DIC Question

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LouiseScot
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DIC Question

#1 Post by LouiseScot » Thu May 20, 2021 4:21 pm

Hi all

Can anyone who has DIC tell me what a prepared/stained slide might look like under DIC - (an example image, if you have one, would be good :) ) ? Failing that, if anyone could recommend a subject for a test slide (preferably inanimate at this stage) that would be helpful. I've been having a go at making a Sanderson Prism DIC setup as per the DIY DIC thread and the paper by Rathi et al. posted about a month ago. I can certainly get an image but I'm not convinced it's as expected as with actual DIC. I've just used a handy Chinese prepared slide of a kidney section as it's fairly easy to focus on. But it may not be the best test subject? I'm also only using a cheap 20x infinity objective so that probably won't be optimal either! Still, I do get an image on a dslr :) I've done my best to set everything up properly so I feel I'm getting close (fingers crossed), but maybe it's still not right yet. At the moment, everything is set up / prototyped horizontally, and using aluminium extrusion t-sections, so not an actual microscope stand.

Many thanks

Louise
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75RR
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Re: DIC Question

#2 Post by 75RR » Thu May 20, 2021 6:44 pm

.
Here is a link to a series of tests done comparing the compatibility of several objectives with DIC sliders.

Most are done with diatoms but some are with 'sections'.

Since the quality of the DIC varies with each objective and on the slider used the images may well prove useful to you.

https://www.photomacrography.net/forum/ ... hlight=dic
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Re: DIC Question

#3 Post by LouiseScot » Thu May 20, 2021 8:00 pm

Ah, yes, thanks. I didn't think frustules would be such good subjects. But my current optics don't compare with the Photomacrography thread:
DiatomsCrop2_0112.jpg
DiatomsCrop2_0112.jpg (75.73 KiB) Viewed 609436 times
Diatoms_011278pc.jpg
Diatoms_011278pc.jpg (65.26 KiB) Viewed 609436 times

I'm not sure whether what I see is simply down to polarization... I'll try with a different objective. I have an Olympus UIS2 40x and a recently acquired 60x. The quoted paper used a 60x.

Louise
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Re: DIC Question

#4 Post by Hobbyst46 » Thu May 20, 2021 9:08 pm

Diatoms are not especially colorful under polarized light (when a polarizer and an analyzer are fitted in the microscope). In that sense, they are distinguished from quartz and mica particles that may be present on the slide.

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Re: DIC Question

#5 Post by LouiseScot » Thu May 20, 2021 9:29 pm

Hobbyst46 wrote:
Thu May 20, 2021 9:08 pm
Diatoms are not especially colorful under polarized light (when a polarizer and an analyzer are fitted in the microscope). In that sense, they are distinguished from quartz and mica particles that may be present on the slide.
Oh ok. The ones in the Photomacrography link above seem quite colourful? DIC is based on polarization, of course. Attempting to build my own DIC is tricky without something to easily compare to - almost a leap of faith! My focus mechanism is very crude - an Allen key and a M4 bolt! The 'tube lens' is from a 50mm astronomy finder - a coated achromat but could be improved. I'd want to know if it's working at all before spending money on a better lens. If I can confirm that DIC has been achieved then I can start optimizing the various components :)

Louise
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Re: DIC Question

#6 Post by PeteM » Thu May 20, 2021 9:44 pm

Tiny and more or less transparent bubbles under a cover slip can make a good DIC subject, since there is an abrupt phase change at the edges. These could be saliva, bubbles in a poorly-mounted slide, or some oil/water/fat mix that ends up with bubbles under the cover slip.

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Re: DIC Question

#7 Post by LouiseScot » Thu May 20, 2021 9:51 pm

PeteM wrote:
Thu May 20, 2021 9:44 pm
Tiny and more or less transparent bubbles under a cover slip can make a good DIC subject, since there is an abrupt phase change at the edges. These could be saliva, bubbles in a poorly-mounted slide, or some oil/water/fat mix that ends up with bubbles under the cover slip.

Funnily enough I had some strange effects from the edge of the diatom slide where the mounting medium hasn't filled properly - but is it DIC?
Diatoms49pc_0109.jpg
Diatoms49pc_0109.jpg (140.54 KiB) Viewed 609408 times
Louise
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Re: DIC Question

#8 Post by PeteM » Thu May 20, 2021 11:28 pm

My inclination is to say no, not DIC and mostly polarization with maybe a prism adding some color.

