Fresh water diatoms

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c-krebs
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Fresh water diatoms

#1 Post by c-krebs » Mon Jun 01, 2020 7:14 am

The first two diatoms were found in an old sample jar I had left outside for a few months. The last one is from a recent marsh sample.

40/0.95 Olympus S Plan Apo. 2.5X NFK photoeyepiece. Canon EOS R. DIC.
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100/1.40 Olympus S Plan Apo. 2.5X NFK photoeyepiece. Canon EOS R. DIC.
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40/0.95 Olympus S Plan Apo. 2.5X NFK photoeyepiece. Canon EOS R. DIC.
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100/1.40 Olympus S Plan Apo. 2.5X NFK photoeyepiece. Canon EOS R. DIC.
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MicroBob
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Re: Fresh water diatoms

#2 Post by MicroBob » Mon Jun 01, 2020 9:10 am

Hi Charles,
Beautiful images!
Your images show nicely the glassy structure of the frustules in combination with the live contents.

Bob

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Wes
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Re: Fresh water diatoms

#3 Post by Wes » Mon Jun 01, 2020 9:40 am

Incredible images! I especially like the second one showing these rather fine details. Thanks for sharing.
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75RR
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Re: Fresh water diatoms

#4 Post by 75RR » Mon Jun 01, 2020 10:18 am

Great images + some very fine detail.

Like the black border as well
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apochronaut
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Re: Fresh water diatoms

#5 Post by apochronaut » Mon Jun 01, 2020 12:02 pm

great images. When highly polished images show up on this forum, it is always a question of how much the final presented image relates to the viewed image , at the point the image was captured. Most microscopists spend 99.9% of their microscope time viewing through the microscope and perhaps the rest capturing and or processing images, if that. A lot of questions on this forum and I also see on the more dedicated photo...forum are about how to present better photos, the answer to at least some of which is in the post processing. I know even my simple pictures can be improved a lot with some fiddling. Cleaning the sensor once in a while for instance. Would there be any chance of showing us one or a few of the original image captures of these, so we can get an idea of how much the p.p. has affected the final outcome?

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Re: Fresh water diatoms

#6 Post by Hobbyst46 » Mon Jun 01, 2020 1:04 pm

I also like "as found" documentation photos. The fourth photo is a beautiful, yet looks very natural of diatoms in the "wild".
Also, the first three (magnificent!) images show clear plain view of the diatoms, without other organisms/debris/remnants that might obscure the diatom by overlap.
I find overlapping particles most annoying. They often happen in overcrowded strew slides, and even in properly occupied slides, since diatoms tend to clump together.
Last edited by Hobbyst46 on Mon Jun 01, 2020 4:17 pm, edited 1 time in total.

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75RR
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Re: Fresh water diatoms

#7 Post by 75RR » Mon Jun 01, 2020 1:14 pm

apochronaut wrote:
Mon Jun 01, 2020 12:02 pm
great images. When highly polished images show up on this forum, it is always a question of how much the final presented image relates to the viewed image , at the point the image was captured.
I do not think most microscopists over worry about this ... but to answer your question I believe you will find that there is more microscopy skill involved than postprocessing.
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Re: Fresh water diatoms

#8 Post by Charles » Mon Jun 01, 2020 2:15 pm

Beautiful Charles! I agree with 75RR. There is a lot of skill getting the shots. The processing just shows the specimen better.

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Re: Fresh water diatoms

#9 Post by apochronaut » Mon Jun 01, 2020 3:41 pm

I hadn't figured that out after 55 years experience with microscopes. Thanks for the insight but the question was actually asked of the poster.

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Re: Fresh water diatoms

#10 Post by Sabatini » Mon Jun 01, 2020 4:32 pm

Wonderful details, you can enjoy in all its immense splendor. so beautiful interior universe.

