Photographing through water (cherry fruit fly larvae)

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Medwar
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Post by Medwar »

My samples are not prepared samles, that's the problem. Often I cannot polish the minerals, there is a need to preserve the natural faces. Usually they are natural crystals with natural faces and with inclusions inside.
Sometimes the minerals can be polished, but usually just to make an even surface, not cutting too much - not to destroy valuable specimen.

So the inclusion can be as deep inside the crystal, as it formed in nature.
Usually I shoot those that are from 1mm to 10mm deep.
If the magnification is lower than 4x, sometimes up to 3 cm deep.
For minerals super-long working distance objectives are the best.

RI of the most common minerals:
quartz 1,544
fluorite 1,434
calcite 1,650 and 1,486 (double refractive)
topaz 1,620

I illuminate with ikea janslo lamps, diffused by a deep polyvinylchloride soup plate cut around the object. Usual reflected light.

Dont know, maybe it's even physycally impossible to shoot with 20x in my conditions?
But I want first get good results wth 10x at least.

Ichthyophthirius
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Post by Ichthyophthirius »

Hi,

Keeping the minerals in the aggregates makes the microscopy very difficult. You simply won't be able to image both with high NA AND deep into the crystals.

First you have to match the immersion RI als closely as possible to the mineral (glycerol, immersion oil, methyleneiodide etc. https://books.google.co.uk/books?id=b-7 ... &q&f=false ) and cover it with a cover glass. This creates a thick mount and most objectives are very limited in the thickness they can compensate for. If you think of the optically dense immersion medium in glass-equivalents, the best you get is around 2.3 mm for oil immersions (Leitz Ks 22/0.65 achromat) and 2 - 2.5 mm for dry objectives with correction collar (Nikon 20/0.40 0-2 mm; Nikon ELWD Plan 40/0.55 0-2.5mm). I don't know of any other objectives with longer working distances that can also compensate for spherical aberration except for multi k$ physiological objectives.

If the mount is thicker than that, you will have to lower the NA of your objective. There is a very useful paper on this (Spinell, Loveland (1955) Optics of the image space in microscopy. http://onlinelibrary.wiley.com/doi/10.1 ... 4454.x/pdf ) which shows formulars and tables with cover glass tolerance depending on NA.

Tests were made using 3 different criteria (Star test; least perceptible degradation in photograph; and maximum acceptable degradation in photograph).

For NA = 0.3, Loveland considers the maximum acceptable degradation at a thickness of 1 mm. Using his formula,

T(?l)/8 = 1/NA^4

- a 10 mm medium thickness is tolerated by a lens of NA 0.16
- a 20 mm medium thickness is tolerated by a lens of NA 0.14

This is in the area that you could get with a 5x or stopped-down 10x objective; or an expensive stereo microscope; or maybe with a reversed prime lens (Rik will know). The last two solutions have the advantage of several cm of working distance! There are also photomicrographic objectives that project directly onto the sensor (no tube lens, no compensating eyepieces) with long working distances and an integrated iris for stopping down. Just an example, the Zeiss Luminar 63 mm with and NA of 0.11 and working distances around 30-50 mm page 69 http://www.science-info.net/docs/zeiss/ ... ystems.pdf

Medwar
Posts: 72
Joined: Mon Apr 16, 2012 1:46 am

Post by Medwar »

Ichthyophthirius, thank you for the valuable info, formula and links.

It is much more clear now.
It seems that immersion objectives are not for my case, too low WD, only specially polished to minimal thickness samples.
I've read that correction collar usually corrects 0.3 to 1.5 mm coverglass, and tend to have low WDs. So I think objectives with collar are not a variant too.

But I am interested in Mitutoyo G Plan APO 20x NA 0.28
They say it is corrected for use with 3.5mm glass between front lens and the object and also may be used in "life science applications" when viewing through water or physiological liquds.

May it be suitable to shoot inclusions that are 1-3 mm under the flat polished crystal surface?
I think the objective is corrected EXACTLY for 3.5mm if glass,
but if the mineal will be shot from above wneh submerged in water (or other liquid) then maybe with manually changing the water level it can be possible to reach almost perfect correction?
Tolerated thickness in case with 0.28 is 1.3 mm - large enough to hit it with manual water level adjustment.
What do you think of that? Is that possible?
The objective may be used at 10-15x, if the NA will appear too low for nominal 20x.