It's tricky, with a sort of experimental setup and less than sharp images, to know if you're getting close.

Between polarization, a prism acting as a wave plate (shifting colors), and a bit of oblique illumination one get get some pretty interesting effects. With standard DIC and two prisms, one can pull out one of the prisms and realize that most of the effect remains (e.g. not DIC). I haven't a clue with "Sanderson" DIC. You and Saul seem to be the pioneers here.

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Re: DIC Question

#9 Post by LouiseScot » Thu May 20, 2021 11:43 pm

PeteM wrote:
Thu May 20, 2021 11:28 pm
My inclination is to say no, not DIC and mostly polarization with maybe a prism adding some color.

It's tricky, with a sort of experimental setup and less than sharp images, to know if you're getting close.

Between polarization, a prism acting as a wave plate (shifting colors), and a bit of oblique illumination one get get some pretty interesting effects. With standard DIC and two prisms, one can pull out one of the prisms and realize that most of the effect remains (e.g. not DIC). I haven't a clue with "Sanderson" DIC. You and Saul seem to be the pioneers here.
Thanks - I'll try and do one with and without one of the prisms but I'd have to also take a polarizer out as it's obviously dark with both polarizers in and no prism(s) :) .

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: DIC Question

#10 Post by 75RR » Fri May 21, 2021 6:54 am

.
Had a quick look (again?) at the Rathi-DIC-plastic-Sanderson-prisms pdf

https://www.quekett.org/wp-content/uplo ... prisms.pdf

I imagine a comparison image (which you can reproduce) would be the way to go for testing.

(Linked image is a little over-magnified, you might want to reduce the size)

https://duckduckgo.com/?t=ffsb&q=bucal+ ... ared-l.jpg
.
Image
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Re: DIC Question

#11 Post by LouiseScot » Fri May 21, 2021 9:03 am

75RR wrote:
Fri May 21, 2021 6:54 am
.
Had a quick look (again?) at the Rathi-DIC-plastic-Sanderson-prisms pdf

https://www.quekett.org/wp-content/uplo ... prisms.pdf

I imagine a comparison image (which you can reproduce) would be the way to go for testing.

(Linked image is a little over-magnified, you might want to reduce the size)

https://duckduckgo.com/?t=ffsb&q=bucal+ ... ared-l.jpg
.
Image
Yeah I tried cheek cells initially but they aren't very 'positive'. Anyway, I suspect my prisms may not be working as expected. I'll keep at 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: DIC Question

#12 Post by MicroBob » Fri May 21, 2021 10:39 am

Hi Louise,
I can't recommend stained sections for evaluation of DIC systems. The colour contrast would be overlayed with the DIC relief effect and the result would not look improved and difficult to evaluate. I would suggest typical phase objects, transparent stuff that is only different to it's surrounding in it's refractive index. The cheeks cells are probably already one of the best test objects.

Bob

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Re: DIC Question

#13 Post by LouiseScot » Fri May 21, 2021 10:54 am

MicroBob wrote:
Fri May 21, 2021 10:39 am
Hi Louise,
I can't recommend stained sections for evaluation of DIC systems. The colour contrast would be overlayed with the DIC relief effect and the result would not look improved and difficult to evaluate. I would suggest typical phase objects, transparent stuff that is only different to it's surrounding in it's refractive index. The cheeks cells are probably already one of the best test objects.

Bob
Thanks, though I think I'll stick to the good old diatoms for now as they are easy to focus on, and easily reproducible for before and after tests. There does seem to be some difference between having one prism in and both in. It's almost black with no prisms. There are a number of variables though - the pressure on each of the prisms and their orientation relative to the cross polarizers and other things, no doubt!
Cheers
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: DIC Question

#14 Post by apochronaut » Fri May 21, 2021 11:26 am

You didn't mention, I don't think , whether you are viewing your results through a properly corrected eyepiece or whether your results are being entirely interpreted as photographed through your tube lens?

