Smooth, non-shiny mounting surface for small stuff?

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Chris S.
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Smooth, non-shiny mounting surface for small stuff?

Post by Chris S. »

I'm guessing that microscopists have answered this question long ago; if so, perhaps someone can tell me what the answer is.

I want to photograph mushroom spores with what microscopists would call "epi-illumination"--that is, not light passed through the spores, but light bouncing off the spores. A microscope slide works poorly for this, as the glass creates reflections of whatever is upon it. (I haven't actually tried this with spores, but have done so with other small things, and very much dislike these reflections--so would rather have a better approach before I start.) I could reduce reflection with cross-polarization, but this would greatly constrain my ability to diffuse the light source, so I would rather start with a non-reflective surface. Ideally, the surface would not be clear or black, but some neutral shade of gray. It should be very smooth, so that surface features on the mounting substrate do not distract from the spore. For example, ordinary paper, at the necessary magnifications, is not at all flat or featureless, as the fibers become very large.

I've thought of painting polished metal with flat paint, or perhaps painting microscope slides. Not sure that either approach would work--for example, brush marks might show. I suspect that dipping a slide in some substance to coat it might work, but have no idea what substance to try.

Advice?

(At the moment, am looking to photograph ascii from various members of the morchellaceae. Or in other words, take pictures of the the small sacks that contain the spores of the various morel mushrooms. Much disagreement exists regarding the taxonomy of this group of fungi--though great enthusiasm does exist regarding the culinary characteristics of some of its members. And of course in mycology, naked-eye observations of fruiting bodies are often insufficient to distinguish species. I suspect that DNA analysis will eventually shed light on this, but I don't do DNA analysis. So I'd like to build a photographic catalogue of morel asci, ascospores and ascocarp phenotypes, and see what, if anything, can be learned.)

So if anyone can suggest an opaque, smooth, non-shiny surface on which to place the ascospores, I'd appreciate it. Thanks!

--Chris
Last edited by Chris S. on Tue May 03, 2011 12:39 am, edited 1 time in total.

Pau
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Post by Pau »

Chris, not a real answer to your question, only a general impression:
I think your problem is difficult to solve because any non specular reflective material I did observe under high magnification has surface irregularities that scatter the light. The more featureless and smoth is a surface it becomes more reflective.
Material science microscopes usually don't use brightfield for epi illumination, but cross polarization, DIC or darkfield.
Pau

Chris S.
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Post by Chris S. »

Thanks, Pau.

As an example of what I'm thinking, carpet is not shiny because of its many surface irregularities. Similarly, sandpaper is not shiny because of its surface irregularities. But the surface irregularities in these two materials are differ in size by orders of magnitude. An ideal substrate for my current project would likely require surface irregularities, but is there a material whose surface irregularies are an order of magnitude or two smaller than the spores I'm wishing to photograph? The spores in question are on the order of 10-20 microns.

Your response was great--this kind of thing helps jar the mind into new thoughts. A thought that occurs to me on reading your kind reponse is that some forms of carbon soot deposit particles much smaller than these spores, and can form a surface that is highly non-reflective. If there is not a better protocol already established, maybe I can try coating a microscope slide in the soot from a flame source, to obtain non-reflective coating with very small particle size. If it worked, it would not be gray, but deep black--but maybe it would work?

Thanks again for your very useful thoughts.

Best,

--Chris

elf
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Post by elf »

I agree with Pau, the smoother and flatter you get the surface, the more reflective it becomes.

You could try ground glass and use different grits of abrasive to get the amount of flatness relative to reflectivity required. Control the color by reflecting some light off colored panels. Snoots, grids, and barn doors can also be used to place the light precisely where you need it.

I think I'd actually go the opposite way and use a highly polished first surface mirror.

Another option is to use a glass table and light the subject from below or the opposite and shoot the subject through the glass.

