First photos and image editing

Starting out in microscopy? Post images and ask questions relating to the microscope and get answers from our more advanced users on the subject.

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cordyceps
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Joined: Thu Apr 11, 2019 7:00 am

First photos and image editing

Post by cordyceps »

Hello everyone,
I want to ask you, for some microphotography and image editing tips because it is my first week of microphotography and I have never edited photos before. I have some experience in microscopy and I have good equipment for that, but I my knowledge about microphotography and photography in general is very low. I took three photos of some creature: 1st - bight field, 2nd and 3rd I used simple polarisation (polarizing foil + transparent plastic from CD box). I am using Olympus BHT with 100W halogen lamp. I connected Canon EOS 550d with photo bellows to trinocular head, using tape. I want to order some intermediate rings soon, I am aware that tape isn't the best way to do that ;)
I took that three photos with Olympus SPlan Apo 20x, and Olympus photo eyepiece NFK 2,5 LD.
I edited those photos in GIMP 2.10 with few steps:
1. Colour levels
2. Saturation
3. Spot heal
4. Sharpness

1st photo - without editing
Image
1st photo - after editing
Image
2nd photo - without editing
Image
2nd photo - after editing
Image
3rd photo - without editing
Image
3rd photo - after editing
Image

My goals: in microphotography I am looking for visual sensations of the photos, so I am more intrested in the aristis side of microphotography, then documentation of different species.
I know that this and other forums are great source of knowledge, but posts are very random and sometimes chaotic, so it is hard for me to learn directly from different forum posts. Specially if I am a beginner, and don't even know when and how to start. If you have any type of advices, about my set-up, photos, photos editing, books I can read, I will be really grateful.
I hope you are all doing alright,
Karol

Ichthyophthirius
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Re: First photos and image editing

Post by Ichthyophthirius »

Hi Karol,

Welcome to the Forum.

There are two issues that you need to deal with from the start: Spherical aberration and depth of focus.

1) Spherical aberration https://www.olympus-lifescience.com/de/ ... spherical/ prevents you from getting a sharp image of your subjects. It is worst with high NA objectives and thick mounts (= lots of water between the cover glass and the copepod). The SPlanApo 20/0.70 requires perfect mount preparation with a very thin layer of water (the objects need to touch the underside of the cover glass), otherwise your images won't be sharp. It is very difficult to use it with thick objects like this copepod.

For thick objects in water, you're much better off using SPlan(Apo) 4 and 10 objectives which have lower NAs and are more forgiving!

2) When using high NA objectives, the depth of focus is very small, so only small areas of your copepod are actually in focus. To get a better view of the entire copepod, you need to either move into focus stacking or again, use the SPlan(Apo) 4 and 10 objectives which have lower NAs.

You can get some inspiration here; many of the images were made with partially crossed polar filters, stacking and an SPlanApo 10 objective: https://www.flickr.com/photos/mrsansiba ... otostream/

Regards, Ichty

NikonUser
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Re: First photos and image editing

Post by NikonUser »

The Olympus (1990) catalogue does not recommend SPlan objectives for the BHT
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

Scarodactyl
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Re: First photos and image editing

Post by Scarodactyl »

Presumably just because it didn't have a superwode head (by default anyway) so there wouldn't be much point to getting superwide objectives for it. Eyepiece corrections are not different between the splan and dplan objectives, just field coverage.

Alan Wood
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Re: First photos and image editing

Post by Alan Wood »

NikonUser wrote:
Tue Aug 11, 2020 5:40 pm
The Olympus (1990) catalogue does not recommend SPlan objectives for the BHT
The Olympus BHS/BHT brochures only show options for D Achromat and D Plan Achromat objectives with the BHT stand, but that is NOT the same as not recommending S Plan Achromats, or even S Plan Apochromats.

Olympus were happy to sell BHT and BHS stands with any combination of eyepieces, objectives and condensers.

http://www.alanwood.net/olympus/downloads.html

Alan Wood

cordyceps
Posts: 11
Joined: Thu Apr 11, 2019 7:00 am

Re: First photos and image editing

Post by cordyceps »

Thanks everyone for feedback!

