LED Lighting and optical resolution questions

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LouiseScot
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Re: LED Lighting and optical resolution questions

#61 Post by LouiseScot » Mon Nov 16, 2020 2:11 pm

I did some comparisons on the Swift between two cameras - the Canon 1100d and a ToupTek 5MP eyepiece camera. Imaged the captive diatom at 40x and did some on-screen measurements at 100% crop:

Canon 1100d
Diatom40xCrop100pcScalemeasure.jpg
Diatom40xCrop100pcScalemeasure.jpg (79 KiB) Viewed 8937 times
Touptek:
Diatom2_40xDead_1358cropScaleMeas.jpg
Diatom2_40xDead_1358cropScaleMeas.jpg (37.89 KiB) Viewed 8937 times
Both gave a length of 63um for the diatom, which was reassuring :) The Touptek gave a bigger image by a factor of 2.36 which is the ratio of the pixel sizes - 5.19/2.2 = 2.36. The striae on the bigger Touptek were slightly clearer. and a measurement of the average distance between striae gave a value of 0.91um (if my calcs were correct!). I've no doubt a properly prepared diatom would give clearer striae. A guesstimate of the potential resolution using the Touptek with the 40x objective was maybe 2/3 of that i.e. ~600nm (but open to discussion!).
Anyone happen to recognise the diatom species? A type of Frustulia?
Anyway, I think I'd conclude that there may well be an advantage to using a camera with smaller pixels than the Canon1100d.

Thanks
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

MichaelG.
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Re: LED Lighting and optical resolution questions

#62 Post by MichaelG. » Mon Nov 16, 2020 2:42 pm

LouiseScot wrote:
Mon Nov 16, 2020 2:11 pm
Anyway, I think I'd conclude that there may well be an advantage to using a camera with smaller pixels than the Canon1100d.
A point which has been previously discussed [sometimes with a little difficulty] on this forum.
Keep at it, Louise ... Your results will be a benchmark in future debates I’m sure.

MichaelG.
Too many 'projects'

hans
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Re: LED Lighting and optical resolution questions

#63 Post by hans » Mon Nov 16, 2020 7:33 pm

It looks like the ToupTek software may be boosting contrast quite a bit more than Canon? Also looks like the Canon image may be suffering more from noise and subsequent noise reduction? What ISO setting are you using on the Canon? I do not get the impression that pixel size is currently the limiting factor in the Canon image.

LouiseScot
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Re: LED Lighting and optical resolution questions

#64 Post by LouiseScot » Mon Nov 16, 2020 8:11 pm

hans wrote:
Mon Nov 16, 2020 7:33 pm
It looks like the ToupTek software may be boosting contrast quite a bit more than Canon? Also looks like the Canon image may be suffering more from noise and subsequent noise reduction? What ISO setting are you using on the Canon? I do not get the impression that pixel size is currently the limiting factor in the Canon image.
I think that's probably just my post processing. Having said that I'm wondering whether I'm doing the auto exposure control in the Canon right. I'll try using a centre weighted evaluation next time. The Toupview software also does auto exposure but it can seemingly take ages for it to work it out (I'm impatient...). Also the camera only allows certain exposure times. On the other hand the Toupview does allow variable gain. I'm thinking about either getting a better dedicated camera for the Swift or maybe an actual dslr adapter e.g. https://www.aliexpress.com/item/3268924 ... 2257eJuJOv. Neither of which helps me improve the images I'm getting from the rail system so I'll have to keep working on that! I think I'll try one of my mono astro cameras out, just to compare some more. I have a 6MP one which should give good images.
Thanks
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: LED Lighting and optical resolution questions

#65 Post by Hobbyst46 » Tue Nov 17, 2020 12:43 pm

LouiseScot wrote:
Mon Nov 16, 2020 8:11 pm
...I think I'll try one of my mono astro cameras out, just to compare some more. I have a 6MP one which should give good images.
Since astro cameras, per definition maybe, are supposedly highly light-sensitive, relative to common inexpensive microscope eyepiece cameras, the results should be very interesting.