Thank you

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Re: Fresh water diatoms

#11 Post by c-krebs » Mon Jun 01, 2020 9:18 pm

apochronaut wrote:
Mon Jun 01, 2020 12:02 pm
great images. When highly polished images show up on this forum, it is always a question of how much the final presented image relates to the viewed image , at the point the image was captured. Most microscopists spend 99.9% of their microscope time viewing through the microscope and perhaps the rest capturing and or processing images, if that. A lot of questions on this forum and I also see on the more dedicated photo...forum are about how to present better photos, the answer to at least some of which is in the post processing. I know even my simple pictures can be improved a lot with some fiddling. Cleaning the sensor once in a while for instance. Would there be any chance of showing us one or a few of the original image captures of these, so we can get an idea of how much the p.p. has affected the final outcome?
Good questions. As a kid, my first microscope "project" was making a collection of pollen slides which I photographed through the eyepiece with a clunky Polaroid B+W camera. (I hate to think about it, but that was about 60 years ago!). Put the microscope away after a few years, but stuck with photography. I got "hooked" again when digital photography became viable... in large part because of the ability to tackle the nemesis of crazy shallow DOF by using image stacking. The images here, and frankly the vast majority of images I post are focus stacked. While I am very much aware of the pitfalls of stacking, I feel that the final images (can) present a far more satisfying view of the subject than a single frame that has perhaps 1/100th the DOF. (And if there is any question about detail representation I still retain all the "raw" camera files that can be scrutinized if need be). When you examine a subject visually under the microscope, you are usually constantly tweaking the fine focus to put the section you are looking at in best focus. So there is much less of a sense inadequate DOF as when you look at a single image still photograph. Looking at a high magnification photograph (single frame, not stacked), especially one made with a high NA objective can be frustrating for a viewer since they don't have the ability to turn the fine focus and see detail in sections where they would like to.

Focus stacking is major "post processing". The resulting image will usually require some digital cleanup. Sometimes a great deal of it. For example, in the third image above the diatom was moving (rotationally and laterally). The stacking software (Zerene in this case) did an admirable job of aligning the layers. But that means that every speck of dust on the sensor, every little bit of debris on the slide get repeated in the background in a "track" mimicking the diatoms motion. (For the first three images above, I was very meticulous in selecting the individual diatoms and placing them in a very clean wet mount with distilled water, so I avoided the normal "debris". They are seldom this clean). Below I have put some of the original "source" files. In the first triptych you can see one from the beginning of the stack, one from the middle and one from near the end. To me, none of these individual frames is completely satisfying if I want to see the entire subject photographed. The stack is (Image #2 above). I have also included single frames from the other images originally posted. Again (to me at least) not as interesting or effective as the stacks.

But this is not just an argument for stacking. With digital we have the ability to present images without the air bubbles, coverslip or slide defects, fibers and unrelated debris that made it into the mount. I guess a purist might say that's all part of the reality. But if it is foreign to the subject and adds no useful context (it rarely does), I don't like it ;-) .

And there is overall correct color balance, proper tonality. (I find it fascinating to look through some of the old 'microscope photography" texts to see all the techniques film photographers needed to use to accomplish what can be done now with some basic post-processing. The "pre-processing" in those days was often quite daunting!) Slide preparation is rarely perfect. The images that any camera spits out (film or digital) are not perfect renditions of the scene before it. If you have an interest in presenting microscope images to people, you are, IMO, doing yourself and your viewers a disservice if you do not have at least some understanding and capability of basic post-processing.

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Re: Fresh water diatoms

#12 Post by viktor j nilsson » Mon Jun 01, 2020 10:52 pm

I found it fascinating and inspiring to see those raw photos, Charles. I mean, they don't actually look all that different from my own sad attempts at photomicrography! :oops: It suddenly felt a bit more attainable to achieve something like your finished result one day. You definitely didn't NEED to show the raw files, but it was very educational.

This discussion also made me think of your flash-illuminated photos of bdelloid rotifers etc. These must nearly always be single shots, right? Somehow I never get the feeling that I want to "fine-focus" up and down from those. I guess the key is to focus on the exact right spots to keep the eyes from wandering to the parts that are unsharp.

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Re: Fresh water diatoms

#13 Post by c-krebs » Mon Jun 01, 2020 11:53 pm

viktor j nilsson wrote:
Mon Jun 01, 2020 10:52 pm
This discussion also made me think of your flash-illuminated photos of bdelloid rotifers etc. These must nearly always be single shots, right? Somehow I never get the feeling that I want to "fine-focus" up and down from those. I guess the key is to focus on the exact right spots to keep the eyes from wandering to the parts that are unsharp.
Everything does not need to be stacked front to back... nor is it possible in many cases. The rotifer with flash shots you mention are a good example. In many of those the subject is in constant motion and changing its position and shape rapidly. There is no chance to acquire a stack, so you just take lots of pictures varying focus slightly on the portion of interest, and then pick the best result. Occasionally you will come across a rotifer or sessile protozoan where the main body is relatively stationary but the cilia and head are constantly moving. Sometimes it is possible to take a few shots to get a good "body" outline and then concentrate on taking many cilia shots. With a program like Zerene Stacker (good editing capabilities) you can sometimes then make a combination of the desired images. Not really a complete "stack" but if done carefully can look really good.