NikonUser I am aware of that, but I do agree with Scarodactyl.
Olympus SPlan Apo series has a F.N. up to 26.5 when DPlan Apo series has F.N up to 20 (according to infomations on Alanwood.net webside). When standard Olympus trinocular head BH2-TR30 has maximum F.N 21. So it is a little bit uselss to use superwide objectives when trinocular head will crop the view anyway. More then that, I use a olympus NFK 2.5x photo eypiece which has F.N about 21.6. So I lose all the extra F.N from my objectives in trinocular head or photo eyepiece (If I understand it right). But I think the field number is the only think i lose using these objectives. It would be probably smarter to use D series. Although I have never seen DPlan Apo objectives on eBay or other websites. When I saw any DPlan Apo auction it was DPlan Apo UV. At first i wanted to buy DPlan series, but I got in my opinion very good offer for SPlan Apo objectives and decided to take it.
So summarising do you think by using SPlan Apo objectives i lose some image quality or only F.N.?


Ichthyophthirius thank you for your thoughts! I will use lower power objectives for thick objects. So is it possible to use even higher NA objectives for example 40x on copepods and get a sharp image? I understand that mount preparation has to be perfect, but assuming it is, at what NA does copepods becomes just to thick, even at perfect slide, to get sharp image? Or is just requires stacking and its fine?

I want to start stack my photos soon but I have a question about stacking itself. I understand the idea, you take a lot of photos where each of it has a different sharp part of object, and then focus program choses sharp parts from different pictures and fold them together to get one sharp image. So using micrometer screw we are able to change focus and see different parts of microbe in focus, but some of the microbes are transparent, and changing focus we are able to see "inside them". So in my head we can get a picture when "skin" of the microbe is in focus, and then we can see his internal organs. How do I stack these photos, so I can get sharp image od whole microbe from outside and different sharp "layers" of microbe doesn't blur together. I hope you understand what I am trying to say here.

Ichthyophthirius
Posts: 1117
Joined: Thu Mar 07, 2013 5:24 am

Re: First photos and image editing

Post by Ichthyophthirius »

cordyceps wrote:
Wed Aug 12, 2020 4:18 am
So summarising do you think by using SPlan Apo objectives i lose some image quality or only F.N.?

So is it possible to use even higher NA objectives for example 40x on copepods and get a sharp image? I understand that mount preparation has to be perfect, but assuming it is, at what NA does copepods becomes just to thick, even at perfect slide, to get sharp image? Or is just requires stacking and its fine?
Hi,

I have seen several users saying the SPlan and SPlanApo are corrected better overall, compared with the D-Series. So you loose FN and those super widefield heads are expensive anyway. However, lower NA can sometimes be an advantage as we just discussed! Some of the DPlanApos were also specially designed for fluorescence applications, so best to have all of them :D But in all seriousness, your Olympus objectives are outstanding; their performance won't limit your photography.

Thick mounts get difficult very quickly with higher NA; 20/0.70 is already very demanding. You will get the best images at the very top of the copepod, where the cuticula is pressed against the cover slip. The SPlanApo 20/0.70 performs best with very thin mounts (algae and protists in very thin mounts; and thin sections and histology slides).

Ways around this are objectives with correction collars (some improvement) or oil immersion objectives (big improvement). But for the start I recommend just working with the SPlan 10 for thick mounts.

You can limit the thickness of the mount - without crushing the copepod - by using sticky tape, two further coverslips or a ring of Vasiline as spacers: https://www.cherrybiotech.com/wp-conten ... -vitro.png

Regards, Ichty

cordyceps
Posts: 11
Joined: Thu Apr 11, 2019 7:00 am

Re: First photos and image editing

Post by cordyceps »

Ichthyophthirius wrote:
Wed Aug 12, 2020 5:12 am
But in all seriousness, your Olympus objectives are outstanding; their performance won't limit your photography.
That is what I thought but I am glad to hear that :D
As I said in the first post I simple taped my bellow to the trinocular head to get started, but I really disliked this way and I was thinking how to change the connection. I read that I can connect my camera with intermediate rings or if I want to use a bellow I can use a tripod. I am enjoying using my bellow so I would rather prefer the second option. I borrowed tripod from a friend, but it was really poor quality, camera with a bellow was to heavy for it and I really could not set it up in a stable way. Good quality tripods are not that cheap, and I do not have many space in my apartment so I would prefer some way to connect head with a bellow but without tripod. I come up with and idea but I would love to hear some feedback from you.

