Using a laser to illuminate.

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
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DonSchaeffer
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Using a laser to illuminate.

#1 Post by DonSchaeffer » Mon Mar 22, 2021 11:24 am

The other day I tried using a ruby laser as side illumination--even in combination with white light. The results were puzzling--with the laser you don't see the subject at all but you see a strange glowing pattern that looks like it forms a wide circle (you only see a small section of arc). Can anyone explain that? Is it useful?

Placozoa
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Re: Using a laser to illuminate.

#2 Post by Placozoa » Mon Mar 22, 2021 1:41 pm

I will try to answer but I cant vouch for the accuracy of this.

I think it has to do with quantum mechanics. Light is absorbed or emitted by electrons jumping up or down one or more shell. The shells are determined by harmonics of the electronss wavelength (QM says "stuff" is both a wave and a particle) in 3 dinensions around whatever atom or molecule it is orbiting. Since the shells are fixed distances from each other, it is always one photon of a fixed wavelength and energy (a quanta) that is emitted in this fashion. In a laser, most of the electrons are excited, then as a photon passes it stimilates that electron to drop as well. The end result is whats called a collimated beam. The beam is in phase and polarised and heading the same direction.

This can be bad news if it gets in your eye, as it may burn the retina and destroy it, permanently, in whatever spot it hits. If it hits something that needs a photon of shorter wavelength to work, say a solar panel, it simply will not interact with it or else it will heat the atom itself. For this reason, shining a red laser on a solar panel will generate no power despite laser light being way brighter than sunlight. I think thats what Einstien got his nobel prize for was discovering that. For this reason too, a red laser will only interact with very specific samples, a random sample is unlikely to glow its natural color doe to absorbing a photon and reemitting another one.

It will likely just get reflected from the surface and bounce across the room, or maybe heat the sample. Lasers are coherent so they move as a team, although some bits bounce off of different stuff (a laser beam is not infinitely thin in real life), and may find themselves bouncing off the inside of the tube or in your eye. These will be simple reflections, so they will just look like glare, which is more or less what you described. Red is also a really long wavelength so the resolution seen by it will be terrible, its not a good color for illumination in any case.

Hopefully others can add more to this answer, its just a quick answer, and I do not have first hand experience to back it up. Its too dangerous, imho.

Ps. I also left out a lot, most on purpose. My QM is not solid enough to teach it so people could understand it, not by a long shot.

DonSchaeffer
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Re: Using a laser to illuminate.

#3 Post by DonSchaeffer » Mon Mar 22, 2021 2:22 pm

wow. Thanks a lot. It gives a sense that my experience is as it should be.

Hobbyst46
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Re: Using a laser to illuminate.

#4 Post by Hobbyst46 » Mon Mar 22, 2021 2:23 pm

Can be described in simple terms.
First, the chief advantage of lase light is that it is monochromatic. If your laser is actually a diode laser, it will be less monochromatic, but still a very narrow bandwidth.
For microscopy, the chief advantage of the monochromaticity is for fluorescence. If it excites fluorescence. A ruby laser does not excite much VISIBLE fluorescence since its wavelength is 694nm, at the end of the visible spectrum.
So, the red light that you shine on a specimen will behave just as ordinary lamp, except that the beam is very narrow. Maybe as narrow as your specimen. yet, this light is reflected, scattered, absorbed, just like white light, qualitatively. Quantitatively, if the specimen reflects or scatters more in the blue and green parts of the spectrum than in the red part, your sideways illumination with the laser will not be effective.
I fully agree with the safety concern in the previous response. Stray laser reflections reach unplanned targets. A concentrated laser beam that emerges from a lens can be very energetic (although very narrow). For the conventional hobby microscopy, a laser has no significant advantage IMHO. Had you used ONLY the laser and not white light, all the images you would get - if they form - would be red, whatever the color of the specimen... Research microscopes fitted with powerful lasers are equipped with halogen/LED illuminators as well for visual inspection of the samples.

DonSchaeffer
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Re: Using a laser to illuminate.

#5 Post by DonSchaeffer » Mon Mar 22, 2021 6:44 pm

In my naivete I was influenced by an ad for an advanced microscope system that apparently uses laser light to get dramatically better resolution. That's why I tried it.

Placozoa
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Re: Using a laser to illuminate.

#6 Post by Placozoa » Mon Mar 22, 2021 7:20 pm

Again, double check this, I am not 100% on it.

Flourescence is caused by short wavelengths, like ultra violet. Standard black lights put out ultra violet that is just off the visible spectrum, These things can excite lots of things to give off visible light since each photon DOES have enough energy to do it. Thats why your clothes glow at a dance club but you you dont go blind.

Bulbs that put out light that is more ultra violet, that is light that is further into the ultra violet spectrum, can be had relatively cheaply. These lights will be damaging the dna in live samples. This will cause ciliates to conjugate a lot to fix their dna, this will also kill many types of bacteria outright, and the danger to your eyes looking through the eyepieces is also substantial.

