Photographing through water (cherry fruit fly larvae)

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rjlittlefield
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Photographing through water (cherry fruit fly larvae)

Post by rjlittlefield »

At http://www.photomacrography.net/forum/v ... hp?t=27702, I posted some pictures of cherry fruit fly larvae mouth hooks. As noted there:
The high mag images are shot with Mitutoyo 10X on Raynox 150 tube lens, Canon T1i camera, illumination with diffused Jansjo LED lamps, mostly directed at a piece of white card under the specimen, with a small black card held in place as a partial darkfield stop. Vertical setup, shot through a thin layer of water with the specimen stuck into a drop of sticky water-soluble transparent "personal lubricant".
Here are photographs of the setup:

Image

Image

It worked out that at ISO 200, this provided an exposure time of 1/6 second. That could be an issue with mechanical first shutter curtain, especially with the "water window". But with electronic first shutter, no problem at all.

Focusing was done with the Olympus CHT block that I documented HERE and HERE. Focus stepped at 5 microns using settling time of 5 seconds, 0.3 seconds shutter pulse, 1.5 seconds after pulse, for a total of 6.8 seconds per frame. For this stack, I shot RAW+JPEG Fine, with images immediately uploaded to the laptop I was using to control the StackShot.

The bellows is a new arrangement that I currently have in prototype. It started life as an ordinary Olympus bellows. Then I cut out the middle section of the bellows rail, and mounted the two end pieces through spacers to a couple of Arca-Swiss clamps. For this application, the main advantage is that the spacers give me full freedom to rotate any of my cameras into any position I want. But I'm pretty sure that this arrangement will be easily modifiable to also do automated stepping of the rear mount by coupling that to a StackShot rail. That would have been handy a couple of times in the past.

A particular challenge when photographing in liquid is "mounting" the specimen so that it stays in the desired position to be photographed. Because of the thin water layer, tilting the stage is not an option. And because the specimen is both soft and wet, gluing or pinning it to any sort of adjustable mount didn't seem practical either. I had been told by other people that they had gotten good results with "personal lubricant", which is a mixture of water, glycerin, hydroxyethylcellulose, and some other minor ingredients. Basically it's sticky, transparent, and slowly water soluble. I had never used the trick before, but it worked great. Squeeze out a small drop of sticky stuff, use a toothpick to form it into a flat fairly thin drop, then gently press the specimen into that, position as desired, and finally flood with just enough water to cover it.

The specimen holder is a 3"x3" microscope slide with a thin section of PVC plumbing pipe glued to it.

Diffusers over the Jansjö lamps are finger ends from latex gloves.

I offer thanks to all those many people whose ideas I borrowed for this project. There's basically nothing original here, with the possible exception of the hacked bellows.

--Rik

Edit: fix typo, "spaces" --> "spacers"
Last edited by rjlittlefield on Mon Aug 10, 2015 9:34 pm, edited 1 time in total.

RobertOToole
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Post by RobertOToole »

Thanks for sharing the set up Rik.

I like the bellows modification, it looks like this set up would give you tilt ability for one right?

Thanks for the lube - subject trick!

Robert

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Post by Charles Krebs »

Nice Rik. The images came out great!

This is an excellent example of what I meant in Robert's MM-11 post when I said that it was really nice to have a large, plain, uncluttered base to work off. It really makes "freestyle" lighting much easier to accomplish. So often you need to experiment and tweak the lighting in unusual ways for best effect.

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Post by zzffnn »

What an elegant rig and nice photos!

Rik and others:

Sorry for my newbie questions, please kindly comment:

1) why is a thin layer of water necessary? Is water used as mounting medium to enhance light transmission and evenness?

2) Would images degrade significantly when only personal lubricant is used (how about submerge the subject entirely in lubricant and flatten liquid surface with a smaller ring)?