First I used masking tape and wrapped it around my head to not scratch the surface.
Image

Then I made a "holder" with metal clamping ring.
Image

I connected bellow with a holder and head.
Image

And this is how it looks with a camera.
Image

It looked good for me but it was a little bit shaky so I decided to add more ring, after all 1 was enough. It doesn't move at all right now and feels pretty stable.
Image

I am satisfied with that solution, but I want to add special ring, between bellow and camera so I will be able to rotate my camera around it own axis so I will be able to manipulate my frames better.

But maybe I am forgetting something and this will not work that well. The only thing I can think are pulses, but I assume it is the same with intermediate rings. Can you please give me your thoughts about that? Is it acceptable or I should better invest in a good tripod.

Scarodactyl
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Joined: Sat Apr 14, 2018 10:26 am

Re: First photos and image editing

Post by Scarodactyl »

Your canon shouldn't have serious vibration issues. I'd recommend directly coupling it using a mldified Olynpus PM adapter as outlined here: http://www.microbehunter.com/microscopy ... f=9&t=9093
It removes some potential variables on positioning.

NikonUser
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Location: southern New Brunswick, Canada

Re: First photos and image editing

Post by NikonUser »

It is obvious that you are not happy with your Cyclops image; and I am a loss as to why it turned out so poorly.
I took this image years ago with my BHS and DPlan objective and 2.5x NFK. I have forgotten what power objective I used.
This is the quality I would expect you to get with your equipment.
Attachments
cyclops-DPlan.jpg
NU.
student of entomology
Quote – Holmes on ‘Entomology’
” I suppose you are an entomologist ? “
” Not quite so ambitious as that, sir. I should like to put my eyes on the individual entitled to that name.
No man can be truly called an entomologist,
sir; the subject is too vast for any single human intelligence to grasp.”
Oliver Wendell Holmes, Sr
The Poet at the Breakfast Table.

Nikon camera, lenses and objectives
Olympus microscope and objectives

cordyceps
Posts: 11
Joined: Thu Apr 11, 2019 7:00 am

Re: First photos and image editing

Post by cordyceps »

NikonUser wrote:
Wed Aug 12, 2020 2:43 pm
It is obvious that you are not happy with your Cyclops image; and I am a loss as to why it turned out so poorly.
I took this image years ago with my BHS and DPlan objective and 2.5x NFK. I have forgotten what power objective I used.
This is the quality I would expect you to get with your equipment.
I am sure that equipment is not everything. These photos I posted are one of my first, and I feel okay about them, because I know I will improve. Obviously I know nothing about microphotography but I am willing to learn. This is why I wrote this post, I thing that I will improve faster with help of others, more experienced users then I would do by my own. I find your post a little bit ridicules, that you expect from me such a high quality photos in my first week of photomicrography.

rjlittlefield
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Re: First photos and image editing

Post by rjlittlefield »

cordyceps,

NikonUser's image appears to be focus stacked, and using some variety of from-the-side illumination that does a great job of separating the subject from dark background.

Your images look pretty typical for un-stacked single shots using a brightfield condenser.

The biggest concern I have with your first couple of shots are that the color balance is way off. That's simple to fix with levels adjustment. I'm happy to you show what I mean if you like, or maybe you intended them to be the way they are.

To avoid issues with vibration that originates in the camera, you should be shooting in Live View mode so that your Canon EOS 550D will be using electronic first shutter curtain (EFSC). That will eliminate all mechanical vibration except for a little bit at the end of exposure, due to mechanical shutter closing.

Your mechanical adapter is clearly home-brew, but it should work fine. I have fastened things together with tape more than a few times.

--Rik

rjlittlefield
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Re: First photos and image editing

Post by rjlittlefield »

cordyceps wrote:
Wed Aug 12, 2020 4:18 am
I want to start stack my photos soon but I have a question about stacking itself. I understand the idea, you take a lot of photos where each of it has a different sharp part of object, and then focus program choses sharp parts from different pictures and fold them together to get one sharp image. So using micrometer screw we are able to change focus and see different parts of microbe in focus, but some of the microbes are transparent, and changing focus we are able to see "inside them". So in my head we can get a picture when "skin" of the microbe is in focus, and then we can see his internal organs. How do I stack these photos, so I can get sharp image od whole microbe from outside and different sharp "layers" of microbe doesn't blur together.
Showing just the surface layers of a transparent subject is pretty challenging. I am not aware of any stacking software that will do a good job of that automatically. To get a clean result usually requires a lot of human judgement to decide which frames show the surface (including areas with not much detail to indicate that it is the surface), then using the software's retouching tool to preserve just those and not other structures behind them. For some discussion of this in a different domain, see http://www.photomacrography.net/forum/v ... hp?t=11271 , "Surface of dragonfly eye".