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Re: DIC Question

#15 Post by LouiseScot » Fri May 21, 2021 11:38 am

apochronaut wrote:
Fri May 21, 2021 11:26 am
You didn't mention, I don't think , whether you are viewing your results through a properly corrected eyepiece or whether your results are being entirely interpreted as photographed through your tube lens?
Hiya

I've constructed a setup which is similar to the one in Rathi paper (Fig 4) (Linked above by 75RR's post) - except I don't have an Olympus IX81 to start with! So I'm imaging through an objective plus tube lens plus the 4F relay optics. It seems to work surprisingly well though I'm still working on achieving DIC. It's a challenge to do it all in my living room and without a proper optical bench...

Louise
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Re: DIC Question

#16 Post by apochronaut » Fri May 21, 2021 12:16 pm

So everything is going to a sensor through that relay lens? You aren't actually viewing your d.i.y. D.I.C. set up through a pair of properly corrected eyepices independent of the relay lens?

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Re: DIC Question

#17 Post by LouiseScot » Fri May 21, 2021 12:28 pm

apochronaut wrote:
Fri May 21, 2021 12:16 pm
So everything is going to a sensor through that relay lens? You aren't actually viewing your d.i.y. D.I.C. set up through a pair of properly corrected eyepices independent of the relay lens?
Through the tube lens + 4F relay - Yep. And nope. No need for corrected EPs. I'm just using a dslr + generic infinity 20x objective on my 'bench' at the moment. I will be trying it with an Olympus UIS2 40x objective later but that doesn't require corrected EPs either. I'm just printing some holders which will allow rotation of the prisms in a similar manner to the polarizers.

Louise
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Re: DIC Question

#18 Post by deBult » Fri May 21, 2021 12:35 pm

To my understanding the Olympus UIS2 requires a correction via the Telan lens in the modern Olympus scopes (actually I assume the Telan lens is in the Bino\Trino head).

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Re: DIC Question

#19 Post by apochronaut » Fri May 21, 2021 12:46 pm

LouiseScot wrote:
Fri May 21, 2021 12:28 pm
apochronaut wrote:
Fri May 21, 2021 12:16 pm
So everything is going to a sensor through that relay lens? You aren't actually viewing your d.i.y. D.I.C. set up through a pair of properly corrected eyepices independent of the relay lens?
Through the tube lens + 4F relay - Yep. And nope. No need for corrected EPs. I'm just using a dslr + generic infinity 20x objective on my 'bench' at the moment. I will be trying it with an Olympus UIS2 40x objective later but that doesn't require corrected EPs either. I'm just printing some holders which will allow rotation of the prisms in a similar manner to the polarizers.

Louise
So you aren't looking through eyepieces, you are trying to do d.i.y. D.I.C. using the objective doublet from a spotting scope as a tube lens? Don't objective lenses from spotting scopes happen to have pretty high levels of ca? They are all marked coated achromat in 2021, which makes them only marginally better than an uncoated achromat, which is the bottom of the barrel when it comes to lens corrections, unless you include magnifying glasses.

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Re: DIC Question

#20 Post by LouiseScot » Fri May 21, 2021 12:59 pm

apochronaut wrote:
Fri May 21, 2021 12:46 pm
LouiseScot wrote:
Fri May 21, 2021 12:28 pm
apochronaut wrote:
Fri May 21, 2021 12:16 pm
So everything is going to a sensor through that relay lens? You aren't actually viewing your d.i.y. D.I.C. set up through a pair of properly corrected eyepices independent of the relay lens?
Through the tube lens + 4F relay - Yep. And nope. No need for corrected EPs. I'm just using a dslr + generic infinity 20x objective on my 'bench' at the moment. I will be trying it with an Olympus UIS2 40x objective later but that doesn't require corrected EPs either. I'm just printing some holders which will allow rotation of the prisms in a similar manner to the polarizers.

Louise
So you aren't looking through eyepieces, you are trying to do d.i.y. D.I.C. using the objective doublet from a spotting scope as a tube lens? Don't objective lenses from spotting scopes happen to have pretty high levels of ca? They are all marked coated achromat in 2021, which makes them only marginally better than an uncoated achromat, which is the bottom of the barrel when it comes to lens corrections, unless you include magnifying glasses.
It's an achromatic doublet from an astro guide scope. I figured it would be ok for prototyping since Rathi et al. used achromatic doublets for their extended optics and most of my objectives are just achromats too. If I can determine that the DIC is actually working then I'll get a better tube lens :) .
Louise
I'm only trying to get the prisms to apparently work. I can maybe optimize the optics later.
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: DIC Question

