular filter, LP530nm, diameter 18mm, thickness 2mm. It is shown as a thick orange horizontal line in the drawing. In a later stage, I added below it a 500nm interference filter (dichroic mirror), 1mm thickness. This filter was rectangular, 18x24mm. Hence, to attach it to the first (top) filter, I laid it flat against the orange filter and attached with pieces of cellotape. Really crude.

The LED was a 5500mcd, 5mm package, 15degrees angle, 470nm bright blue LED. It was fed from a 6V power supply, through a 125 Ohm resistor. The specimen was a one-year old mounted leaf moss in glycerin. To illuminate it, I held the LED very near the objective, pointing diagonally downward. Turned
off the room lights and observed.

Using the orange filter alone, the moss appeared green or blue-green, with a faint red glow around some of the "leaves". The FOV was fairly uniformly illuminated. Blue arrows in the drawing are excitation, red arrows - fluorescence (perhaps the true Red Arrows shine as well - I do not know )

Using the combination of two filters, the red fluorescence on dark/black background was visible, without blue or green interfering light. It was not possible to use the interference filter alone, because of its geometry, I could not attach it to the objective without leakage of the blue light through the sides or internal reflections.

Conclusions:
1. The LED light was sufficiently intense to excite visible fluorescence. The fact that red fluorescence was weak in this case is because the of peak wavelength of 470nm is fairly remote from the optimum, which IMO is at 400-430nm.
2. The challenge to the method, as suspected, is elimination of the strong excitation from the FOV. This was only achieved with the interference filter (dichroic mirror). So, at least for a DIY apparatus, I strongly recommend an interference filter. If it can be attached to the objective bottom hermetically, that is, without light leakage, I believe that a single interference filter will do the job, and the additional orange filter will be superfluous.
If I can safely replace the blue LED with a violet or UV LED, there is a chance of stronger fluorescence against black background, and a chance to obtain photos of the FOV.

[billbillt]

Here is a link to a paper where it is done on the cheap... Note the LED wavelengths marked on the interchangeable units....

http://www.microscopy-uk.org.uk/mag/ind ... luoro.html

BillT

[MichaelG.]

That sounds perfectly logical as a 'proof of concept'

I assembled a simple illumination setup for a biological, not stereo microscope. It is shown in the drawing.
[ ... ]
The LED was a 5500mcd, 5mm package, 15degrees angle, 470nm bright blue LED.
[ ... ]
If I can safely replace the blue LED with a violet or UV LED, there is a chance of stronger fluorescence against black background, and a chance to obtain photos of the FOV.

Thanks for trying this ... Your results are, I think. 'much as we might expect' and provide a very useful benchmark for future experimentation.

I have violet LEDs available, and also a [very tempting, but almost certainly too dangerous*] violet laser-pointer.

MichaelG.
.

[*] 405nm wavelength is, reasonably successfully, used in low-end laser engraving machines !!
It will burn wood, paper, and some plastics ... so would probably do instantaneous and very serious damage to a careless eye. ...There MAY be a way of beam-spreading that renders it harmless, but I have yet to find it.

[MichaelG.]

Here is a link to a paper where it is done on the cheap... Note the LED wavelengths marked on the interchangeable units....

http://www.microscopy-uk.org.uk/mag/ind ... luoro.html

BillT

Thanks for the link, BillT
Yes, I remember seeing that.

MichaelG.

[Hobbyst46]

I have violet LEDs available, and also a [very tempting, but almost certainly too dangerous*] violet laser-pointer.
[*] 405nm wavelength is, reasonably successfully, used in low-end laser engraving machines !!
It will burn wood, paper, and some plastics ... so would probably do instantaneous and very serious damage to a careless eye. ...There MAY be a way of beam-spreading that renders it harmless, but I have yet to find it.

Lasers are used in confocal microscopy for example, with a variety of wavelengths, but the eyesight protection is built in these machines, neither do they etch anything there.

MichaelG, Please, please, please, resist any temptation to shine a laser pointer/diode near the microscope. Not just the 405nm, but ANY laser/diode beam. Note that a laser or laser/diode beam can be reflected by any reflective surface around you, without losing much power.

