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Looking for help with specimen contrast / lighting
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JohnyM



Joined: 24 Dec 2013
Posts: 380

PostPosted: Thu Apr 26, 2018 10:18 am    Post subject: Looking for help with specimen contrast / lighting Reply with quote

Hello.
I've run into troubles when running stacks with ULWD 20/0.4


Im quite happy with specimen contrast when it's flat against the lens:


But it drastically fall when angled.
Bottom


Maybe besides getting a better lens ( old finite olympus pushed down without compensating eyepiece ) lighting could be somehow improved to bring that contrast?

Setup im using is 4 jansjo's (flashes available if that would help somehow) against plastic cup on petri dish placed on white paper:

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rjlittlefield
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PostPosted: Thu Apr 26, 2018 11:04 am    Post subject: Reply with quote

Have you tried with a darker background? One common problem is that glare from surface reflections become stronger as the surface tilts.

Another possible problem is one similar to what is described at http://www.photomacrography.net/forum/viewtopic.php?p=135042#135042 . Shown there is the case of a flat background being degraded by foreground that is seen by part of the objective's aperture. But a similar problem can occur with subjects that are very tilted, so that one side of the aperture gets a clear view but the other side is blocked by the tilted subject. An NA 0.4 objective has an entrance cone that extends about 23 degrees on either side of center (=arcsin(0.4)), so that's the critical angle where this effect starts to happen.

--Rik
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JohnyM



Joined: 24 Dec 2013
Posts: 380

PostPosted: Thu Apr 26, 2018 11:44 am    Post subject: Reply with quote

Thank you for this insight. I'll try to shoot against darker background (cant remove it from gray'ish agarose as it would eventually kill it).

Subject is almost perfect cylinder so that indeed might be the case. So only solution would be to shoot the stack with lower NA objective when imaging plane get to steep angle?
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rjlittlefield
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PostPosted: Thu Apr 26, 2018 11:55 am    Post subject: Reply with quote

JohnyM wrote:
So only solution would be to shoot the stack with lower NA objective when imaging plane get to steep angle?

You could try to fix it in post-processing, with a curves adjustment applied locally with masking. Other people have had success with that method.

A third "solution" is to just figure out what's going on and then decide to live with it. To be honest, the image that you have looks good to me as it is.

By the way, what is this thing, and why is it growing in agarose? Looking toward future specimens, is it possible to darken the agarose?

--Rik
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JohnyM



Joined: 24 Dec 2013
Posts: 380

PostPosted: Sat Apr 28, 2018 7:09 am    Post subject: Reply with quote

Thanks for the hint. I've had no success with processing so far.

I know that i need proper lens for the job, this Olympus lens does cover A7RII frame, but IQ is acceptable only withing center of the frame.
I've bought Nikon CFI PlanApo VC 20x/0,75 which seems to cover A7RII sensor with excellent image quality across the frame, with 135mm lens, but have only 2mm working distance.
Now im looking toward Nikon CF ELWD 20/0.4 which have 13mm working distance, and CF ELWD 50x/0,55 with 8mm WD for close-ups. But im not sure how much it can be pushed down for full frame.
I know that Mitutoyos are considered an "ultimate" solution, but it's very difficult to buy it officially, and it's too expensive for me to purchase it privately (i could buy Nikon CF set of 10x, ELWD 20x and ELWD 50x for single Mitutoyo lens). So it's risking my own money on things that might or might not work.

Subject is Cyathea delgadi InVitro eksplantat. Those little green "blobs" are new embryos. It is not possible to alter agarose color as we're using same culture media for years of study, just slightly altering some ingridiends for study purpose. I could remove it from medium and place on black card, but that would remove one of the biggest advantage of optical method.

Until now, we were using environmental SEM with some degrees of success, but optical method allows to capture same subject in longer time period, without killing it. And 20x pushed down provides good field of view and just adequate resolution (at least in the center of the frame). Nikon BE Plan 10x/0,25 images proved to be just below acceptance limit (althrough images looked ok'ish, we've asked multiple researchers to count and measure cells on multiple images and results were too different).
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rjlittlefield
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PostPosted: Sat Apr 28, 2018 9:02 am    Post subject: Reply with quote

Thanks for the further info.

Quote:
Subject is Cyathea delgadi InVitro eksplantat. Those little green "blobs" are new embryos.

So, this thing, or something very much like it? https://en.wikipedia.org/wiki/Cyathea_delgadii

What does "eksplantat" mean?