#21 Post by Greg Howald » Fri May 21, 2021 1:16 pm

Prepared animal slides like skin, spinal cord, etc. really pop under DIC and make very good test slides that can always be referred to.
Greg

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Re: DIC Question

#22 Post by 75RR » Fri May 21, 2021 1:20 pm

.
Have you been able to observe the interference pattern as shown in the image below? (from post #10 link)
.
Attachments
Interference pattern.png
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Re: DIC Question

#23 Post by LouiseScot » Fri May 21, 2021 1:22 pm

Greg Howald wrote:
Fri May 21, 2021 1:16 pm
Prepared animal slides like skin, spinal cord, etc. really pop under DIC and make very good test slides that can always be referred to.
Greg
MicroBob (#12) says not? Anyway, I'll stick to the diatoms for now :)

Louise
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Re: DIC Question

#24 Post by apochronaut » Fri May 21, 2021 1:24 pm

LouiseScot wrote:
Fri May 21, 2021 12:59 pm
apochronaut wrote:
Fri May 21, 2021 12:46 pm
LouiseScot wrote:
Fri May 21, 2021 12:28 pm


Through the tube lens + 4F relay - Yep. And nope. No need for corrected EPs. I'm just using a dslr + generic infinity 20x objective on my 'bench' at the moment. I will be trying it with an Olympus UIS2 40x objective later but that doesn't require corrected EPs either. I'm just printing some holders which will allow rotation of the prisms in a similar manner to the polarizers.

Louise
So you aren't looking through eyepieces, you are trying to do d.i.y. D.I.C. using the objective doublet from a spotting scope as a tube lens? Don't objective lenses from spotting scopes happen to have pretty high levels of ca? They are all marked coated achromat in 2021, which makes them only marginally better than an uncoated achromat, which is the bottom of the barrel when it comes to lens corrections, unless you include magnifying glasses.
It's an achromatic doublet from an astro guide scope. I figured it would be ok for prototyping since Rathi et al. used achromatic doublets for their extended optics and most of my objectives are just achromats too. If I can determine that the DIC is actually working then I'll get a better tube lens :) .
Louise
I'm only trying to get the prisms to apparently work. I can maybe optimize the optics later.
Achromatic doublets are corrected by oculars in finder scopes and even with that they show coloured borders. I'm thinking a bunch of your chroma and distortion in your system is coming from that lens.
Achromatic lenses are only a a-chromatic in the sense that they are less chromatic than one that is chromatic. That's why apochromats exist or systems that correct for an achromatic doublet's deficiencies, usually known as corrective optics in microscopes.

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Re: DIC Question

#25 Post by LouiseScot » Fri May 21, 2021 1:40 pm

75RR wrote:
Fri May 21, 2021 1:20 pm
.
Have you been able to observe the interference pattern as shown in the image below? (from post #10 link)
.
I can certainly see the stress/polarization effects in a prism:
StressedPC_Crop_0122.jpg
StressedPC_Crop_0122.jpg (88.87 KiB) Viewed 609280 times
I'm unsure whether I have correct/sufficient bending pressure on the prisms. I'll have to experiment a bit :)

I'll have to check whether there are interference fringes with the two prisms. I'm not doing quite the same as Sanderson in that I'm aiming to focus on the objective BFP via the relay optics. I'm just waiting for a second prism rotation holder to print out and then I'll see what I can see if the prisms are rotated in respect of each other and also the polarizers..

Louise

ps I'll try and do a test with the prisms either side of just the relay optics - I'm not sure exactly how Rathi et al. did their gradient test. I don't think I have a dark central band. I've kindof skipped over some bits that I should have put more effort into... I don't actually have a 6mm bar as per Rathi and Sanderson. I went straight to making two 3x12mm prisms. I have now ordered some 6mm polycarbonate so I'll then be able to duplicate the open frame setup. Probably a good idea ha ha.
Last edited by LouiseScot on Fri May 21, 2021 2:08 pm, edited 1 time in total.
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Re: DIC Question

#26 Post by LouiseScot » Fri May 21, 2021 1:46 pm

apochronaut wrote:
Fri May 21, 2021 1:24 pm
LouiseScot wrote:
Fri May 21, 2021 12:59 pm
apochronaut wrote:
Fri May 21, 2021 12:46 pm