Brightness is the amplitude of the waves, but the wavelength of the wave is what determines the photons energy. Shorter waves have more energy, and get enough energy and it starts exciting thise electrons so much that they jump right off of whatever molecule they are on. Once this happens its no longer a molecule, just a couple of atoms drifting around. In other words chemical damage has occurred.

Another thing about the brightness of UV is that you cant see it, since your eyes dont detect it. You really have no idea how bright it really is, worse, your eyes wont know either. This means your pupils wont constrict to protect your eyes making it that much more dangerous.

Research labs use UV and lasers routinely, I am not completely sure how they do it safely, maybe they dope the sample with uv while NOT looking through the eyepieces then look after, maybe they use cameras as detectors instead of eyeballs, maybe they use a lens that blocks UV but lets visible light through. I really dont know, but I feel that a hobbyist like me should use caution, since my safety is my own responsibility and I can follow (or not follow) whatever restrictions I want.

Again, I purposely said a few things that are half truths to keep it simple. I would like to add that just how short of a wavelength you can go safely, just how far into the ultraviolet spectrum, I do not know exactly and its likely a fuzzy yet fine line.

Placozoa
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Re: Using a laser to illuminate.

#7 Post by Placozoa » Mon Mar 22, 2021 7:54 pm

Hobbyst46 wrote:
Mon Mar 22, 2021 2:23 pm

For microscopy, the chief advantage of the monochromaticity is for fluorescence. If it excites fluorescence. A ruby laser does not excite much VISIBLE fluorescence since its wavelength is 694nm, at the end of the visible spectrum.
This makes a lot of sense to me. If you stain a live sample the stains end up in specific places, and if the stain can fluoresce at some wavelength, then hitting it with a laser of an appropriate wavelength would make it really glow, and you could probably detect stuff way below what you could actually resolve.

Hobbyst46
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Re: Using a laser to illuminate.

#8 Post by Hobbyst46 » Mon Mar 22, 2021 8:06 pm

Placozoa wrote:
Mon Mar 22, 2021 7:54 pm
Hobbyst46 wrote:
Mon Mar 22, 2021 2:23 pm

For microscopy, the chief advantage of the monochromaticity is for fluorescence. If it excites fluorescence. A ruby laser does not excite much VISIBLE fluorescence since its wavelength is 694nm, at the end of the visible spectrum.
This makes a lot of sense to me. If you stain a live sample the stains end up in specific places, and if the stain can fluoresce at some wavelength, then hitting it with a laser of an appropriate wavelength would make it really glow, and you could probably detect stuff way below what you could actually resolve.
The point is, that the wavelength of the fluorescence is ALWAYS higher than that of the exciting laser/lamp, usually by several nm at least. A 694nm light will excite fluorescence of 700+ nm. That is, outside the range of human visibility - no matter how strong the laser light is. A green laser will excite many molecules, for example will cause red fluorescence from a leaf. But it is neither straightforward nor safe setup on the microscope at home.

Placozoa
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Re: Using a laser to illuminate.

#9 Post by Placozoa » Mon Mar 22, 2021 8:22 pm

I agree completely, and simple quantum mechanics makes it clear why. When most people say "quantum mechanics" the next few lines should be viewed with skepticism. A lot of skepticism. Hopefully I didnt say anything too ridiculous, and if I did I genuinely want to know. :)

QM maies clear a few things with triple crossed polars and also DIC. Microscopy is actually one of the few places QM actually has any noticable effects, another reason I find it interesting.

jmp
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Re: Using a laser to illuminate.

#10 Post by jmp » Mon Mar 22, 2021 8:35 pm

In my naivete I was influenced by an ad for an advanced microscope system that apparently uses laser light to get dramatically better resolution. That's why I tried it.
Laser light is indeed used in microscopy to dramatically increase resolution by techniques such as confocal laser microscopy, super-resolution, etc. But those are applications of fluorescence microscopy that use specialized hardware, software and techniques to 'overcome' the resolution limit of conventional light microscopes (kind of a loop hole, the optical resolution limit is still the same, but the way in which photons are collected and processed allows a much finer spatial localization of them). For example, confocal microscopy uses a laser and a rotating disk with a pin hole to excite a fluorescent dye in a small point of the sample, while measuring the resulting light emission from that sole point excited in the sample. By repeating this process over the whole sample (i.e. scanning the sample) a whole series of images are collected and processed by software to build the final image. This is something that you can not achieve by simply shining laser light to a sample on a conventional microscope.

As others I also second the warning regarding lasers. Even if they do not emit UV light they can still be harmful for your eyes. Just take the appropriate precautions if you want to keep on experimenting with lasers. Perhaps use a digital camera instead of looking with your eyes through the microscope. Worst case you'll damage the camera sensor, not your eyes.

The ibiology microscopy course is a great resource to learn about microscopy. The lectures cover different relevant topics including super-resolution, confocal, and other techniques with good examples and a good introduction to the relevant theory. Its available on youtube and worth checking out.