LouiseScot
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Re: LED Lighting and optical resolution questions

#66 Post by LouiseScot » Tue Nov 17, 2020 12:54 pm

Hobbyst46 wrote:
Tue Nov 17, 2020 12:43 pm
LouiseScot wrote:
Mon Nov 16, 2020 8:11 pm
...I think I'll try one of my mono astro cameras out, just to compare some more. I have a 6MP one which should give good images.
Since astro cameras, per definition maybe, are supposedly highly light-sensitive, relative to common inexpensive microscope eyepiece cameras, the results should be very interesting.
I'll let you know! I'm going to have a go today :) I'll probably have to dial down the LED brightness a bit. I'll try it with blue and green narrowband filters. I've been looking at Arturo Agostino's DIC images - I can't compete with them!
I've just ordered a 2x dslr adapter off Ebay. It probably won't improve my picture quality but should overcome the vignetting on the Swift. Fingers crossed I don't get stung for vat/import duty... ha ha.
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

LouiseScot
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Re: LED Lighting and optical resolution questions

#67 Post by LouiseScot » Tue Nov 17, 2020 4:17 pm

So here are some images taken with the ToupTek cam on the Swift 380T, then with the mono astro cam (Sony 178m sensor), and then with the astro cam + a 486nm narrowband filter. I couldn't find the previous diatom I imaged - I know it's there somewhere! Anyway, found this other one. I should mention I've been using a cavity slide plus cover slip - not sure if a cavity slide has a negative effect on image quality?

Touptek cam 40x:
Diatom4_Toup_40xG38Crop.jpg
Diatom4_Toup_40xG38Crop.jpg (38.85 KiB) Viewed 8885 times
at 50%:
Diatom4_Toup40x50pcCrop.jpg
Diatom4_Toup40x50pcCrop.jpg (6.64 KiB) Viewed 8885 times
Astro cam 40x:
Diatom4_178m_40x_00001Crop.jpg
Diatom4_178m_40x_00001Crop.jpg (22.56 KiB) Viewed 8885 times
at 50%:
Diatom4_178m40x_00001_50pcCrop.jpg
Diatom4_178m40x_00001_50pcCrop.jpg (5.2 KiB) Viewed 8885 times
Astro cam narrowband 486nm:
Diatom4_178m40x486nm_00002Crop.jpg
Diatom4_178m40x486nm_00002Crop.jpg (28.79 KiB) Viewed 8885 times
There you go

I suppose these are about as good as I'm going to get with the Swift though I'd like to think I could somehow get better images. Apart from the colour, obviously, no big difference between the Touptek and the Astro cam, as far as I can see. I think cropping images at 100% size is maybe not such a good idea - they always appear to come out blurry whatever camera or objective I use. I did a calculation for the images I posted yesterday (with the scales). The diatom was 63um in length, the length on the screen of the Touptek image was 146mm so that's an enlargement of 146/.063 = 2317x which seems rather a lot! The actual magnification was 40x and it does seem tiny looking at it via the eyepieces!

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: LED Lighting and optical resolution questions

#68 Post by Hobbyst46 » Tue Nov 17, 2020 4:49 pm

In this case I think that the astro camera with or without filter performed less sharply than the other camera. When I convert the color photo of the diatom to grayscale, it is less blurred than the astro camera images.
Live diatoms usually move in water. I would suspect that is the chief reason of the blurry. The cavity slide only promotes swimming :)

viktor j nilsson
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Re: LED Lighting and optical resolution questions

#69 Post by viktor j nilsson » Tue Nov 17, 2020 5:18 pm

I would suggest you redo your tests with regular slides, and try to wick away as much water as possible (or use prepared slides). A thick layer of water between the cover glass and the subject will deteriorate the image substantially, and the effect will be stronger for high NA objectives (like the 20x 0.75) than lower NA ones.

I'm personally pretty bad at sample prep, I always put too much gunk on the slide, so I always use my Klaus Kemp 8 form slide when I want to perform standardized tests.

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75RR
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Re: LED Lighting and optical resolution questions

#70 Post by 75RR » Tue Nov 17, 2020 5:51 pm

LouiseScot wrote:
Tue Nov 17, 2020 4:17 pm
I should mention I've been using a cavity slide plus cover slip - not sure if a cavity slide has a negative effect on image quality?
Very much so! You want a flat slide, a cover slip that is 0.17mm or very near to that and minimum water between the diatom and the bottom of the cover slip.