I too have 5mm package violet and long UV LEDs available, and even with these relatively humble light sources, I will arrange precautions before installation. I believe that a super bright ordinary LED will do the job - again, the proof is in the barrier filter.

[MichaelG.]

... MichaelG, Please, please, please, resist any temptation to shine a laser pointer/diode near the microscope. Not just the 405nm, but ANY laser/diode beam. Note that a laser or laser/diode beam can be reflected by any reflective surface around you, without losing much power.

It's O.K. we are 'on the same page'
My comment was intended as a warning to others.
... I only have one pair of eyes, and already have artificial lenses in them [Cataract surgery]
... and I intend to keep them !!

MichaelG.

[billbillt]

Another interesting paper on this subject...

http://www.microscopy-uk.org.uk/mag/ind ... oray5.html

BillT

[billbillt]

And another....

http://www.microscopy-uk.org.uk/mag/ind ... oray6.html

BillT

[Hobbyst46]

Thanks BillT for the links. Never knew about these articles by David Walker. Nice to read about transmittance light fluorescence, what they used in the very old days.

In the article:
http://www.microscopy-uk.org.uk/mag/ind ... oray6.html
Walker demonstrated a different setup from what I thought: he used an interference excitation filter on top of the LED, and an orange absorption filter as emission filter. In principle, both options can work, depending on the narrowness of the LED wavelength range and the wavelength of the produced fluorescence.

[crb5]

I recently discovered the MicrobeHunter forum which I find very educational and inspiring. I have been using fluorescence microscopy for academic research for 30 years, but in the last couple of years have been exploring ways to adapt basic bright-field microscopes for observing plankton with community scientists and college students. I “rediscovered” polarization, dark field and oblique illumination methods, topics that I now see have been well covered in the forum. However, I found little on fluorescence microscopy. I note this topic was raised back in October 2018, but the discussion petered out after a month and several ideas seemed to have gone untested.

We have used a simple LED flashlight (torch) to excite fluorescence in microplastics in sea water samples stained with Nile Red. This we achieved with a $8 blue (455 nm) flashlight and low specification color filters/film, using stereo microscopes and low power (up to 10x objective) compound microscopes. The set-up is described in some YouTube videos:

https://www.youtube.com/watch?v=w1tG2wj_TAU&t=6s

https://www.youtube.com/watch?v=BYvU9sOAuFM


A key element is to add a second focusing lens to the flashlight so that the focused spot size is reduced to a few millimeter square (the image of the LED chip) to gain sufficient excitation intensity and an appropriate field of illumination for moderately high magnifications using a stereo scope. The flashlight is held at around 60 degrees using a table-top tripod. With higher power objectives on a compound microscope, the angle must be shallower, to avoid the objective blocking the beam, and this gives a more elliptical illumination spot with lower intensity. In this case, a laser pointer source gives better results. Research grade emission filters are required for the lowest background fluorescence, but useful results can be obtained with yellow and orange plexiglass filters attached to the stereo objective housing or inserted in the body of compound microscope beneath the removable ocular head.

Previous discussions have raised a couple of important questions. (1) Is it generally useful for the hobby microscopist and (2) is it safe? Obviously not many samples are highly fluorescent and usually dyes must be added. The latter may not be readily available to individuals, but schools and colleges should be able to order dyes from companies. Chlorophyll fluorescence is one form that can readily be observed in Nature and best excited around 400 nm. So called black light LED flashlights are available that emit in the near uv and just into the visible region and these are sufficient. As the light is not directly entering (or reflecting into) the microscope objective and the beam is strongly convergent on the sample (and hence divergent at distances beyond), I do not see any safety issues beyond those encountered in everyday use of these flashlights. As far as laser pointers go, similar considerations apply. The main problem here is that some violet (405 nm) laser pointers exceed the 5 mW limit and the hobbyist does usually not have access to a power meter to check. If the focused beam ignites a match then beware!

Our work should soon appear in J Chemical Education, and I can provide more details to anyone who is interested.

[Scarodactyl]

Would there be any particular problem with just putting a barrier filter in the light path inside the microscope? I assume it would assure safety if it were a good quality one, though maybe the visual impact would be severe.