This process can be simplified to some extent by using Zerene Stacker's "slabbing" facility to reduce a lot of very thin layers to a relatively fewer but thicker layers, without suffering the resolution loss that you would get from stopping down the condenser.

If stereo presentation is an option, then consider also using Zerene Stacker's "synthetic stereo" capability, described at https://zerenesystems.com/cms/stacker/d ... eticstereo . Examples of this applied to a transparent small subject (fruit fly larva) can be seen at http://www.photomacrography.net/forum/v ... 17#p171017 .

--Rik

cordyceps
Posts: 11
Joined: Thu Apr 11, 2019 7:00 am

Re: First photos and image editing

Post by cordyceps »

rjlittlefield wrote:
Wed Aug 12, 2020 7:22 pm

The biggest concern I have with your first couple of shots are that the color balance is way off. That's simple to fix with levels adjustment. I'm happy to you show what I mean if you like, or maybe you intended them to be the way they are.
I would be grateful :)

Thank you Rik. Your messages are really helpful.

I had some time today to work with microscope. First I started with cleaning my microscope, I was pretty sure that it is clean, but it I thought it its always better to check twice. I did not find any dirt other that some dust, which I blowed off. But then I thought about my glass slides and cover glasses. I bought them long time ago, both were the same brand and rather cheap. After a closer look my glass slides looked fine, but cover glasses were dirty. Now when I prepare a slide I clean them the same way I clean my objectives, and I think the quality of image is better. I did not think of that before because most of the time I used my microscope to look at ready slides of animal tissue, and did not make a lot of slides myself.

Unfortunately I was not able to get some interesting water to look at, so I focused on photo editing and focus stacking as you advised. I downloaded ZereneStacked (trial version) and compared it to FOCUS projects 3 professional, which was free to download some time ago. I am happy I did that, because i liked ZS stacks much more, at least at default setting. I used photos I took the same day I wrote first post here. This is a stack of wasp's wing/12 photos/ 4x Objective
Image

This is another stack I made: 12 photos/ 10x Objective
Image
I know I should take more photos, some parts are still out of focus, but I think its better then the first one I posted.

And this are some agae's I photographed today. I really enjoyed their placement, I am pretty sure I caught on camera microscopic UFO ;) stack/3 photos/ 40x Objective
Image

Unfortunately I looks fake to me, like most of UFO's pictures :P There were a lot of dirt on that frame, which I tried to remove, but I think my lack of experience in photo editing really showed off here.
There is original version:
Image

What is your advise about frames like that, should I try to edit the dirt out like I tried, and when I get better it may look more natural, or I should leave them and try to find better ones?
I appreciate your help guys.
Regards,
Karol

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Re: First photos and image editing

Post by rjlittlefield »

Here are images that I corrected by levels adjustment in Photoshop. The way I do this is to add an adjustment layer, bring up a histogram, focus my attention on the three peaks that represent background color (which is almost everything here), then separately adjust the upper limit of two R/G/B bands so as to move the left two peaks over to match the right. Notice that in the uncorrected version there are three clearly separated peaks, one each R, G, B, but in the corrected version there's only a single peak.

I see that I also brightened the image when I color-corrected it. That's because cameras' metering expects the scene to average 20% gray, so brightfield microscopy images that are "properly" exposed according to the camera are routinely too dark.

OriginalPlusHistogram.jpg

ColorCorrectedPlusHistogram.jpg


Then, here is the color corrected version with another adjustment layer for increased saturation.

ColorCorrectedAndSaturatedPlusHistogram.jpg


I'm sorry that I cannot tell you exactly which buttons to press in GIMP. I use GIMP too seldom to be good with it. But the concepts should be the same -- overlap the peaks in the histogram.

The water creature in your latest post looks very good -- much more like the sharpness that we saw in NikonUser's image.

As for removing dust/debris that overlaps your subject, that's pretty much a lost cause. You would be better off to just get the color right and leave that dark cloud in place. (Perhaps those long skinny things are projectile launchers shooting at the UFO, and that's a cloud of smoke...)

--Rik

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