Placozoa
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Re: Using a laser to illuminate.

#11 Post by Placozoa » Mon Mar 22, 2021 8:49 pm

jmp wrote:
Mon Mar 22, 2021 8:35 pm
In my naivete I was influenced by an ad for an advanced microscope system that apparently uses laser light to get dramatically better resolution. That's why I tried it.
As others I also second the warning regarding lasers. Even if they do not emit UV light they can still be harmful for your eyes. Just take the appropriate precautions if you want to keep on experimenting with lasers. Perhaps use a digital camera instead of looking with your eyes through the microscope. Worst case you'll damage the camera sensor, not your eyes.
This response is pure gold, this is why I come here, to learn stuff. :)

I would like to add that lasers can also damage the optics of the microscope. I am fairly sure I have seen pictures of objective lenses with laser holes burned in them (damaged, not burnt through), and, after my eyes, that would be my next worry. I am not sure how strong or what wavelength of a laser that requires, but I would rather hear about it than see it.

jmp
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Re: Using a laser to illuminate.

#12 Post by jmp » Mon Mar 22, 2021 9:01 pm

I am fairly sure I have seen pictures of objective lenses with laser holes burned in them
One can only hope that whoever was messing with those lenses and a laser knew what he was doing! I'd imagine that you'd need some serious laser to do so, though apparently an 8W laser would be able to let you engrave glass. Still 8W is way more than the few mW that you'd typically get from consumer grade stuff (like a laser pointer). But as usual, safety first.

LouiseScot
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Re: Using a laser to illuminate.

#13 Post by LouiseScot » Tue Mar 23, 2021 7:01 pm

I used to do laser scanning confocal microscopy. As mentioned, it uses expensive, specialised hardware. Absolutely no viewing via laser light! It's all done by computer and camera. It's used to show different coloured fluorescent antibody staining. I think there are some newer even higher resolution techniques around now but I'm a bit out of touch. An actual true ruby laser in itself is a dangerous piece of kit which shouldn't be operated without safety glasses and precautions. I suspect, though, this wasn't a true ruby laser? Mind you, even laser diode pens can be dangerous - they can actually be higher power than the nominal 1 mW they are sold as, so never assume they are safe.

Louise
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crb5
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Re: Using a laser to illuminate.

#14 Post by crb5 » Tue Mar 23, 2021 10:00 pm

by DonSchaeffer The other day I tried using a ruby laser as side illumination
Ruby lasers tend to be high powered pulse lasers, not the kind of toy to play with. Or do you mean a ruby-colored laser pointer which is relatively safe with a power < 5 mW. I would not expect a red laser pointer to be a very suitable illumination source as the light is coherent (photons are in phase) which means reflections tend to be in or out of phase and the image is obscured by bright and dark interference fringes. As has been pointed out, any fluorescence will have a longer wavelength and appear in the invisible infra red region. Blue laser pointers are suitable for fluorescence illumination and I have used them with a $200 microscope to image fluorescence from chlorophyll, nile red-stained microplastics and DAPI-stained cell nuclei. The set up and safety issues are discussed in https://drive.google.com/drive/u/0/fold ... t33-dGnXrQ

Hobbyst46
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Re: Using a laser to illuminate.

#15 Post by Hobbyst46 » Tue Mar 23, 2021 10:22 pm

crb5 wrote:
Tue Mar 23, 2021 10:00 pm
by DonSchaeffer The other day I tried using a ruby laser as side illumination
Ruby lasers tend to be high powered pulse lasers, not the kind of toy to play with. Or do you mean a ruby-colored laser pointer which is relatively safe with a power < 5 mW. I would not expect a red laser pointer to be a very suitable illumination source as the light is coherent (photons are in phase) which means reflections tend to be in or out of phase and the image is obscured by bright and dark interference fringes. As has been pointed out, any fluorescence will have a longer wavelength and appear in the invisible infra red region. Blue laser pointers are suitable for fluorescence illumination and I have used them with a $200 microscope to image fluorescence from chlorophyll, nile red-stained microplastics and DAPI-stained cell nuclei. The set up and safety issues are discussed in https://drive.google.com/drive/u/0/fold ... t33-dGnXrQ
Thanks for the link ! the idea of a simple, inexpensive DIY fluorescence microscope, in particular a stereo microscope, has been discussed on the MH forum at least once in recent years.

microb
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Re: Using a laser to illuminate.

#16 Post by microb » Wed Mar 24, 2021 5:29 pm

DonSchaeffer wrote:
Mon Mar 22, 2021 6:44 pm
In my naivete I was influenced by an ad for an advanced microscope system that apparently uses laser light to get dramatically better resolution. That's why I tried it.
Are you trying to do light sheet?

https://www.youtube.com/watch?v=Y7VFAoUo8Fs

DonSchaeffer
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Re: Using a laser to illuminate.

#17 Post by DonSchaeffer » Wed Mar 24, 2021 7:11 pm

I didn't have any particular goal in mind. I just wanted to see what would happen.

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