[crb5]

No problem for an infinity corrected microscope, but most hobby scopes form an intermediate image in the microscope tube and adding a filter inside the microscope gives a small shift in focus and a bit of spherical aberration but barely noticeable with a 10x objectives. This is exactly what we do in the case of a compound scope. A 37 mm diameter camera filter sits in the port where the ocular head is attached and is readily removed with a pair of tweezers. It can be left in place for bright field imaging as long as a yellow or orange image is acceptable. For our purpose of scanning samples for microplastics it works well, but the field is not that evenly illuminated for those seeking high quality photographs. My stereo scope does not come apart that easily, so here we put the emission filter between the sample and objective lenses - either screwed into a filter stepping ring glued to the housing or attached with a small magnet.

[MichaelG.]

I note this topic was raised back in October 2018, but the discussion petered out after a month and several ideas seemed to have gone untested.

We have used a simple LED flashlight (torch) to excite fluorescence in microplastics in sea water samples stained with Nile Red. This we achieved with a $8 blue (455 nm) flashlight and low specification color filters/film, using stereo microscopes and low power (up to 10x objective) compound microscopes. The set-up is described in some YouTube videos:

Thanks for the input ... I will follow your links etc. with great interest.

To be frank: my own interest petered out when I discovered the UK price of Nile Red
... Can you suggest an affordable source ?

MichaelG.
.

Edit: Just to demonstrate what a cheapskate I am ... This was my price reference:
https://www.fishersci.co.uk/shop/produc ... s/11472502

[crb5]

Yes, getting hold of Nile Red is the hard part. As you note, it costs around $30 to $40 for around 10 to 50 mg minimum order. We use around 20 micro liters of 1 mg/ml stock NR dissolved in methanol or rubbing alcohol stain for 5 ml of sea water. So one order goes a long way. In our case, we had a group of around 10 volunteers working on the project so it was cost effective, but doesn't work for an individual who just wants to try it out. Alternative cheaper fabric dyes for staining plastics are discussed by Karakolis et al but I haven't tested them

https://pubs.acs.org/doi/10.1021/acs.estlett.9b00241

The main reason why I posted on the forum was to point out that cheap LED flashlights are suitable as a light source for fluorescence and work for chlorophyll excitation where no dye is required. The focusing arrangement approximates more to critical rather than Koehler illumination, to maximize the intensity, so it may not appeal to those seeking professional looking photomicrographs, but as an ID tool for picking out chloroplasts it works fine. More powerful light sources are required to evenly illuminate fields of view of several centimetres diameter, but if they comprise multiple LEDs they cannot be optically coupled to smaller fields of view efficiently.

The original demo using the royal blue Nightsea excitation source depended on intrinsic fluorophores added as colorants during the manufacturing process of the plastic.

https://www.emsdiasum.com/microscopy/te ... fa-30.aspx

so the intensity and optimal excitation wavelength will likely vary from sample to sample.

[MichaelG.]

Yes, getting hold of Nile Red is the hard part. As you note, it costs around $30 to $40 for around 10 to 50 mg minimum order. We use around 20 micro liters of 1 mg/ml stock NR dissolved in methanol or rubbing alcohol stain for 5 ml of sea water. So one order goes a long way. In our case, we had a group of around 10 volunteers working on the project so it was cost effective, but doesn't work for an individual who just wants to try it out. Alternative cheaper fabric dyes for staining plastics are discussed by Karakolis et al but I haven't tested them
< etc. >

.
Thanks for the information ... This might re-kindle my interest

MichaelG.

[crb5]

Our paper describing the polarization and fluorescence modifications is now on-line: https://pubs.acs.org/doi/10.1021/acs.jchemed.0c00518

Supporting information is free to download (section III has more details of the optics). Message me if you want a copy of the main text

[Hobbyst46]

Our paper describing the polarization and fluorescence modifications is now on-line: https://pubs.acs.org/doi/10.1021/acs.jchemed.0c00518

Supporting information is free to download (section III has more details of the optics). Message me if you want a copy of the main text

Thanks for the article and for the link here ! downloaded for reading.
Indeed, simple fluorescence microscopy (not epi-fluorescence, which depends on expensive accessories) has been briefly discussed on the forum.
For example, this technique might facilitate playing with live diatoms on a stereo or low-mag biological microscope. Micro-aquarium, flow system etc.
Thanks for mentioning the safety aspects. I would still take care to avoid spurious reflections of the beam - ESPECIALLY if the light source is a laser of any sort.