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Post by ChrisLilley »

Chris S. wrote:An ideal substrate for my current project would likely require surface irregularities, but is there a material whose surface irregularies are an order of magnitude or two smaller than the spores I'm wishing to photograph? The spores in question are on the order of 10-20 microns.
10-20 microns is 10,000 to 20,000 nanometres. One order of magnitude below that is 1,000 to 2,000 nanometres. The wavelengths of visible light are 400 to 700 nanometres. Surface irregularities much smaller than a quarter of a wavelength, say, will be be seen as 'smooth'. So there is not much wiggle room between 'smooth enough to not show texture' and 'effectively a flat specular mirror, as far as that range of wavelengths is concerned'.

Another way to avoid surface texture being seen is to take advantage of the extremely shallow DOF and arrange some way that the spores are not directly on the surface but lifted off it a little (a few microns).

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Post by Chris S. »

elf wrote:I agree with Pau, the smoother and flatter you get the surface, the more reflective it becomes.
But is this inexorable? The wavelength of visible light is measured in nanometers, while these subjects are measure in microns. Could there not be some substance whose surface irregularities stand between these two regions, so that under appropriate magnification, reflection will be minimal, but the surface will appear smooth?
elf wrote:You could try ground glass and use different grits of abrasive to get the amount of flatness relative to reflectivity required.
Interesting approach! But I must confess I'd prefer dipping the slide in something that would adhere in particles with irregularity in the proper range--if possible, might be much easier.
elf wrote: Control the color by reflecting some light off colored panels.
But this would also color the spores, and the color of fungi spores is diagnostic.
elf wrote: Snoots, grids, and barn doors can also be used to place the light precisely where you need it.
I routinely do this--but with a 10-20 micron subject, this gets difficult. If I wasn't clear in my first post, my question is not about photographing the mushrooms--that is very easy--but in photographing their spores. Many photographs of such spores exist, but most are silhouettes. I'm looking to do nice, front-lit, spore portraits.
elf wrote:I think I'd actually go the opposite way and use a highly polished first surface mirror.
Ed, I don't follow you here--sorry for being thick. Do you mean to suggest using the mirror as a surface on which to support the spores? If so, in other experiments with small structures, I have not found reflection to be a good thing--it tends to be artistic, but not illustrative. Here, I'm not trying to creat art, but to record useful observational data.
elf wrote:Another option is to use a glass table and light the subject from below or the opposite and shoot the subject through the glass.
Ed, here, again I don't follow you--sorry for misunderstanding. I could easily see this approach for photographing mushrooms, but spores? Here we're talking about something almost dead flat, and very small. so I don't get how this approach would work--sorry again for being thick!

Thanks for your insights, Ed!

--Chris

elf
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Post by elf »

I look at it as a problem of how to control the light on the subject rather than a problem of getting a flat non-reflective surface. It doesn't matter how reflective the surface is if there is no light reflecting from it or the reflected light isn't hitting the lens.

Here's an example of shooting through a light table: http://www.pixiq.com/article/how-to-sho ... ment-39752

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Post by Pau »

Chris, thanks for your comments
After re-reading the topic I realized that I probabily misunderstood your post. Are you illuminating the sample trough the objective (this is what microscopists usually call epi-illumination) or from outside the objective like usually we do in macro?
In the first case reflection may be unavoidable without DIC or X-pol, But if now I understand correctly you really want to avoid the specular image of the spores itselves on the background, isn't it?
A possible aproach may be to play with the angles between the slide, the light surce and the optical axis (I will think more in this topis later).
But I think that the simpler approach will be a conventional microscope with darkfield or Reinberg illumination.
Pau

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Post by rjlittlefield »

How about this?

Get a piece of really good anti-reflection coated glass. Deposit the spores on the glass and place whatever you want for background behind the glass, far out of focus.

Then you get close to no reflections and no texture either. Make the background gray, and whatever small reflections the coating allows will get washed out to invisible contrast by the background light.

This approach should also be quick, cheap, and easily reproducible by other people who might like to see their spores the same way they appear in your catalogue.

