Another annoying stacking question

[oxkarthemighty]

Bwaa ha ha! It's me again with a 100x image that is a 108 image stack from an oil immersion objective.
This is a section of the tarsus on a cockroach that sadly did not make it across the bathroom sink. Poor guy, or gal.

I had to sharpen the snot out of this photo and raise the contrast 20% to get it close to looking decent without overdoing it. I do realize that I have to be practical and understand the diffraction and effective stop I am shooting at..However it might be of a benefit to keep the images stacked at 100x (or larger) to a smaller size. I suppose the real question here is am I correct in my assumption that this is what I should be getting from a 100x at this image size?



I feel like I have the 40x down to a degree that is pleasing to my eyes anyway, here is the same section of leg at a 40, which was a 41 image stack. (probably overdoing it a little)

[rjlittlefield]

If I got that image from 100X I would be lark happy. But I'll be interested to hear other comments from other people with more experience in that regime.

I do see some haloing around that small curly fiber that might possibly be reduced with better tuning of the sharpening. Or maybe it's an optical effect. Light does pretty weird stuff down at this scale. I also see some broad "echos" around the left bristle, looking almost like a shock wave, that I suspect are diffraction effects in OOF frames, captured by PMax. Perhaps some of our more experienced 100X users can offer suggestions about how to address those.

The 40X definitely looks good to my eye.

--Rik

[Charles Krebs]

I've been doing this for some time now and I don't think I have ever even attempted to do a 108 image stack with a 100X objective. It is really not an easy thing to do. A great deal depends on the subject and its preparation. Living subjects and thick 3-dimensional subjects are very difficult. A decently prepared, stationary, and relatively flat subject could work, as you have shown here.

But I think I understand what you are asking. When you start out using a microscope you need to make a "mental" adjustment as to what constitutes poor, good, and excellent image quality at different magnifications since it will be significantly different from what you may have become accustomed to in "general" photography. And you really have no basis upon which to make this accommodation. These look very good for a 40X and 100X objective. (Sometimes I think it can actually be trickier to make microscope image quality assessments from larger stacks because of the potential of introducing artifacts and detail from different levels of the subject.)

Looking at your first few shots I would say that the image quality is right where it should be. In fact if you really have just started out using a microscope you are doing exceptionally well. (You must have gotten some sage advice somewhere along the line ). I suspect that you have some very good photographic skills that you have been able to successfully apply to your new microscopy efforts.

[Cactusdave]

These two images are very good indeed and should be gracing the main Photography through the Microscope gallery rather than hiding their light under a bushel here. I'd be delighted if I'd taken either of them and the X40 is excellent, superbly lit and a really nice stack. IMHO there is still a little more interesting and attractive surface detail that can be pulled out of the X40 image by boosting micro contrast more using something like Topaz Detail 3 without the thing looking over-sharpened.

I'd be interested to know how you mounted and lit this leg.

[oxkarthemighty]

If I got that image from 100X I would be lark happy.

This is good news!!! Makes me totally happy!

I do see some haloing around that small curly fiber that might possibly be reduced with better tuning of the sharpening. Or maybe it's an optical effect. Light does pretty weird stuff down at this scale.

Unfortunately this was done in my post processing. I was of the mind that I had a bad shot and was trying to compensate (poorly I might add).

But I think I understand what you are asking. When you start out using a microscope you need to make a "mental" adjustment as to what constitutes poor, good, and excellent image quality at different magnifications since it will be significantly different from what you may have become accustomed to in "general" photography. And you really have no basis upon which to make this accommodation. These look very good for a 40X and 100X objective. (Sometimes I think it can actually be trickier to make microscope image quality assessments from larger stacks because of the potential of introducing artifacts and detail from different levels of the subject.)

Exactly! I had no idea on what constitutes as good. I know what good macro is.....but microscopy is as you know about a 3 day old field to me. One in which is very exciting and frustrating at the same time I have found. (mainly with preparing the samples to get them to do what you want on a slide )

In fact if you really have just started out using a microscope you are doing exceptionally well. (You must have gotten some sage advice somewhere along the line).

I sure did Charles!!! Thanks to you and Mister Rik I feel I am starting to get the hang of this jazz.

These two images are very good indeed and should be gracing the main Photography through the Microscope gallery rather than hiding their light under a bushel here.

Thanks Cactus, I still have a little too many questions at this point to be posting in that part of the forum. I have only been at this type of photography for 3-4 days.

I'd be interested to know how you mounted and lit this leg.

To mount the subject I did this:
http://micro.sci-toys.com/permanent_slides
I used the clear type of Karo syrup as the medium instead of fructose. I would recommend staying away from the white reinforcement and use glue instead. This might take longer, but I have found that when you are lighting your subject that it pushes a white reflection to the left and right of your subject. Sadly I found this out after the 108 image stack and my eyes were too tired to do THAT again. The lighting was a normal simple brightfield lighting. I did however have two IKEA lights on either side, but I don't think that they mattered too much.
Ill check out that Topaz detail 3 business........Looks like it produces really quality results.

[Chris S.]

Oxkar,

I do deep stacks at 100x fairly often, and I think your image is plumb wonderful (this is true of the 40x image, also). Now, I work in air (not oil) with unmounted subjects, using a long working distance Mitutoyo objective with an NA of 0.70. You mounted your subject and almost surely used an oil-immersion objective with a much higher NA than could be used in air. So your 100x situation and mine are so different that observations of mine may apply only barely to you. Your regime can record much more resolution, mine can image specimens that cannot be mounted or immersed. This perhaps softens the blow to myself when I say that your image looks better than any of mine at 100x, and I hope this is entirely attributable to the respective NA’s of our objectives.

I also see some broad "echos" around the left bristle, looking almost like a shock wave, that I suspect are diffraction effects in OOF frames, captured by PMax. Perhaps some of our more experienced 100X users can offer suggestions about how to address those.

I see “echoes” like this all the time, and would just retouch them out. At 100x, “slabbing” in Zerene Stacker is particularly helpful, in that it tends to make this sort of retouching easier. (With slabs, you might be retouching between, say, 10 slabs, instead of 108 images; a particular feature or echo can often be retouched in or out with fewer jumps between input images.)

I also agree with Rik that the haloing around the curly fiber should be fixed. I understand that this was introduced in post processing, and know that this is very easy to do. Unlike Dave, I’m not a fan of the Topaz filters (though to be fair, have not tried them in several years, and likely they have improved). Back when I did try them, my sense was that a person well-versed in sharpening could do a better job than the filters could. "Well-versed,” in this use, would involve familiarity with Photoshop’s smart sharpen filter, high-pass sharpening, use of lab color to permit sharpening the luminosity channel alone (though this seems much less necessary as the smart sharpen filter has evolved), and use of masking and the history brush to selectively place varied levels of sharpening exactly where required. Comparatively, Topaz seemed like a blunt instrument. But perhaps I should revisit it and see how it has evolved.

Anyway, I found your images beautiful and fascinating. Well done!

Cheers,

--Chris