[Greg Howald]

I'm certainly no expert at this so I'll just ask a couple of dumb questions.

1. Why not mount a uv flashlight in the vertical position on the nose of the stereo scope so the light is directed downward and more light will be reflected into the objective?

2. Reflective? What happens if you place a small mirror under the specimen? Will that help or hinder?

That's all I got. The system they are trying to sell looks nice but it is beyond my price range.

Thanks for listening. Greg

[Hobbyst46]

I'm certainly no expert at this so I'll just ask a couple of dumb questions.

1. Why not mount a uv flashlight in the vertical position on the nose of the stereo scope so the light is directed downward and more light will be reflected into the objective?

2. Reflective? What happens if you place a small mirror under the specimen? Will that help or hinder?

To question 1: In general, to observe fluorescence, the excitation light (the UV in your question) must be efficiently separated from the fluorescence, otherwise the latter - which is immensely weaker than the former - will be masked. This is generally achieved in fluorescence (all kinds) by having them perpendicular to each other. Reflected light must be also taken into consideration. In your suggested configuration the UV is reflected back to the image-forming optical parts, so in the image, fluorescence will be masked. This is true regardless of the wavelength of the flashlight beam - UV, green, blue or whatever. A more specific point is that reflected UV into the eyes is very dangerous - so, I would vote against it - but the physics of fluorescence is the dominant challenge here.

To Question 2: And, if I understand correctly your question 2, adding to the configuration in question 1 a mirror under the specimen will only make it worse... enhancing the reflected excitation beam without enhancing the fluorescence...

[Scarodactyl]

This is generally achieved in fluorescence (all kinds) by having them perpendicular to each other.

I think most fluorescence work is with coaxial illumination, and separation is done by filtering out the excitation wavelengths.

[Hobbyst46]

This is generally achieved in fluorescence (all kinds) by having them perpendicular to each other.

I think most fluorescence work is with coaxial illumination, and separation is done by filtering out the excitation wavelengths.

Yes, I only mentioned the general principle. In more detail, it goes like this:
The initial excitation is at a right angle to the optical axis of the microscope; depending on the type of light source, it can pass through a filter. It is then directed downwards by means of the 45 degrees beam-splitter (which further purifies the beam from non-excitation). And hits the specimen. The light emitted and reflected from the specimen goes upwards through a filter, which removes the excitation, and the fluorescence goes upwards towards the sensor, since it passes through the beam splitter.
The reflected residual excitation from the specimen is directed sideways, back to the light source, by means of the beam splitter. So the beam splitter acts to purify the excitation, purify the fluorescence, and separates the excitation from emission. It comes out that there is a short section of the optical train where excitation and fluorescence are coaxial; but this section does not include the final image.
I should have stressed that neither the excitation filter nor the emission filters do a totally complete job of removal of the respective unwanted wavelengths; hence the importance of the beam splitter.

[viktor j nilsson]

Charles Krebs has gotten good results with a Nichia Convoy S2+ flashlight aimed at the slide, and a 420nm emission filter.

http://www.photomacrography.net/forum/v ... hp?t=33123

Only likely to work with long work distance objectives, though.

[Hobbyst46]

Charles Krebs has gotten good results with a Nichia Convoy S2+ flashlight aimed at the slide, and a 420nm emission filter.

http://www.photomacrography.net/forum/v ... hp?t=33123

Only likely to work with long work distance objectives, though.

Yes; that is sideways illumination though, not coaxial. And he took the necessary precautions.
When the spectral difference between the excitation and fluorescence is large, for example - chlorophylls, the separation is easier.

[viktor j nilsson]

I mentioned that as a response to Greg's question:

1. Why not mount a uv flashlight in the vertical position on the nose of the stereo scope so the light is directed downward and more light will be reflected into the objective?