Thank you!
Selling my Canon FD 200mm F/2.8 lens

rjlittlefield
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Post by rjlittlefield »

Thanks for your interest and kind words!
RobertOToole wrote:I like the bellows modification, it looks like this set up would give you tilt ability for one right?
I guess it could be used that way, but it's not designed to be and I don't intend to. Each bellows standard is fastened to its corresponding Arca clamp with a 1/4-20 screw that goes through the clamp, through the spacers, and into a new hole that I drilled and tapped into the sections of old bellows rail. Getting everything aligned nice and square is fiddly, so I want to do that just once, then lock everything down nice and tight so I don't have to worry about it again.
Charles Krebs wrote:This is an excellent example of what I meant in Robert's MM-11 post when I said that it was really nice to have a large, plain, uncluttered base to work off. It really makes "freestyle" lighting much easier to accomplish. So often you need to experiment and tweak the lighting in unusual ways for best effect.
Thanks, Charlie. Indeed, I had to do a lot of playing with the illumination.

It's not evident in the images here, but that clear box I'm using to allow backlighting has three steel screws through one side, here positioned on the bottom. That makes it simple to fasten with magnets to steel positioning plates, like the example shown HERE with a different orientation and a different container. For shooting the fruit fly larvae, I did without the magnets, but if I were going to do it again, I think I'd lay down a steel plate just to make it simpler to move things around without accidentally bumping them out of position.
zzffnn wrote:1) why is a thin layer of water necessary? Is water used as mounting medium to enhance light transmission and evenness?
Whenever you're shooting something in dense medium, it's important that the surface be very smooth. If the surface is non-planar by as little as 1 micron across the entrance cone, it adds aberrations that degrade the image. Think of looking through "privacy glass". When the larva is not covered in water, the overall shape and local irregularities of the cuticle cause problems looking through it. But when the larva is covered with water, the problem pretty much goes away because the interior of the larva has about the same refractive index as water. In addition, the top surface of the water naturally forms a smooth and nearly flat surface, at least near the center of a wide span like I'm using here.

These two shots illustrate the issue. The top one is shot with the top of the larva "dry", meaning it had recently been blotted on a Kleenex tissue. The bottom one is after a spoonful of water had been added to just barely cover the larva. The larva in this case is live and squirming, so these are single shots and the postures aren't quite the same. But I think the difference in how much detail can be seen inside the critter is pretty clear. (Shot with a 2X objective and stopped down using an external iris for more DOF.)

Image
2) Would images degrade significantly when only personal lubricant is used (how about submerge the subject entirely in lubricant and flatten liquid surface with a smaller ring)?
I expect that would work fine, except for the difficulties of getting the specimen fully wetted and cleared of air bubbles. It seemed pretty straightforward to lay down a clean thin layer, but if you work the stuff very much, it quickly accumulates bubbles. You can see a few of them in those last two pictures, where I didn't take time to worry about getting a clean layer.

--Rik

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Post by zzffnn »

rjlittlefield wrote: Whenever you're shooting something in dense medium, it's important that the surface be very smooth. If the surface is non-planar by as little as 1 micron across the entrance cone, it adds aberrations that degrade the image. Think of looking through "privacy glass". When the larva is not covered in water, the overall shape and local irregularities of the cuticle cause problems looking through it. But when the larva is covered with water, the problem pretty much goes away because the interior of the larva has about the same refractive index as water. In addition, the top surface of the water naturally forms a smooth and nearly flat surface, at least near the center of a wide span like I'm using here.
Thank you very much Rik.
I remember seeing similar techniques a few times. For example, when Ted Clarke used dry >=10x objectives to look through/into a drop of water without cover slip, he dropped the water inside a flat metal washer glued to slide to limit aberration.
Selling my Canon FD 200mm F/2.8 lens

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Post by Chris S. »

Rik,

Excellent thread, at multiple levels. =D>

This said, there is a notable aspect of your setup here that strikes me as worthy of detailed discussion of wide interest, and not yet noted:

On your right-most Arca rail assembly, it appears that you've included three Arca-style clamps, where others might have used just one clamp. To my eye, your approach creates two levels of redundancy, greatly reducing the chance that user error would let the camera/lens assembly plummet downward to possible destruction. Specifically, it appears that you've employed two Arca-style clamps (not one) to hold the microscope block, bellows, and camera--so both these clamps, not just one, would need to be loose in order for those components to fall. And in addition, it appears that you've added a third Arca-style clamp below the focus block, to act as a safety stop. This would protect the rig, even if both the other clamps were too loose.