--Rik

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Post by Charles Krebs »

I would try placing the spores on a coverslip (or maybe better a multicoated clear camera filter), and elevating the coverslip some distance from a background of the Protostar or ScopeStuff flocking material. (Do this by placing it on top of a short cylinder also lined with flocking material... this helps to keep the background really black!. Then light it from above, off to the side a bit, paying careful attention to minimize any direct light reflection back to the camera. You will likely encounter some image reflection from the glass, but with a black background it should not be too hard to re-touch out.

If you are using epi-illumination (light through the lens) then, as Pau mentioned, crossed polarization will likely be needed.

There may exist some exotic background material I am not aware of, but most "matte" surfaces are surprisingly "non-matte" when looked at at high magnification.


Dang.... I need to learn to type faster than Rik!

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Post by NikonUser »

Charles Krebs wrote: Dang.... I need to learn to type faster than Rik!
Or move to the Eastern Time Zone :P
NU.
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No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
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Post by lauriek »

I'd vote for Charlie and Rik's suggestions, I don't tend to shoot things as small as fungal spores but I don't see that scale actually matters here, I shoot down onto the subject which is usually sitting on a glass microscope slide on the microscope stage. The background is several inches below the stage. I shoot the lights in at 30-60 degrees and I don't tend to suffer much in the way of reflections. I think as long as you're perpendicular to the glass slide and you're careful with the angle of the lights you should be okay...

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Post by Chris S. »

Thanks for all the advice so far. It’s much appreciated.

I should clarify that I’m not concerned about the reflections from my lights—those are pretty easy to deal with. What I want to avoid is reflections of the subject itself on the glass slide (or cover slip, if I place them on that, instead). Imagine placing a subject on a mirrored surface and photographing it. You’d get a picture of the subject, and also of its reflection on the mirrored surface below. This is of course sometimes a desirable effect, and purposefully done. But in many cases, such as these spores, I don’t want a subject sitting upon its mirror image. With subjects that can be suspended far from apparent support (such as a bug pinned from behind), this is easy to accomplish. For backlight subjects, ditto. But for a number of subjects so far—these spores being the current example—I’ve wanted to place them directly on a very smooth, non-mirrored surface and retain wide freedom to place lights in configurations that convey the shape, color, texture, and reflectivity of the subject. It’s quite easy to produce images of fungal spores that are, effectively, silhouettes, but I would prefer not to do that.

X-pol lighting is also easy—I do it all the time—but as far as I know, while it can be very effective at reducing reflections of the lights on the mounting substrate, it does not eliminate the reflection of the subject on this substrate. And if it did, x-pol sharply constrains the ability to diffuse or bounce light onto the subject; x-pol is an extremely useful technique, but imparts its own limitations.

Sorry—I apparently miss-used the term “epi-illumination”—not surprising, as I’m a photographer working down into smaller subjects, but by no means a microscopist. I’m talking various forms of frontal lighting such as portrait artists might use—not illumination through the objective. The point I wanted to highlight is that I want to capture light reflected off these spores, rather than transmitted through them.

I think I’ll try Rik’s approach first. I knew that anti-reflective coatings existed, but until Rik suggested “really good anti-reflection coated glass,” I had no idea that they worked well. A bit of follow-up reading suggests that, indeed, they can work very well, and that anti-reflective coatings at not as simple as I’d thought. Edmund Optics and Anchor Optics (and probably others) offer what look like pretty nice anti-reflective glass “windows,” some of which include anti-reflective coating on both sides. I’ll likely order a few inexpensive samples to test. As Rik said, if this approach works, there would be some nice benefits, including the ability to tune the background independently of subject lighting, and reproducibility for other researchers. On the downside, morel mushrooms in Ohio are fruiting now, and the season tends to be short—so by the time I receive these materials, the season may well be over. But dried specimens and spore prints may allow me to transfer spores to the antireflective glass. And I expect this to be an annual project that will take a few years before I can collect enough data to suggest anything.