So you aren't looking through eyepieces, you are trying to do d.i.y. D.I.C. using the objective doublet from a spotting scope as a tube lens? Don't objective lenses from spotting scopes happen to have pretty high levels of ca? They are all marked coated achromat in 2021, which makes them only marginally better than an uncoated achromat, which is the bottom of the barrel when it comes to lens corrections, unless you include magnifying glasses.
It's an achromatic doublet from an astro guide scope. I figured it would be ok for prototyping since Rathi et al. used achromatic doublets for their extended optics and most of my objectives are just achromats too. If I can determine that the DIC is actually working then I'll get a better tube lens :) .
Louise
I'm only trying to get the prisms to apparently work. I can maybe optimize the optics later.
Achromatic doublets are corrected by oculars in finder scopes and even with that they show coloured borders. I'm thinking a bunch of your chroma and distortion in your system is coming from that lens.
Achromatic lenses are only a a-chromatic in the sense that they are less chromatic than one that is chromatic. That's why apochromats exist or systems that correct for an achromatic doublet's deficiencies, usually known as corrective optics in microscopes.
Guide scopes just use the lens with a camera - with software, they fix on a star to enable guiding the imaging telescope.
Of course, I know my 20x objective isn't great but I should still be able to spot a difference between DIC and not DIC.

Louise
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Re: DIC Question

#27 Post by Scarodactyl » Fri May 21, 2021 6:02 pm

deBult wrote:
Fri May 21, 2021 12:35 pm
To my understanding the Olympus UIS2 requires a correction
All UIS/UIS2 olympus infinity objectives are fully internally corrected.

The chrimatic aberrations showing up in photos are well beyond what you should be seeing, even with a pretty low end objective. I think getting that more dialled in should be the first step which will make everything else a lot easoer and more rewarding.

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Re: DIC Question

#28 Post by LouiseScot » Fri May 21, 2021 7:03 pm

Scarodactyl wrote:
Fri May 21, 2021 6:02 pm
deBult wrote:
Fri May 21, 2021 12:35 pm
To my understanding the Olympus UIS2 requires a correction
All UIS/UIS2 olympus infinity objectives are fully internally corrected.

The chrimatic aberrations showing up in photos are well beyond what you should be seeing, even with a pretty low end objective. I think getting that more dialled in should be the first step which will make everything else a lot easoer and more rewarding.
My living room setup is rather challenging to work with - the imaging train is a metre long from slide to sensor, so sensitive to any vibrations. My slide holder and focusing mechanism are very crude. The objective I've been using is itself a bit dodgy! I bought it a few years ago off a well-known UK-based Chinese lady importer/seller. It's marked as 20x/0.40 with an infinity symbol but was sold to me at the time as a DIN finite objective. Even stranger, it has an apparent working distance of about 5mm. Not sure how that happens! :shock: I've been using it because of it's apparent long working distance - makes life a little easier, and because I didn't want to risk damaging a decent objective whilst I was messing about (I'm old, my hands shake, I've no grip and I frequently drop things :oops: ). However, I'll definitely put the Olympus 40x on tomorrow. It makes sense to work with a known quantity :) Also, the diatom images I've been posting are very much enlarged. It's surprising I'm getting anything recognisable 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

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Re: DIC Question

#29 Post by deBult » Fri May 21, 2021 7:17 pm

Scarodactyl wrote:
Fri May 21, 2021 6:02 pm
deBult wrote:
Fri May 21, 2021 12:35 pm
To my understanding the Olympus UIS2 requires a correction
All UIS/UIS2 olympus infinity objectives are fully internally corrected.
Hmm: from the Olympus guide on selecting objectives:
“ The UIS2 optical system optimally corrects aberration with a dedicated telan lens and an eyepiece so that the coma aberration and flatness are not degraded even when the telan lens exit pupil position is changed by changing the objective lens and telan distance. ”

http://resources.olympus-europa.com/mic ... _0310B.pdf

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Re: DIC Question

#30 Post by Hobbyst46 » Fri May 21, 2021 7:27 pm

LouiseScot wrote:
Fri May 21, 2021 7:03 pm
It's marked as 20x/0.40 with an infinity symbol but was sold to me at the time as a DIN finite objective. Even stranger, it has an apparent working distance of about 5mm. Not sure how that happens! :shock: I've been using it because of it's apparent long working distance - makes life a little easier,...
If it is a finite optics 20x0.40 objective, 5mm is a long working distance IMO.

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