Poor Mans DIC setup, please offer comments?!

What equipment do you use? Post pictures and descriptions of your microscope(s) here!
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charlie g
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Poor Mans DIC setup, please offer comments?!

#1 Post by charlie g » Thu Oct 27, 2016 5:06 pm

In 3/4/13 I purchased a low cost Olympus Tokyo/Japan trinoc stand with an MPlan 40X objective with it's wolaston prism attachment, and with epi illumination polarizer filter...I was thinking on achieving 'poor mans DIC' microscopy.

Ahh...I wanted transmitted illumination DIC contrast...so for more than the cost of the stand...I purchased a substage Normanski DIC filter compatible with the 40X MPlan objective DIC filter (prism).

Please any and all, when you have time...comment if my trans illumination images have: DIC contrast method?

I oriented the two polarizers to extinction, I oriented the two wolaston prisms...to what I think is 'negative bias DIC contrast.

It's a basic question I ask all...can one sense with my simple image captures of a water flea/ seed shrimp/ Ostracod carapace shell...can you sense DIC contrast as being achieved in my setup?

Thanks all you microscopists with DIC capable stands for your thoughts on my images here.

charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#2 Post by charlie g » Thu Oct 27, 2016 5:10 pm

I really never before thought about how DIC is approached...how it varries in 'degree of DIC contrast' in an image...as one refines all the four elements respective orientations. charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#3 Post by charlie g » Thu Oct 27, 2016 5:16 pm

The substage field illuminator is from my Reichert/Austria Biozet stand, the sustage free standing condenser is NA 1.25, I did not know how to actually cobble a substage condenser to this Olympus Tokyo/Japan eppi stand..there was only a fork substage mirror which simply is removable.
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Re: Poor Mans DIC setup, please offer comments?!

#4 Post by charlie g » Thu Oct 27, 2016 5:18 pm

The specimen is a wet mount slide Ostracod carapace surface.
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Re: Poor Mans DIC setup, please offer comments?!

#5 Post by charlie g » Thu Oct 27, 2016 5:20 pm

So here was my configuration. charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#6 Post by charlie g » Thu Oct 27, 2016 5:25 pm

Is this negative bias DIC simple image capture? No stacking of images are in my tool box at this time. charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#7 Post by zzffnn » Thu Oct 27, 2016 5:29 pm

Charlie G,

I am guessing only:

Your MPlan 40x objective is a no cover objective and the objective prism is a match for that objective (and only that objective), correct?

That epi DIC objective/prism may not work with 0.17 mm cover and transmitted DIC. Are you sure your substage filter is compatible with your objective prism?

It would help, if you post your water flea/ seed shrimp/ Ostracod carapace shell images again. I searched in your profile but did not find them.

I don't think DIC is working in your "109.jpg" and "108.jpg".

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Re: Poor Mans DIC setup, please offer comments?!

#8 Post by charlie g » Thu Oct 27, 2016 5:32 pm

charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#9 Post by charlie g » Thu Oct 27, 2016 5:38 pm

charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#10 Post by charlie g » Thu Oct 27, 2016 5:41 pm

The camera screen view of the ostracod shell wet mount. charlie guevara
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Re: Poor Mans DIC setup, please offer comments?!

#11 Post by hkv » Thu Oct 27, 2016 7:30 pm

I would suggest that you select a very simple subject to test your dic. For example a tiny amount of water under the cover glass forming dry and wet areas, or even simple things like a few dust particles. Then DIC really shows. Air bubbles in small amount of water looks like small mountains in DIC. Then you really can confirm that you have achieved DIC. Here is one example.


Image


With Ostracods, which more or less are non-transparent, you will not see much difference from brightfield. Here is an Ostracod in DIC, but I could probably have achieved a similar image with pure brightfield. This is not a subject where DIC shines.

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Re: Poor Mans DIC setup, please offer comments?!

#12 Post by hkv » Thu Oct 27, 2016 7:42 pm

Please also specify how you have placed the prisms and polarizers. The order should be (bottom to top):

Polarizer - DIC Prism - Condensor - Objective - DIC Prism - Polarizer

If you pull out one of the eye pieces and look down the tube. You should see a white circle with a black stripe in the middle if DIC is properly aligned.

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Re: Poor Mans DIC setup, please offer comments?!

#13 Post by gekko » Thu Oct 27, 2016 8:15 pm

charlie, I agree with hkv: for an example image, my suggestion would be epithelial cheek cells, as they are easy to obtain, and the nucleus would show up nicely in DIC but not so well in brightfield.

