Diatom pictures today - trying to improve res by enhancement techniques 1of2

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cmtalb01
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Diatom pictures today - trying to improve res by enhancement techniques 1of2

#1 Post by cmtalb01 » Mon Feb 19, 2018 3:56 am

I have been trying to learn the true improvements that are gained by neofluar and apochromat objectives and corrected eyepieces. Yesterday great suggestions from the "microscope and optics" section of the forum led to some great reading and more pictures this afternoon of Kemp's Diatom 8 test plate, examining the effects of several enhancement techniques. Resulted in some interesting pictures. Three attached to this post, and two to another post.
Attachments
Use of monochromatic light from a Lomo microscope monochromator (100X apo)
Use of monochromatic light from a Lomo microscope monochromator (100X apo)
Monochromator testing reduced.jpg (175.14 KiB) Viewed 5127 times
Overview with DIC Epi objective and accidental phase ring giving interesting dark field effect.
Overview with DIC Epi objective and accidental phase ring giving interesting dark field effect.
Darkfield testing_reduced.jpg (37.72 KiB) Viewed 5127 times
Overview of the test plate with bright field, crossed pols (CPL) and CPL and green filter
Overview of the test plate with bright field, crossed pols (CPL) and CPL and green filter
Overview CPL.jpg (35.82 KiB) Viewed 5127 times

cmtalb01
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Re: Diatom pictures today - trying to improve res by enhancement techniques 2of2

#2 Post by cmtalb01 » Mon Feb 19, 2018 3:57 am

Second set Hoffman modulation and Phase Contrast. I could not get good results with DIC??
Attachments
100X Plan achromat phase contrast, optovar 2.0X
100X Plan achromat phase contrast, optovar 2.0X
Phase Contrast Testing_reduced.jpg (327.33 KiB) Viewed 5123 times
Hoffman Modulation 40X, Optovar 2.0X, with 438 monochromatic light
Hoffman Modulation 40X, Optovar 2.0X, with 438 monochromatic light
Hoffman Mod tests with 438nm_reduced.jpg (271.86 KiB) Viewed 5123 times
Hoffman Modulation 40X with Optovar at 2.0X
Hoffman Modulation 40X with Optovar at 2.0X
Hoffman Mod tests_reduced.jpg (333.89 KiB) Viewed 5124 times

cmtalb01
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#3 Post by cmtalb01 » Mon Feb 19, 2018 4:00 am

Actual pictures are much better resolved than what is posted due to reduced image resolution.

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zzffnn
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#4 Post by zzffnn » Mon Feb 19, 2018 4:02 am

Can you rule out camera vibration completely (have you tried, for example, 2" exposure)? You apos should at least resolve Frustulia with immersion.

You may want to zoom photos to 100% crop to see more dot details. Maybe you have resolved much more, but were just not seeing them without cropping photos.

einman
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#5 Post by einman » Mon Feb 19, 2018 4:29 am

Based on my observations of the same test slide I can only assume you are getting a lot of camera shake as mentioned.

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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#6 Post by cmtalb01 » Mon Feb 19, 2018 4:57 am

Thanks for the responses. I was using a s second delay with a wireless remote on the camera. I'm not getting the resolution I think I should, but not sure why. These were all focal using a Canon Micro F connection system. I'm waiting on a 35mm lens that should arrive this week. I'll try the afocal method when it arrives to isolate the camera completely.

Attached are images from five Zeiss 100X objectives wihtout any enhancement. My question in the other section is "Why am I not seeing differences between the different objectives?" I am still puzzling over that?

Any and all help appreciated!
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Diatoms 1
Diatoms 1
P. Ang 100X zoomed 200%.jpg (45.53 KiB) Viewed 5111 times

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75RR
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#7 Post by 75RR » Mon Feb 19, 2018 5:20 am

Have you had a look at David Walker's article: Diatom Pleurosigma angulatum - a versatile 'demonstration' subject.

You may find a few pointers there.

http://www.microscopy-uk.org.uk/mag/ind ... -test.html
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
Olympus E-P2 (Micro Four Thirds Camera)

MicroBob
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#8 Post by MicroBob » Mon Feb 19, 2018 7:21 am

Some ideas:
- Are you using electronic first shutter curtain on your camera?
- For Amphipleura pellucida you need all the resolution you can get, so high n.A condenser oiled to the slide
- You might try circular oblique lighting
- You might use a high n.A. darkfield condenser if you have one, maybe decenter it
- Do you know the refractive index of the mounting medium?

It took me quite a while of experimenting to resolve Amphipleura pellucida acceptably. In the end a decentrered darkfield condenser was the solution for me. I enclosed images that show the closest that I came to resolving the single dots. There is still a lot of room for improvement. :D
Attachments
11b  Bob Foto 3.jpg
11b Bob Foto 3.jpg (162.38 KiB) Viewed 5100 times
11 Bob Foto 3.jpg
11 Bob Foto 3.jpg (67.55 KiB) Viewed 5100 times

Hobbyst46
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#9 Post by Hobbyst46 » Mon Feb 19, 2018 7:07 pm

Regarding the first photo, I am afraid that the order of the wavelengths of the yellow/orange and red panels should be changed to agree with the actual colors.

Regarding the second photo, about an "accidental" use of phase contrast to create a darkfield effect.

I would not call it "accidental": The Zeiss manual recommends using the phase rings, especially Ph2 and Ph3, to create darkfield with objectives of low magnification and low NA. The condenser phase plates serve as effective light stops in this case.
Indeed, With my 6.3x/0.16, 10x/0.30 and 16x/0.32 objectives, I get good darkfield at the Ph3 setting (not with the "D" setting)
of the Zeiss turret condenser.

Regarding the third photo, I wonder what caused the vertical alternative bright and dark stripes in the background? was it under any modulated illumination?

einman
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Re: Diatom pictures today - trying to improve res by enhancement techniques 1of2

#10 Post by einman » Mon Feb 19, 2018 8:28 pm

I too have used the phase rings for darkfield. Initially, quite by accident, as I intended to do phase contrast but had the annulus mismatched.

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