There is also another one, Mitutoyo 50x G plan APO NA 0.5, but for NA 0.5 tolerated thickness is 0.2 mm - don't think the above approach can be usable here.

Ichthyophthirius
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Post by Ichthyophthirius »

Medwar wrote:But I am interested in Mitutoyo G Plan APO 20x NA 0.28
They say it is corrected for use with 3.5mm glass between front lens and the object and also may be used in "life science applications" when viewing through water or physiological liquds.

May it be suitable to shoot inclusions that are 1-3 mm under the flat polished crystal surface?
I think the objective is corrected EXACTLY for 3.5mm if glass,
but if the mineal will be shot from above wneh submerged in water (or other liquid) then maybe with manually changing the water level it can be possible to reach almost perfect correction?
Tolerated thickness in case with 0.28 is 1.3 mm - large enough to hit it with manual water level adjustment.
Hi,

I saw that objective but at $4000 new I didn't see it as an option :) But the method you described (adjusting the liquid levels to find a good compromise) should work.

However, the NA of 0.28 might still be too high to go several mm into a crystal. I'm wondering of the exit pupil of this objective is high enough to install an iris to stop it down. That would also work with your Mitutoyo 10x NA 0.28 so ou might not have to buy a new objective.

Regards, Ichty

Medwar
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Joined: Mon Apr 16, 2012 1:46 am

Post by Medwar »

Mitutoyous 10x and 20x certainly can be stopped down and I have an iris that does it.
But stopping will result in loss of resolution, so the resolution may become equal to that of 4x objective. Why use the 10x if there will be no additional detail at 10x?

Before trying to find "G plan" I will of course experiment more with my current 10x 0.28, but my past experiment with stopping down to shoot inclusions gave not good result. Though I never tried shooting through water.

Ichthyophthirius
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Post by Ichthyophthirius »

The Nikon 4x is a Plan and the Mitutoyo 10x is a Plan Apo? That should make a difference.

However, if you haven't done RI matching so far, that will make more of a difference than any change in optics. Don't limit yourself to water and try higher RI liquids as well.

If you have a look at these images http://www.sciencealert.com/gallery-gem ... erfections they were made just with a Wild photomacroscope, so if the photographer had a Apozoom objective for his Wild, the NA would have been 0.116 or less.

rjlittlefield
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Post by rjlittlefield »

Ichthyophthirius wrote:- a 10 mm medium thickness is tolerated by a lens of NA 0.16
- a 20 mm medium thickness is tolerated by a lens of NA 0.14

... or maybe with a reversed prime lens (Rik will know).
For a prime reversed in front of an infinity-focused rear lens, the relationships are NA = 1/(2*f_number) and f_number = 1/(2*NA).

So, NA 0.14 is f/3.6, NA 0.16 is f/3.1. It takes a high quality lens to work well at such wide apertures, and at best the result would only be similar to a Mitutoyo M Plan Apo 5X NA 0.14 .

--Rik

Charles Krebs
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Post by Charles Krebs »

Another possibility that might deserve consideration is the Olympus LUCPLFLN 20/0.45. It has a working distance of 6.6-7.8mm, and has a correction collar for 0-2mm (glass).

http://www.olympus-lifescience.com/en/o ... lfln%2F20x

These newer Olympus plan fluorites are very good. The 0.45 NA is (as Ichthy has brought up) probably too large to work as you intend, but it might be possible to add a rear stop or rear iris (don't know).

The price is much more reasonable than the Mitutoyo G, although the working distance is far less. The 0-2mm correction collar makes me think it might be a lot more versatile (if you can handle the NA for your primary purpose).

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Medwar
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Joined: Mon Apr 16, 2012 1:46 am

Post by Medwar »

Ichthyophthirius wrote: If you have a look at these images http://www.sciencealert.com/gallery-gem ... erfections they were made just with a Wild photomacroscope,
I know Danny's photos well. They are great, but these are made at lower magnifications, up to 4x. These mags are accessible for me also.

Thank you all for your advice. I will return later when I manage to make some more experiments with my current optics.

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