Then there is "optical sectioning" where you can use the very shallow DOF to look into a transparent subject without the surface registering (at least not very obviously) in the view or image. In this case, if conducive for acquiring a stack I will often do that. Later I will determine whether a single image will work best, or perhaps just a few images stacked is best. I recently shot a testate amoeba this way and got two interesting images from the overall stack. One emphasized the outer morphology nicely, and the other utilized the "optical sectioning" to see inside the shell by using only a select portion of the full stack. I'll track that down and post it in a separate topic. I'll stop back and reference it here when I do. It is a good example of what we're talking about.

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Re: Fresh water diatoms

#14 Post by krame » Tue Jun 02, 2020 1:40 am

Thanks for taking the time to explain the answer, I am the newest of the new to microscopy (my first microscope arrived today, but didn't get a chance to unbox) and I had wondered the same thing.

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Re: Fresh water diatoms

#15 Post by viktor j nilsson » Wed Jun 03, 2020 9:52 am

c-krebs wrote:
Mon Jun 01, 2020 11:53 pm
Everything does not need to be stacked front to back... nor is it possible in many cases. The rotifer with flash shots you mention are a good example. In many of those the subject is in constant motion and changing its position and shape rapidly. There is no chance to acquire a stack, so you just take lots of pictures varying focus slightly on the portion of interest, and then pick the best result. Occasionally you will come across a rotifer or sessile protozoan where the main body is relatively stationary but the cilia and head are constantly moving. Sometimes it is possible to take a few shots to get a good "body" outline and then concentrate on taking many cilia shots. With a program like Zerene Stacker (good editing capabilities) you can sometimes then make a combination of the desired images. Not really a complete "stack" but if done carefully can look really good.

Then there is "optical sectioning" where you can use the very shallow DOF to look into a transparent subject without the surface registering (at least not very obviously) in the view or image. In this case, if conducive for acquiring a stack I will often do that. Later I will determine whether a single image will work best, or perhaps just a few images stacked is best. I recently shot a testate amoeba this way and got two interesting images from the overall stack. One emphasized the outer morphology nicely, and the other utilized the "optical sectioning" to see inside the shell by using only a select portion of the full stack. I'll track that down and post it in a separate topic. I'll stop back and reference it here when I do. It is a good example of what we're talking about.
Many thanks for taking the time to explain. It's really valuable to hear about your approach, which I see as both science and art, as you capture the different layers with scientific accuracy, and paint with them as an artist. That testate amoeba is a wonderful example of that!

It makes a lot of sense to me that combining a sharp body outline with details of sharp cilia produces an image that we as humans find relaxing and undistracting to view.

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Re: Fresh water diatoms

#16 Post by RobBerdan » Tue Jun 09, 2020 2:01 am

Very nice crisp DIC images Charles showing lots of internal detail.
Cheers
RB

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Re: Fresh water diatoms

#17 Post by wporter » Tue Jun 09, 2020 2:30 am

Thanks to Apo for the question, and Charles for the excellent detailed reply, and the very illustrative before-and-after images; an excellent presentation.

A much less honest person might have said, "That's the raw shot, of course. Doesn't your DIC look like that?" Lol. But seriously, this is the kind of comparison that beginners need to see: that a lot of work is involved and beautiful images don't just spring fully-formed out of the camera with the first try.

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Re: Fresh water diatoms

#18 Post by IvaniaAreas » Tue Jun 09, 2020 5:07 am

Absolutely stunning photos, thank you for sharing.

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Re: Fresh water diatoms

#19 Post by c-krebs » Tue Jun 09, 2020 7:52 am

In an answer above I referenced a testate amoeba shot I intended to post, and that I would reference here as a continuation of the above discussion. Hopefully if you were interested you found it... but in case not this was the post:

viewtopic.php?f=6&t=9541

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Re: Fresh water diatoms

#20 Post by rmcnelly » Tue Jun 16, 2020 11:22 am

Thank you very much for explaining your focus stack method.

Your photos are stunning!
—Rick

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