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Re: Poor Mans DIC setup, please offer comments?!

#14 Post by charlie g » Thu Oct 27, 2016 10:20 pm

Thanks, zzffnn, hkv, and gekko! I will setup this system and image tiny air bubbles. cheek cells...and I'll view down the tube with occular removed.

You give me specific images to look at, hkv...thank you!

With the epi illumination...that stand was already 'ready' for epi-DIC with the epi lamp shafts polarizer...but I'm trying for transmitted light DIC...and yes, zzffnn, the M40X/0.65 objective probably is 'no cover slip'...but as with the old B&L metalurgical epi stand I have...I've used coverslip mounted pond life preps with that 'no cover slip' set of epi objectives.

charlie guevara BTw...the set up was cobbled 3/13....it now is on the 'back burner' bench in my cluttered study.

I guess the Ostracod subject does not give any viewer here the ability to comment? I sense Fan says:'it's not a DIC image'?

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Re: Poor Mans DIC setup, please offer comments?!

#15 Post by zzffnn » Thu Oct 27, 2016 11:01 pm

Charlie G,

Your MPlan 40x NA 0.65 won't tolerate 0.17 mm well. At NA lower than and equal to NA 0.4, 0.17mm can be tolerated very well. So it depends on NA.

Sorry, your ostracod shell image is not high resolution enough for my poor eyes to see. And as others said, it is not a good subject to judge DIC effect (it is naturally three dimensional / contrasty enough to stand out even in brightfield). Cheek epithelial cells from cheek swaps would work better, as gekko mentioned.

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Re: Poor Mans DIC setup, please offer comments?!

#16 Post by 75RR » Fri Oct 28, 2016 12:57 am

Hi charlie g,

I find that DIC is very intolerant of even minor departures from a correct setup.
It takes a bit of practice and good procedure to achieve good DIC.

I would agree that a simple subject (Cheek cells or a bubble) is the way to go.
A bubble, looking at hkv's excellent image would be my first choice.

I set the levels and fiddled with shadows/highlights on your ostracod image. (Hope you do not mind)
There is definitely some 3D effect, but as has been said, it is not the best test subject around.

Image
BTw...the set up was cobbled 3/13....it now is on the 'back burner' bench in my cluttered study.
I think it is definitely worth the effort to dig it out and have another go at it.
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Re: Poor Mans DIC setup, please offer comments?!

#17 Post by zzffnn » Fri Oct 28, 2016 1:45 am

I remember 75RR published some very nice DIC images shot with his 40x NA 0.65 objective. One thing that contributed to his success was minimizing the water gap between subject and cover slip.

That water gap is around 30-45 microns thick, based on my experience. With a thick water gap, DIC images did not look that great.

If a no cover objective is used for 0.17mm cover, the optical error is 170 microns. That would be too much optical error for NA 0.65, since even a 45 micron error can significantly reduce image quality there. One may still get some DIC effect, but that objective may be performing at only 10-20% of its full potential.

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Re: Poor Mans DIC setup, please offer comments?!

#18 Post by charlie g » Fri Oct 28, 2016 3:50 am

Fan and 75RR, thanks for your comments...I especially am glad that ( as a DIC practitioner) 75RR 'fiddled with my image..thank you, both!

Err, Fan...your comments on 'poor resolution' of my Ostracod image leave me puzzeled. As I view and enjoy live water flea microscopy...it totally elludes me for you to sense my images lack resolution! With a kind lower magnification image of an Ostracod water flea supplied by hkv in this thread...it totally puzzels me on how you can sense the images I posted...lack resolution!

I thank you for your sense that my images are not DIC contrast, Fan...but do understand that the revealed Ostracod carapace images are quite detailed...I enjoy these water flea shells at all levels of magnification...resolution is not an issue for these images.

So we agree...as with a lot of subjects...first image capture simple targets...then sense if DIC contrast is apparent.

I guess I,m hearing that it is not possible to offer opine on when an image is: COL contrast, CX pol, simple oblique contrast...or DIC contrast...with no series of treating a target object/organism to each of these contrast methods.

You all have given me specific target objects to generate images with...thank you all for your comments. charlie guevara

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Re: Poor Mans DIC setup, please offer comments?!