Ideally, as mentioned, you want a Test Slide ... bite the bullet
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LouiseScot
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Re: LED Lighting and optical resolution questions

#71 Post by LouiseScot » Tue Nov 17, 2020 6:26 pm

Hobbyst46 wrote:
Tue Nov 17, 2020 4:49 pm
In this case I think that the astro camera with or without filter performed less sharply than the other camera. When I convert the color photo of the diatom to grayscale, it is less blurred than the astro camera images.
Live diatoms usually move in water. I would suspect that is the chief reason of the blurry. The cavity slide only promotes swimming :)
Although there is water still there all the diatoms are dead - they are ex diatoms! I've messed up that particular slide now so will have to make up a fresh one and with a flat slide. I find the diatoms very difficult to focus on with the Swift. Since I had to change focus for the different cameras it may just be a difference in focus rather than quality.
Thanks
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

LouiseScot
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Re: LED Lighting and optical resolution questions

#72 Post by LouiseScot » Tue Nov 17, 2020 6:37 pm

viktor j nilsson wrote:
Tue Nov 17, 2020 5:18 pm
I would suggest you redo your tests with regular slides, and try to wick away as much water as possible (or use prepared slides). A thick layer of water between the cover glass and the subject will deteriorate the image substantially, and the effect will be stronger for high NA objectives (like the 20x 0.75) than lower NA ones.

I'm personally pretty bad at sample prep, I always put too much gunk on the slide, so I always use my Klaus Kemp 8 form slide when I want to perform standardized tests.
Yeah, I thought that might be a problem so I'll use flat slides for any other goes. I'm only using the Swift for now. I looked up Klaus Kemp - he's not taking any new orders at the moment. I only have the 3 vintage slides with max NA of 0.65 for the 40x achro. There are other diatoms in my culture on the window sill :)
Thanks
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

LouiseScot
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Re: LED Lighting and optical resolution questions

#73 Post by LouiseScot » Tue Nov 17, 2020 6:40 pm

75RR wrote:
Tue Nov 17, 2020 5:51 pm
LouiseScot wrote:
Tue Nov 17, 2020 4:17 pm
I should mention I've been using a cavity slide plus cover slip - not sure if a cavity slide has a negative effect on image quality?
Very much so! You want a flat slide, a cover slip that is 0.17mm or very near to that and minimum water between the diatom and the bottom of the cover slip.

Ideally, as mentioned, you want a Test Slide ... bite the bullet
Ok. As mentioned I have the vintage test slides. I'll have a look to see if I can find some contemporary ones but, if there are any, they are probably rather expensive....
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: LED Lighting and optical resolution questions

#74 Post by Hobbyst46 » Tue Nov 17, 2020 7:17 pm

LouiseScot wrote:
Tue Nov 17, 2020 6:40 pm
75RR wrote:
Tue Nov 17, 2020 5:51 pm
LouiseScot wrote:
Tue Nov 17, 2020 4:17 pm
I should mention I've been using a cavity slide plus cover slip - not sure if a cavity slide has a negative effect on image quality?
Very much so! You want a flat slide, a cover slip that is 0.17mm or very near to that and minimum water between the diatom and the bottom of the cover slip.

Ideally, as mentioned, you want a Test Slide ... bite the bullet
Ok. As mentioned I have the vintage test slides. I'll have a look to see if I can find some contemporary ones but, if there are any, they are probably rather expensive....
Louise
PM sent.

LouiseScot
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Re: LED Lighting and optical resolution questions

#75 Post by LouiseScot » Wed Nov 18, 2020 5:22 pm

I've just had a quick try on the Swift of using oblique illumination. With the Pleurosigma vintage slide at 40x and Touptek camera.

50% crop:
40x_obliquePleurosigma1adj50%crop.jpg
40x_obliquePleurosigma1adj50%crop.jpg (132.28 KiB) Viewed 8824 times
100% crop:
40x_obliquePleurosigma1adj100%crop.jpg
40x_obliquePleurosigma1adj100%crop.jpg (136.6 KiB) Viewed 8824 times
The little adjacent diatom is rather nice. The oblique lighting certainly increases contrast! I'll have to look further into how best to use it.

Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: LED Lighting and optical resolution questions

#76 Post by 75RR » Thu Nov 19, 2020 7:26 am

Nice!

My take is that Brightfield should only be used as default illumination i.e. the first step in setting up the microscope.

This includes finding and focusing on the specimen, adjusting initial lamp brightness and setting up Köhler.

At that point one should then bring in one of the main illumination methods such as Darkfield, Oblique, COL, Phase, DIC etc

The most rewarding cost wise is of course Oblique, the only downside is the lack of consistency/repeatability in its effects, as it depends greatly on the skill of the microscopist.
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
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Re: LED Lighting and optical resolution questions

#77 Post by LouiseScot » Thu Nov 19, 2020 8:47 am

75RR wrote:
Thu Nov 19, 2020 7:26 am
Nice!

My take is that Brightfield should only be used as default illumination i.e. the first step in setting up the microscope.

This includes finding and focusing on the specimen, adjusting initial lamp brightness and setting up Köhler.

At that point one should then bring in one of the main illumination methods such as Darkfield, Oblique, COL, Phase, DIC etc

The most rewarding cost wise is of course Oblique, the only downside is the lack of consistency/repeatability in its effects, as it depends greatly on the skill of the microscopist.
I wish someone had mentioned that to me before! But, never mind - live and learn! I'm going to try and 3d print a COL filter today :)
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: LED Lighting and optical resolution questions

#78 Post by viktor j nilsson » Thu Nov 19, 2020 9:04 am

Agree, looking good! Quite a lot of chromatic aberration, but that's expected with basic achromats. Should be easy to improve with some post processing. But sharpness and resolution looks good. Nicely illuminated, good use of oblique. Will be interesting to see a comparison with your better objectives, now that you've found a good test slide to use.

LouiseScot
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Re: LED Lighting and optical resolution questions

#79 Post by LouiseScot » Thu Nov 19, 2020 9:59 am

viktor j nilsson wrote:
Thu Nov 19, 2020 9:04 am
Agree, looking good! Quite a lot of chromatic aberration, but that's expected with basic achromats. Should be easy to improve with some post processing. But sharpness and resolution looks good. Nicely illuminated, good use of oblique. Will be interesting to see a comparison with your better objectives, now that you've found a good test slide to use.
I'm not worried about small amounts of CA. I shall continue with my investigative experiments :) Oh, it's the same Pleurosigma vintage slide I used before.
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: LED Lighting and optical resolution questions

#80 Post by viktor j nilsson » Thu Nov 19, 2020 10:14 am

LouiseScot wrote: Oh, it's the same Pleurosigma vintage slide I used before.
Louise
Oh, I should've noticed that. :oops:

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Re: LED Lighting and optical resolution questions

#81 Post by LouiseScot » Thu Nov 19, 2020 10:19 am

viktor j nilsson wrote:
Thu Nov 19, 2020 10:14 am
LouiseScot wrote: Oh, it's the same Pleurosigma vintage slide I used before.
Louise
Oh, I should've noticed that. :oops:
There's quite a few Pleurosigma on that slide, plus a few other species, so each time I use it I'll get a different view :)
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

Hobbyst46
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Re: LED Lighting and optical resolution questions

#82 Post by Hobbyst46 » Thu Nov 19, 2020 11:16 am

viktor j nilsson wrote:
Thu Nov 19, 2020 9:04 am
Agree, looking good! Quite a lot of chromatic aberration, but that's expected with basic achromats. Should be easy to improve with some post processing. But sharpness and resolution looks good. Nicely illuminated, good use of oblique. Will be interesting to see a comparison with your better objectives, now that you've found a good test slide to use.
I like these images.
I think that with diatoms, light refraction, and even reflections, in the glass-like frustule is more significant than the type of objective. I get the same chromatic effects on diatoms with Neofluars. Never gave it a serious attention, though.