--Rik
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Cactusdave



Joined: 09 Jun 2009
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Location: Bromley, Kent, UK

PostPosted: Sat Apr 28, 2018 9:32 am    Post subject: Reply with quote

Quote:
What does "eksplantat" mean?.


I'm guessing Johny M means explant, in other words in vitro cultivation of a part of the plant in an agarose medium.

There seems to be a surprising amount of noise in the images as well as some overexposure and lack of contrast that could be improved in post processing by use of curves.

Looking at your setup I am a bit concerned about tube length. You don't seem to have anything like the total 160mm extension needed for the kind of objectives you are using.

Quote:
old finite olympus pushed down without compensating eyepiece



You can't push magnification up or down by altering tube length without introducing problems, as you are dragging the lens away from what it is designed to do. Especially important at higher magnification.

I think diffusing your lights would also help.
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JohnyM



Joined: 24 Dec 2013
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PostPosted: Sat Apr 28, 2018 10:17 am    Post subject: Reply with quote

rjlittlefield wrote:
So, this thing, or something very much like it? https://en.wikipedia.org/wiki/Cyathea_delgadii

What does "eksplantat" mean?

--Rik


Yes, that's exactly what you've linked.
Sorry about "eksplantat" i've switched to my native language here, but the word is very similar. Cactusdave explanation is accurate.
We basically cut parts of the plant, then cultivate it into "mini bonsai" version of it, but altering medium and checking results.


@Cactusdave
Quote:
There seems to be a surprising amount of noise in the images as well as some overexposure and lack of contrast that could be improved in post processing by use of curves.

Whole stack of ~250 frames was shot at ISO100 from RAW and sharpened to 50 and radius 1,5 in camera raw. Stack was created by pyramidal method. Im sure you know what that does to image noise.

Anyway, those images are not supposed to be pretty. They are exposed and sharpened so important parts are clearly distinguishable for statistical analysis. In fact, we're often using skeleton filter, im sure you wouldnt like the looks of it after that.

Im just not gonna spend hours in photoshop to make it look nice, when 99% of those images are gonna be forever stored and probably never looked upon again, and one gonna be printed in paper with ~3" size.
For pretty image it's less hassle to just put it into SEM after we're done with it.

Quote:
You can't push magnification up or down by altering tube length without introducing problems, as you are dragging the lens away from what it is designed to do. Especially important at higher magnification.

I am well aware of that, in fact that's why i've mentioned need for infinite lens to do the job. Right now im using the tools available.

Quote:
I think diffusing your lights would also help.

You got any suggestions? Adding ping-pong ball seems to do nothing besides eating more light in this case. Before Rik explanation of halo effect, i've been experimenting with more directonal light on subject perifery, but it brought no improvement at all.
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Cactusdave



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PostPosted: Sun Apr 29, 2018 6:12 am    Post subject: Reply with quote

For diffusion I suggest you try a suitable sized cylinder of paper. Tracing paper is a good place to start, but it is worth experimenting with alternatives. If it eats too much light the answer should be more light. Diffusion is pretty much always important with incident lighting. It looks like you are using some kind of plastic cylinder for diffusion in your setup photo, with some kind of ridged surface structure. This may not be optimal for your application. Time spent experimenting with diffusion is nearly always worthwhile.

Probably you have already thought about it, but I wonder if some kind of tilting stage, even a simple home made one, would help with 'seeing round' the edges of your cylinder shaped specimen, if that is important to your evaluation.
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Lou Jost



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PostPosted: Sun Apr 29, 2018 6:39 am    Post subject: Reply with quote

Another suggestion is to apply the strong sharpening AFTER stacking. I don't know if this will help, but for images that are noisy to begin with, sharpening individual frames makes the noise more prominent and that gets exaggerated in stacking.

(I have this plant or a related tree fern growing wild next to my house.)
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mawyatt



Joined: 22 Aug 2013
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Location: Clearwater

PostPosted: Sun Apr 29, 2018 7:11 am    Post subject: Reply with quote

Cactusdave wrote:
For diffusion I suggest you try a suitable sized cylinder of paper. Tracing paper is a good place to start, but it is worth experimenting with alternatives. If it eats too much light the answer should be more light. Diffusion is pretty much always important with incident lighting. It looks like you are using some kind of plastic cylinder for diffusion in your setup photo, with some kind of ridged surface structure. This may not be optimal for your application. Time spent experimenting with diffusion is nearly always worthwhile.