Would you care to elaborate?

Also, on your left-most Arca-style rail, would you recommend adding a third clamp, below the others, as a safety stop?

--Chris

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Post by ChrisR »

It's the single clamp on the wooden block which bothers me rather, from a rigidity point of view.
I expect there's some movement at the camera, if you touch it?

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Post by rjlittlefield »

Chris S., thanks for prompting me to think carefully about the various clamps. When I assembled this vertical setup my attention was focused on other issues, so I have had to go back and reconstruct what I was probably thinking about with the clamps.

You're correct, there are two clamps holding the focus block to the vertical rail. Why two? Mainly because it was simple to do, using clamps that I had on hand, and I figured that I might as well reduce one possible source of compliance or misalignment.

There is a third clamp on the same rail, underneath the whole focus block assembly. That one serves a dual role as safety stop and repositioning stop, making it simpler to lift the whole assembly to give more clearance for changing objectives. That one I do remember thinking about.

There is no second clamp on the rail that holds the bellows, only because I didn't take time to put one on. But there's more to the story than that.

When I moved the camera assembly upright into this vertical configuration to shoot this one series, I had no intention of touching any of the clamps associated with the bellows. Those had been put in appropriate places while the rig was still horizontal, then cinched down tight and just left there.

If I intended to adjust any of that stuff while the rig is vertical, I would have to rethink several aspects. As it sits, there are five clamps -- three Arca-Swiss and two more on the bellows standards -- that could allow equipment to fall if loosened. Offhand I'm not sure how to mitigate all of those, but I am sure that doing just one of them addresses only part of the safety issues.
ChrisR wrote:It's the single clamp on the wooden block which bothers me rather, from a rigidity point of view.
I expect there's some movement at the camera, if you touch it?
There is, but only a small amount of the movement is associated with that clamp.

Instead, "movement when touched" seems to be due to compliance that is distributed across the whole assembly.

At this particular moment I have reorganized my equipment into a horizontal setup that is built on a Newport TD-12 optical breadboard sitting on SA2-FT Sorbothane feet, with the specimen positioning stack fastened to a Newport Model 70 optical rod (1.5 inch diameter) that is bolted to the breadboard. As seen in Live View, touching the camera makes the image move. Touching the specimen positioning controls also makes the image move. Touching the body of the focus block makes the image move. Touching any part of the optical rod makes the image move. For that matter, touching any corner of the breadboard makes the image move, and by "touching" I mean light fingertip pressure like typing a key on a keyboard.

These effects are most obviously seen when the pressure is a shock, because then the image vibrates for a little while.

But there is an easily detectable and stable shift of the image depending on whether a roughly 4" section of M42 tubes that weighs only 106 grams is or is not sitting on one corner of the breadboard.(!) This surely represents a tiny bit of flexibility in the breadboard, magnified by lever arms due to elevated equipment that is mounted at different points on the breadboard, further magnified by lenses and tiny photosites that take 0.4 micron of physical displacement between subject and lens, and turns that into a full pixel at 10X.

ChrisR recently wrote elsewhere on the forum that "Even with something apparently rigid I'm always surprised how much differential movement there is." To that, I can only respond "Me too!"

When I had the main platform built of wood, I used to look at the amount of compliance and think to myself how much more stable it surely would be if everything were metal instead.

Now that it is metal, and I'm still observing qualitatively the same effects, I have given up on the idea of the image not moving when anything is touched.

Instead, it seems quite sufficient that vibration damps quickly and that the equipment always comes to rest in the same position after that happens.