I may have misunderstood, but I thought Greg was thinking about mounting a UV flashlight on the outside of the microscope pointing downward toward the sample at an angle (=sideways illumination).

I agree with all you have said about why coaxial epi-illumination with dichroic beam splitters is superior. I just wanted to point out that it's possible to get good results with sideways illumination.

[crb5]

Just some comments on recent posts about side illumination for fluorescence excitation. Thanks Viktor
for the link to a previous impressive article by Charles Krebs on another forum using a similar arrangement to that I am using,

I use an external blue LED mounted about 30 to 45 degrees to the objective of an upright microscope with low power objectives (4x to 10x). A larger angle is required for short working distance objectives in order to illuminate the sample, but it is just about manageable with a 40x 0.65NA objective. The circular spot from the LED at the image plane becomes more elliptical as the angle increases and the intensity (photons/unit area) is reduced, so the smallest angle possible works best. Mounting the LED on the nose of the objective of a stereo scope should work, provided it is at a sufficient angle (say 10 to 20 degrees) to illuminate the sample without casting a shadow from the nose and the directly reflected beam from the slide/Petri dish does not enter the objectives. It is important to reduce scattering from the sample and associated surfaces into the microscope objective as this increases the background light. Mounting the slide/Petri dish on a spacer (e.g. 9.5 cm Talenti gelato jar lid https://www.pinterest.com/thaslon/repur ... enti-jars/ with a hole drill in it) helps reduce the scatter from the stereo microscope diffuser plate beneath. On a compound scope, it is useful to lower the condenser lens, so that any reflection is shifted away from the optical axis.

Purpose-built fluorescence microscopes generally use a dichroic mirror in the body of the microscope to reflect the excitation light through the objective lens (i.e. epi illumination). This is an efficient way of illuminating the sample and allows ready switching between objective lenses. In addition, an excitation and emission filter are used in the filter cube to block the unwanted wavelengths. The dichroic mirror helps in this discrimination but is not the main source of blocking - it may transmit 5 to 10 % of the excitation light, whereas the emission filter should transmit <0.01%. While epi fluorescence is by far the most common arrangement in research-grade microscopes, it does not produce the best signal-to-background ratio owing to stray light within the filter cube. External sources are often used for single fluorophore detection microscopy https://pubmed.ncbi.nlm.nih.gov/11106962/.

Finally, a word on using a laser pen as an excitation source. With due care, I consider this operation less dangerous than attending a presentation by an inconsiderate speaker using the same pen and who waves the beam in the direction of the audience. One problem is that some laser pens exceed the stated 5 mW limit and without a power meter, it may be difficult to know this. However, when used in conjunction with a microscope there are 3 aspects that reduce the intensity emerging through the eyepiece to safe levels. (1) an emission filter should be present in the body of the microscope (or in front of the objective lenses of a stereo scope) which should reduce the intensity to <0.1%. (2) Any directly reflected light from the slide surface will be of the order of 5%. Also, if the laser pen is positioned at an angle to miss the objective barrel, the directly reflected beam will also miss the objective when the slide is horizontal on the stage. Scattered light from surfaces, although may dominate the fluorescence signal, is of much lower intensity than the directly reflected beam which retains its laser characteristics. It would NOT be a good idea to use a mirror beneath the specimen to increase the effective excitation intensity, as this has the potential to direct the beam into the objective lens. (3) A 50 mm focal length lens can be placed in front of the laser pen, which will cause the beam to converge to a diffraction-limited spot and then diverge to a diameter greater than the eye pupil, so reducing the fraction of light that could enter the eye of an observer some distance away. Also, such a divergent beam would not be brought into a sharp spot on the retina. Such a divergent beam allows the area of the spot to be adjusted to match the field of view of the specimen. These 3 factors should be first checked by pointing the laser pen at a wall and seeing the effect of introducing the emission filter (take care it may reflect most of the laser light), slide or focusing lens in the beam path.

I understand anyone who considers using a laser pen is not worth the risk for a hobby. I have the same reaction to my rock climbing friends who assure me it is safe because they are attached by ropes. However, I do consider it useful for students who go on the use high power lasers in their research, to learn the characteristics of laser light using a laser pen.