Ed, thanks for linking to that interesting tutorial—I may use that technique for other things. But for spores, I would prefer to avoid the blown-out background it’s designed to create. And in the example used in the tutorial, the subject lighting struck me as being counter to my beliefs in what good lighting should accomplish, as quite a bit of visual information about the shape and texture of the subject was lost. Could it be done better? Maybe, but with all that light flooding up from beneath the subject, very precise light control on the subject would be very difficult.

By the way, yesterday I quickly created a layer of black carbon on a slide, by holding the slide in an oil lamp. The area of this soot was very black, and under quick inspection seemed to have dramatically reduced reflectivity. Light-colored spores might show up well against this background, though dark spores would not. My tube lens came back from the fabricator last night, so I’ve been spending my time setting that up, rather than following up on this post.

Thanks again, all! Cheers,

--Chris

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Post by rjlittlefield »

Chris,

One other point to note is that obvious reflections of the subject on a glass substrate only occur when the lens is looking obliquely onto the glass.

If the glass is perpendicular to the optical axis, and the lens is telecentric, then any reflections will be directly behind the subject that creates them, so you won't see them.

As previously discussed, at high magnifications DOF is so small that you can treat the lens as telecentric even if it isn't really. (Un-check scale adjustments in your favorite stacking software.) In addition, some objectives are very close to telecentric just because of their design. The Nikon CFI60 10X Plan achromat is one of those. I don't know about your Mitutoyo's.

Considering all this, I'm not sure that the issue you're concerned about would be a problem in reality. Given the tight schedule, the very first thing I would do is to run some tests with an ordinary glass slide and see how things work out that way.

--Rik

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Post by Chris S. »

Thanks, Rik.

One of the things I like to do with the Bratcam is pull away from what I think of as the “tyranny of the flat.” Well-known microscopic approaches already reveal wonderful detail, so long as the specimen is kept perpendicular to the optics. However, with a number of small subjects, I’ve enjoyed “flying” around the subject and viewing it from various angles. It’s like the difference between looking straight down when using Google Earth, versus adding tilt in order to see mountains, valleys, and plains more as a hiker would view topography.

But when doing this with subjects placed on a microscope slide, I’ve repeatedly encountered the problem of having the subject form a mirror image on the slide. Your point is well taken that when one places the glass perpendicular to the optical axis, this problem disappears. But for some subjects, I don’t want to look straight down.

The fungal ascospores of my immediate interest are likely a poor example, as they are pretty flat, and therefore likely tractable to traditional photomicroscopic techniques, as mentioned by Charlie and others. Since this is a self-assignment, I can talk about it. But I have other subjects covered under non-disclosure agreements that might benefit from your suggestion to use a piece of glass with really good anti-reflective coating as a substrate. For those projects, I’ll definitely pursue that approach. Thanks!

BTW, my first experiment tonight in photographing the spores of morels and their allies was disappointing. Of the several species I set out for spore prints (M. semilibera, M. elata (complex), M. esculenta (complex), and Verpa bohemica (actually looked to be about halfway between V. bohemica and V. conica), only the M. semilibera dropped substantial spores. (This is interesting, as I have a conjecture that collectors looking to eat these species tend to pick fruiting bodies before they have a chance to disperse spores, which may be detrimental to the species. I have a study area where nobody else collects, so I have the luxury to let specimens mature in situ; as a first pass, I collected only a few specimens, and chose each based on apparent signs of maturity.)

So I imaged only the ascospores of M. semilibera, with a 100x Mitutoyo, with x-pol. I was disappointed in the image, and sent it to a friend who is a highly competent mycologist. Will await his comments, and act on them if possible, before posting anything.

I intend to go out into the field again tomorrow, and if I find decent specimens, will place sections on plain microscope slides, on coverslips, and on black-carbon-coated slides. Then will take pictures and see what develops. (On the downside, Ohio has had a few days of cold temperatures, and the morel mushrooms seem to fruit better after warm nights.)

Thanks again, and cheers,

--Chris

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