LouiseScot
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Re: LED Lighting and optical resolution questions

#83 Post by LouiseScot » Thu Nov 19, 2020 11:18 am

viktor j nilsson wrote:
Thu Nov 19, 2020 9:04 am
Agree, looking good! Quite a lot of chromatic aberration, but that's expected with basic achromats. Should be easy to improve with some post processing. But sharpness and resolution looks good. Nicely illuminated, good use of oblique. Will be interesting to see a comparison with your better objectives, now that you've found a good test slide to use.
A quick go with COL. I think the colour fringing is a consequence of the oblique technique rather than the property of the lens per se.
40x again, but limited to 50% of the image size. Seems to be better than 100%.

First one, focus is biased towards the top of the Pleurosigma:
Pleurosigma40x_col2_top50pc.jpg
Pleurosigma40x_col2_top50pc.jpg (104.45 KiB) Viewed 8781 times
I find it difficult to get a sharp focus with the Swift. Of course, I'll also try this out on the rail system with the better lenses.

Louise
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Last edited by LouiseScot on Thu Nov 19, 2020 12:22 pm, edited 1 time in total.
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: LED Lighting and optical resolution questions

#84 Post by 75RR » Thu Nov 19, 2020 11:53 am

LouiseScot wrote:
Thu Nov 19, 2020 8:47 am
I'm going to try and 3d print a COL filter today
It would be interesting to see what that entailed and the source you got it from.

By the way, your images labeled Pleurosigma40x_col2_top50pc.jpg and Pleurosigma40x_col2_sideadj50pc.jpg look the same to me. Mislabeled?
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
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Re: LED Lighting and optical resolution questions

#85 Post by LouiseScot » Thu Nov 19, 2020 12:32 pm

75RR wrote:
Thu Nov 19, 2020 11:53 am
LouiseScot wrote:
Thu Nov 19, 2020 8:47 am
I'm going to try and 3d print a COL filter today
It would be interesting to see what that entailed and the source you got it from.

By the way, your images labeled Pleurosigma40x_col2_top50pc.jpg and Pleurosigma40x_col2_sideadj50pc.jpg look the same to me. Mislabeled?
Yeah, I accidentally saved one on top of the other, if you know what I mean! D'uh. Have now deleted the second one...
Actually, in the end for the COL I just used an iris diaphragm plus a an opaque central stop (as used for dark field) which I'd previously 3D printed. It might be better with a slightly smaller stop than I used. I'll have to experiment to see what works best. There is some info about COL here:
http://www.microscopy-uk.org.uk/mag/artdec02/pjcol.html
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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Re: LED Lighting and optical resolution questions

#86 Post by 75RR » Thu Nov 19, 2020 2:12 pm

LouiseScot wrote:
Thu Nov 19, 2020 12:32 pm
Actually, in the end for the COL I just used an iris diaphragm plus a an opaque central stop (as used for dark field) which I'd previously 3D printed. It might be better with a slightly smaller stop than I used. I'll have to experiment to see what works best. There is some info about COL here:
http://www.microscopy-uk.org.uk/mag/artdec02/pjcol.html
Paul James is my go to person on COL as well

Don't know if this will be usefull to you, they increase in size by 1mm

download/file.php?id=335
Zeiss Standard WL (somewhat fashion challenged) & Wild M8
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Re: LED Lighting and optical resolution questions

#87 Post by LouiseScot » Thu Nov 19, 2020 2:21 pm

75RR wrote:
Thu Nov 19, 2020 2:12 pm
LouiseScot wrote:
Thu Nov 19, 2020 12:32 pm
Actually, in the end for the COL I just used an iris diaphragm plus a an opaque central stop (as used for dark field) which I'd previously 3D printed. It might be better with a slightly smaller stop than I used. I'll have to experiment to see what works best. There is some info about COL here:
http://www.microscopy-uk.org.uk/mag/artdec02/pjcol.html
Paul James is my go to person on COL as well

Don't know if this will be usefull to you, they increase in size by 1mm

download/file.php?id=335
Thanks - though they are simple to 3D print and I already have a number :)
Louise
A Nikon CF plan 20x; A Swift 380T; A DIY infinity corrected focus rail system with a 40x/0.65 Olympus Plan, a 10x/0.30 Amscope Plan Fluor, and a 20x/0.75 Nikon Plan Apo

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