Probably you have already thought about it, but I wonder if some kind of tilting stage, even a simple home made one, would help with 'seeing round' the edges of your cylinder shaped specimen, if that is important to your evaluation.


Dave,

I've found the standard white styrofoam drink cups work well as diffusers for flash/strobes, but eat a lot of light (which isn't a problem with strobes). They don't seem to introduce much of a color shift either, but a white balance is usually a good idea with any diffusion or light source changes. Also made some larger "cones" for diffusers with cheap stiff white paper stock from the $ Store. This worked OK but did have a large color shift, certainly requiring a WB correction.

Best,
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JohnyM



Joined: 24 Dec 2013
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PostPosted: Sun Apr 29, 2018 8:20 am    Post subject: Reply with quote

Lou Jost wrote:
Another suggestion is to apply the strong sharpening AFTER stacking. I don't know if this will help, but for images that are noisy to begin with, sharpening individual frames makes the noise more prominent and that gets exaggerated in stacking.

(I have this plant or a related tree fern growing wild next to my house.)

Thanks you, this is very valuable insight. I was tinking that it's best to do as much in RAW as possible, but your suggestion is very reasonable and im gonna try it with next subject.

Quote:
Probably you have already thought about it, but I wonder if some kind of tilting stage, even a simple home made one, would help with 'seeing round' the edges of your cylinder shaped specimen, if that is important to your evaluation.

I dont want to do 250 shots for every angle. In fact, if you notice, specimen is already mounted on ballhead.

Quote:

For diffusion I suggest you try a suitable sized cylinder of paper.... ...Time spent experimenting with diffusion is nearly always worthwhile.

Thanks for suggestions, but it's not what im interested in.
I've tried rolled paper, ping-pongs, plastic cups and Lee diffusion sheets. Pictured plastic cup is least hassle to set-up. All of those materials worked equally well for me, and combining them did nothing for PROBLEM mentioned in first post. In fact, more diffusion is reducing contrast.
I'll repeat myslef: im absolutely fine with some blown highlights in the image, as i mentioned it's NOT an art. Most of my coworkers doesnt know what's histogram, and say "im not counting cells in this imgage, i hate the composition and it have too much noise for my taste".

Quote:
If it eats too much light the answer should be more light

I do have strobes, but i would like to avoid using them - too much hassle.
Adding more LEDs is dangerous for specimen preservation. Again, im not looking for pretty images, just need contrast / resolution on steep specimen borders. Which i undestand is very hard/ might be impossible to achive.
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manu de hanoi



Joined: 11 May 2018
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PostPosted: Fri May 11, 2018 6:54 am    Post subject: Reply with quote

-are the angled parts blurry just in the final stack or are they blurry too in individual layers ?
-DSLR being closer to the objective as pictured should help you getting good depth of field at the expense of magnification/resolution.
-250 pics for a stack sound like a lot, sometimes less is more (but that depends on you answer to my 1st question)
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ChrisR
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PostPosted: Fri May 11, 2018 5:05 pm    Post subject: Reply with quote

Quote:
-DSLR being closer to the objective as pictured should help you getting good depth of field at the expense of magnification/resolution.
I don't think so? That's what a reduced aperture would do.

JM, things you've probably already considered:
  • "Gobos" (small pieces of black paper, adjusted by eye to try to put a shadow where you need it, to show relief,
  • flocking inside the microscope head. I had an Oly which was very shiny. Looking by eye, no problem, but a camera saw flare.
  • Crossed polars - may do nothing,
  • Coloured lights - like Hoffman modulation, but incident,
  • A tiny hole in the white sleeve - highlights can show curves at cell boundaries,
  • UV illumination - Jacek's mosses show cell boundaries well (Chlorophyll > red)

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rjlittlefield
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PostPosted: Fri May 11, 2018 6:08 pm    Post subject: Reply with quote

ChrisR wrote:
Quote:
-DSLR being closer to the objective as pictured should help you getting good depth of field at the expense of magnification/resolution.
I don't think so? That's what a reduced aperture would do.

There is a subtlety here. The aperture will be reduced somewhat (smaller effective NA) because the focused subject will move away from the objective as the DSLR is moved closer to it.

This is the same reason that many old books counseled that to get more DOF at any particular f-number, you should move away from the subject and compensate for the loss of magnification by enlarging more.

Now much the effect will matter is difficult to predict because it is different for different objectives.

--Rik
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