Even that much -- coming to rest in the same position -- cannot be taken for granted. You cannot see them in the setup photo posted here, but on the front of that bellows there are several drops of superglue that guarantee the M42-to-Olympus-bayonet adapter does not shift sideways in the bellows standard. I discovered the need for those when I was trying to measure tiny focus steps and discovered that my observing equipment was reporting small back and forth movements from one shot to the next, even when I had taken pains to make sure that the focus block was in fact not moving. That problem turned out to be that the shock of mirror/shutter action was propagating from the camera to the front bellows standard, where it was causing the M42 adapter to shift much less than a hair sideways in what was then just a spring-loaded bayonet socket. A few drops of gel superglue applied to the junction, and hey presto, the little movements went away and the data became clean.

--Rik

Edit #1: to clarify who wrote what.
Edit #2: to document weight of the 4" section of tubes
Last edited by rjlittlefield on Mon Aug 10, 2015 10:59 pm, edited 2 times in total.

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Post by Charles Krebs »

I am enjoying this little "set-up" side discussion.

First aspect... search out all possible locations of movement, and, within reason, remedy them.

The reason I say "within reason" is because I'm convinced that no matter what you do, a versatile set-up that will allow you to work effectively with a variety of optics, subjects, and lighting arrangements will show some motion when you touch it as Rik has described. This certainly does not imply that you should not work toward the most stable arrangement possible, but sometimes very useful versatility features might require some additional connections and components that could potentially be a source of minute flexure.

The second aspect that comes to mind is that no matter how obsessive you are with the hardware set up, you must use shooting techniques that eliminate those last vestiges of vibration. I really have sympathy for some folks that live in places where they need to deal with constant external environmental causes of vibration... this is tough. But I think the majority of us work in places where we can avoid environmental causes of vibration. It may mean turning off the washer/'dryer, or working at times when other household members are not running around upstairs. I've created equipment arrangements that, in retrospect, looked as if movement might be a nightmarish problem, and also set-ups that were extraordinarily stable. As long as the appropriate shooting techniques were used they were all fine and yielded excellent results.

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Post by Medwar »

Very interesting topic. Rik, thank you for the explanations!

My question is not only about photographing in water, but this topic seems to be closely related to my subject – photography of objects in dense medium and in water as immersion liquid.

I shoot minerals and inclusions inside crystals. When I shoot minerals in air, I make photos that satisfy me up to 20x.
My optics are: Canon MP-E 65mm, Nikon 4x NA 0.13, Mitutoyo 10x NA 0.28 and 20x NA 0.42
Camera: Sony NEX-7.
I have difficulties with shooting inclusions at high magnification. When I shoot inclusions at below 4x, everything goes fine, more or less. But when I go up to 5x and more, the quality degrades. With Mitutoyo 20x it seems that there isn’t any resolution improvement over 10x.

I suppose there may be 3 possible reasons for that.

1.1) The first reason. Look at this picture. Sorry for awful quality.
Image

Marginal rays cone is narrower when shooting in medium.
Does it mean that the effective aperture becomes narrower? If so, then diffraction is the reason for quality degradation.
I tried to shoot with Mitutoyo 20x at 10x (with 100mm tube lens), and still the quality was much worse then expected.

1.2) Before it was the simpliest case. More complex case is shooting at an angle to the crystal surface. Simetimes such shots have to be done because there is a need for another point of view at inclusion, or there is a crack directly in front of it. And in most cases I can’t repolish the crystals at the angle I need, e.g. crystal may belong to a museum. In this case I can’t even suppose, what maths can be applied to count the effect of aberrations depending on magnification, and of angles which are allowed without much degradation.

2) The second reason. The surface of the crystals that I shoot is not always polished, and often can’t be polished. In this case of course it doesn’t have a smooth relief.
Is there a way to count how much curvative or roughness is allowed depending on magnification?

3) The third reason. Most crystals are anisotropic. They have double refraction, that effects in doubling the image in certain crystalographic directions. I use polarizer to remove one of the rays. But the anisotropy may also be somehow concerned with a loss of resolution.
But image quality of inclusions degrades even when I shoot them inside isotropic crystals (fluorite).

Now what I plan to do.
I want to experiment with shooting through water. Now I am making a few small aquariums for the specimens. I plan to try shooting both horizontally through aquarium glass, and vertically through water surface (this variant seems better because of no additional glass in between. But I don’t know whether I will face problems with meniscs and surface tension distortions).