#19 Post by zzffnn » Fri Oct 28, 2016 11:54 am

charlie g wrote:The camera screen view of the ostracod shell wet mount. charlie guevara
Sorry, Charlie G. Please excuse my poor choice of wording. I should also have referred to the exact image that I commented on.

I was merely trying to say "my poor eyesight does not allow me to see your '059.jpg' above in detail".

I am sure others can help.

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Re: Poor Mans DIC setup, please offer comments?!

#20 Post by charlie g » Fri Oct 28, 2016 7:54 pm

Indeed Fan your comments are helpful to me...again thanks. The silly image of my camera screen (Canon rebel xt with simple relay lens...yup my image captures are simple)..that image was included simply to show how I determine best focus...some day I may get a computer at my microscope bench...sighh...'// $ \\' .

Now why didn't I think to use air bubles or cheek cells?! charlie guevara

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Re: Poor Mans DIC setup, please offer comments?!

#21 Post by woyjwjl » Mon Aug 15, 2022 4:55 pm

I don't quite understand if you are achieving EPI illumination or transmission?

If it is EPI, there is only one set of Nomarski prisms in the system, as the light passes through it twice.

Attached is a comparison picture I just made, which may help you
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Re: Poor Mans DIC setup, please offer comments?!

#22 Post by charlie g » Tue Aug 16, 2022 4:09 pm

Thanks lilly and woyj, for your comments. Woyj...somewhere early on in the series of images I posted..you can clearly see I'm using transmitted illumination for this image capture.

I think it was in the Nikon microscopy tutorial it was mentioned:' easy to achieve trans mitted DIC..set up polarized illumination..crossed -pol, then ad the Normanski prisms'. This is why I purchased the Normanski -prism.


75RR...I hear you..devils in the details. What was I thinking..why did'nt I use cheek cells or air bubbles?! I thought air bubbled more ellusive to image ( guess I.m wrong)...here are bubbles in bright field...I compare these to the bubbles kindly submitted earlier in this thread. Why did,nt I think to use cheek cells?!!

Attention good forum members..I am looking to purchase a finite-transmitted DIC setup. If any has one to sell..a good working system..please contact me. Thanks, charlie g
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Re: Poor Mans DIC setup, please offer comments?!

#23 Post by LouiseScot » Tue Aug 16, 2022 6:00 pm

Hi Charlie

I was reading your posts with interest. I had a go setting up DIC with a Nikon Optiphot nosepiece incorporating prisms earlier in the year. I tried to match the nosepiece up with a Zeiss DIC condenser but couldn't get it to work properly. I got a bit distracted and then put it all to one side. However, I thought I'd revisit it and have another go. I also decided that since the nosepiece is really intended for use with m plan objectives and reflected light, then that's what I should do. That doesn't mean it's not possible to set up a transmitted light DIC with the nosepiece, but... So I'm going to go back a step and try and get the nosepiece to work using reflected light and with an m-plan objective which I don't currently have, but I've ordered one from China :) I probably should build an LED epi illuminator with polarizer etc. So will have a go with that. I'll post an update if/when I might make some progress.

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Re: Poor Mans DIC setup, please offer comments?!

#24 Post by charlie g » Tue Aug 23, 2022 12:57 am

Thanks, louise for alerting us to your upcoming DIC project. I too will ( within a few weeks I guess) drag out my cobeled setup..and target cheek cells for an attempt at 'poor mans transmitted DIC'.

I eagerly await your set up and findings, louise! best to all, charlie g.

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Re: Poor Mans DIC setup, please offer comments?!

#25 Post by LouiseScot » Tue Aug 23, 2022 11:27 am

charlie g wrote:
Tue Aug 23, 2022 12:57 am
Thanks, louise for alerting us to your upcoming DIC project. I too will ( within a few weeks I guess) drag out my cobeled setup..and target cheek cells for an attempt at 'poor mans transmitted DIC'.

I eagerly await your set up and findings, louise! best to all, charlie g.
I'm working on making an epi illuminator at the moment. A simple 'proof of concept' one seemed to work ok with a 3W LED light source. I'm now trying to fabricate a better one with iris diaphragms and lenses, to (hopefully!) achieve decent Kohler illumination. I'm short on design info so having to rely on heuristic methods! If anyone has any universal epi optical design info, that would be very useful! I'm wondering if I need to incorporate a secondary tube lens in the epi unit for the infinity objectives? Anyone know?

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: Poor Mans DIC setup, please offer comments?!