At first glance, shooting in water or in glycerin should remove or lessen the effect of reasons (1.2) and (2). But for reason (1.1) – I don’t have an answer.

I decided to gather all my questions in one place below my post:
1) Am I right in 1.1 that the real aperture will be narrower when the object is inside dense medium (crystal)?
2) Maybe you know other reasons why shooting inclusions at magnification above 5x gives bad image?
3) Does anyone have the maths how to count what magnifications/apertures will be optimal for shooting inclusions, depending on crystal’s refraction index, and immersion liquid refractive index?
4) Does anyone have maths how to count max possible surface unevenness depending on magnification.
5) What happens if I submerge the front lens of Mitutoyo objective in distilled water to shoot through?
First, does anyone have information, is it waterproof or not? :)
Second, will it give any positive effect in compare to shooting through water surface? The objective was designed to work in air, may there be any improvement when it is placed in water?

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Post by Pau »

5) What happens if I submerge the front lens of Mitutoyo objective in distilled water to shoot through?
First, does anyone have information, is it waterproof or not? Smile
Second, will it give any positive effect in compare to shooting through water surface? The objective was designed to work in air, may there be any improvement when it is placed in water?
Don't do it!

The objective may or may not be waterproof (I lean to think not) but i'ts calculated to work in air. For immersion you need a water immersion objective, they are rare but Nikon makes some models for physiology experiments, although the WD could be relatively small.
Pau

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Post by rjlittlefield »

Medwar wrote:1) Am I right in 1.1 that the real aperture will be narrower when the object is inside dense medium (crystal)?
The angle will be narrower, but the diffraction will not be worse. This is because the wavelength shortens in proportion to the narrowing. The narrowing and the shortening cancel, so it is a wash for diffraction. Shooting in dense medium does offer more real DOF, however. See http://www.photomacrography.net/forum/v ... 025#137025 for more about that.
2) Maybe you know other reasons why shooting inclusions at magnification above 5x gives bad image?
In general, the problem is the NA. The wider the entrance cone, the more opportunity there is for roughness and inhomogeneities to introduce wavefront errors.
3) Does anyone have the maths how to count what magnifications/apertures will be optimal for shooting inclusions, depending on crystal’s refraction index, and immersion liquid refractive index?

4) Does anyone have maths how to count max possible surface unevenness depending on magnification.
I do not.
5) What happens if I submerge the front lens of Mitutoyo objective in distilled water to shoot through?
What Pau said!

--Rik

Medwar
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Post by Medwar »

Thank you very much for the answers!

I will follow your wise advice not to submerge the Mitutoyo.
Short WD is not suitable to shoot inclusions, so water immersion objectives will are not a variant for me.

When my small aquariums will be ready, I will try to shoot inclusions through water surface. Maybe it will lessen those wavefront errors at crystal surface.

On the other hand, now even if I shoot inclusions through a polished face, there is still degradation in compare to minerals shot through air.
Need to experiment more.

Rik, in one of the threads you wrote, if I remember correctly, that wavefront errors from coverglass at 10x are still too small, and can be ignored.
But crystal in front of inclusion + water in front of the crystal are often much thicker than the coverglass.
How to calculate wavefront error effect depending on magnification andaperture, to calculate theoretically, through how deep medium you can shoot?

Your information about increased DOF for free surprises!
I already knew that the DOF increases when you shoot inclusions, but I always thoght that I loose sharpness because of more diffraction also.
That should meanthat image formed after refracton though a perfect surface of a dense medium will be as detailed as the image formed through air, but for now I don't see that in practice.

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Post by Ichthyophthirius »

Medwar wrote:But crystal in front of inclusion + water in front of the crystal are often much thicker than the coverglass.
Hi; Can you give us an idea of the thickness of your prepared samples and if the RI of the minerals? What minimum working distance do you absolutely require with a 20x objective (prepared sample)? How do you illuminate your sample?

Regards, Ichty

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