#26 Post by Greg Howald » Tue Aug 23, 2022 2:18 pm

Louise,
I dint believe you will need a secondary tube. My epi scope has only one.
Greg

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Re: Poor Mans DIC setup, please offer comments?!

#27 Post by LouiseScot » Tue Aug 23, 2022 2:40 pm

Greg Howald wrote:
Tue Aug 23, 2022 2:18 pm
Louise,
I dint believe you will need a secondary tube. My epi scope has only one.
Greg
Oh ok - thanks. I was a little unsure as infinity optics always confuse me! At the moment I'm working towards an image of the LED to be focused on the objective BFP and the field iris image to be projected on to the sample plane via the objective, of course. Hopefully, I'll be in a position to test it by the weekend. Fingers crossed!

Louise
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Re: Poor Mans DIC setup, please offer comments?!

#28 Post by Greg Howald » Tue Aug 23, 2022 5:08 pm

Good luck. Center. Center. Center

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Re: Poor Mans DIC setup, please offer comments?!

#29 Post by rogeliomoreno » Wed Aug 24, 2022 5:34 am

Charlie,

With DIC we have to consider the following:

1. The objective's prism and the condenser's prism must match. For example: Nikon M condenser's prism must be used with objective's prism of the same family/version. I did try to use Nikon objective's prisms (made for the N2 condenser's prism) with M condenser's prism and they did not work. Maybe you are lucky and your prims match.

2. The condenser's prims are made to be mounted on the condenser at precise distance from the condenser's lens. I saw that you put the condenser's prism on the exit of your light, that prism is made to be inside the condenser (close the focal plane of the condenser's lens, the condenser's iris is at the focal plane so the prism is made to be between the iris and the lens. Your prism is too away from the condenser lens.

3. The prisms have to be oriented 45 degrees from polarizer direction (normally the polarizer is oriented left-right and the analyzer back-front of the microscope), the nosepiece (when the nosepiece has the slot for the objective's prisms) has the slot so when the prisms are inserted they are oriented correctly. First you need to found how the objective's prism is oriented: removed the both prisms from the light train and put the polarizer and analyzer crossed (when the light that you see through the eyepieces is darkest), now put the objective's prism in the light train and remove one of the eyepieces and check for the black line: it should be oriented / or \ (at 45 degrees, note: not all prims show the black line when put between crossed polarizers (the Nikon N1, N2 family of DIC prisms do not show the line); but mostly do).
Second you need to orient the condenser's prism (the black line) in the same direction (/ or \) as the objective's prism, remove the objective's prims from the light train and insert the condenser's prism, check for the black line and turn the prism until the black line is in the same direction as the black line of the objective's prism.

Rogelio

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Re: Poor Mans DIC setup, please offer comments?!

#30 Post by Sure Squintsalot » Fri Aug 26, 2022 5:27 am

rogeliomoreno wrote:
Wed Aug 24, 2022 5:34 am
Charlie,

With DIC we have to consider the following:

1. The objective's prism and the condenser's prism must match. For example: Nikon M condenser's prism must be used with objective's prism of the same family/version. I did try to use Nikon objective's prisms (made for the N2 condenser's prism) with M condenser's prism and they did not work. Maybe you are lucky and your prims match.

2. The condenser's prims are made to be mounted on the condenser at precise distance from the condenser's lens. I saw that you put the condenser's prism on the exit of your light, that prism is made to be inside the condenser (close the focal plane of the condenser's lens, the condenser's iris is at the focal plane so the prism is made to be between the iris and the lens. Your prism is too away from the condenser lens.

3. The prisms have to be oriented 45 degrees from polarizer direction (normally the polarizer is oriented left-right and the analyzer back-front of the microscope), the nosepiece (when the nosepiece has the slot for the objective's prisms) has the slot so when the prisms are inserted they are oriented correctly. First you need to found how the objective's prism is oriented: removed the both prisms from the light train and put the polarizer and analyzer crossed (when the light that you see through the eyepieces is darkest), now put the objective's prism in the light train and remove one of the eyepieces and check for the black line: it should be oriented / or \ (at 45 degrees, note: not all prims show the black line when put between crossed polarizers (the Nikon N1, N2 family of DIC prisms do not show the line); but mostly do).
Second you need to orient the condenser's prism (the black line) in the same direction (/ or \) as the objective's prism, remove the objective's prims from the light train and insert the condenser's prism, check for the black line and turn the prism until the black line is in the same direction as the black line of the objective's prism.

Rogelio
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