3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

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3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#1 Post by josmann » Wed Jan 26, 2022 9:01 pm



This is a video I put together recently describing a technique which seems to have been largely overlooked for decades. It presents simultaneous stereo pairs through the eyepieces of a traditional compound microscope. Unlike half moon apertures on the eyepieces, this has no finnicky alignment or consequences for the image dimensions. You get two full field image circles of slightly different perspectives. Some of you will recognize that this is essentially a mod to turn a compound binocular microscope into a common objective stereoscope. You of course necessarily toss out a lot of light in the process (but I've found a 1W LED still gives plenty) and there is a resolution impact although I think the images still look plenty clear!

An adjustment must be applied for Siedentopf heads (brownie points for those who know why) and there are some other interesting quirks related to S and P polarization and the optics of the binocular head - I'll be covering those in future videos.

If you haven't heard of this one before - you should try it! I think you're gonna love it.
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#2 Post by marcel_ » Wed Jan 26, 2022 10:58 pm

I happen to have seen your video and already posted about it here:
viewtopic.php?f=28&t=14787

I think it is a very nice mod. Want to try it myself.
I have some questions though.
1) You already mentioned it more or less: isn't the quality of the image not affected by this procedure?
2) Aren't the halfway cuts of the polarisation filter not giving all sorts of artifacts?
3) in combination with darkfield, it seems like this there could be hardly any light left to actually get a picture with a reasonable resolution. Or is that not really the case?


Of course any drawbacks are compensated by the fact that you will have an awesome 3d view instead.
Looking forward for more of your video's.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#3 Post by josmann » Wed Jan 26, 2022 11:44 pm

Hi Marcel,

The images are very clean - there is no artifacting although you will start to get a double image if the eyepiece polarizers are rotated from optimal (tilted 90 degrees they will project the view belonging to the other eyepiece).

Necessarily there will be an impact on numerical aperture and therefore resolution because we’re obscuring the objective light by half. If my understanding is correct, horizontal resolution will be diminished while vertical resolution should be mostly unaffected. In my experience, there is still plenty of NA to form a sharp image capable of resolving fine details like the striations along a spirostomum’s body. Though stepping up to a .40 NA or higher 10x is definitely nice.

It does indeed waste a lot of light, but if you set up your darkfield correctly, use a patch stop of minimal diameter, and crank the light to maximum, there is more than enough light for good viewing on an amateur scope with a 1W illuminator. Scopes with Koehler illuminators should have even more top end output.

One thing to try when you get your setup is, after you align the objective and get a stereo view, try rotating the objective a further half turn and swapping the left and right eyepieces. You may find there’s a significant brightness difference (and maybe even color temperature) with the objective oriented one way versus the other. This is because of the S and P polarization effects I alluded to above :)

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#4 Post by zzffnn » Thu Jan 27, 2022 12:12 am

I encourage other members to try this and follow their heart/eyes (as to whether or not to adopt it).

For my eyes though, I don't like this half darkfield effect.

Half oblique sort of works for some samples, though due to pronounced shadow artifacts, my eyes prefer well implemented COL.

Your eyes can be different; I understand everyone sees differently.

Half mask oblique is much easier to implement near condenser iris.

I also wonder how well Shinya's method would work at NA 0.65 and higher.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#5 Post by josmann » Thu Jan 27, 2022 1:36 am

zzffnn wrote:
Thu Jan 27, 2022 12:12 am
I encourage other members to try this and follow their heart/eyes (as to whether or not to adopt it).

For my eyes though, I don't like this half darkfield effect.
I think you may be misunderstanding the mod I’ve described. There is no half darkfield here - it is not a “3D shading effect” like oblique filtering - it is an actual 3D stereoscopic view like using a stereoscope. You can use it with darkfield, brightfield, COL, Rheinberg, etc illumination. All this mod does is change the projection angle of the image coming out the back of the objective such that it is different for the left and right sides. Whatever you set up before it should be preserved with the possible exception of directional oblique methods which may have too much angular bias. I think you’d want the illumination to be left/right symmetric but I haven’t done nearly enough experimentation!

I encourage you to give it a shot!
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#6 Post by zzffnn » Thu Jan 27, 2022 2:29 am

I guess I did misunderstand it. So camera at trinocular tube would not "see" such effect.

I did watch the entire video (I am your subscriber). It was just the shadow effect towards the end of your video (granted video was taken from one single eye tube, which gave me the wrong impression); it reminded me of half darkfield effect that I tried and did not like.

I heard that, in general, we should not put optically inferior material after objective lens (menaing in between objectives BFP and eyepiece) though, because putting filters before objectives affects image quality much less.

Have you looked at a test diatom (for example, Frustulia) with and without Shinya mod and compared the image quality degradation, if there is any?

I should have some polarization filters left from years ago, if I can still find them.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#7 Post by BramHuntingNematodes » Thu Jan 27, 2022 2:42 am

The polarizing media shouldn't reduce the resolution much if it's new and not smudged. You won't see the junction as half of the image that.butts.up against it is blacked out.
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#8 Post by josmann » Thu Jan 27, 2022 3:04 am

zzffnn wrote:
Thu Jan 27, 2022 2:29 am
I guess I did misunderstand it. So camera at trinocular tube would not "see" such effect.

I did watch the entire video (I am your subscriber). It was just the shadow effect towards the end of your video (granted video was taken from one single eye tube, which gave me the wrong impression); it reminded me of half darkfield effect that I tried and did not like.

I heard that, in general, we should not put optically inferior material after objective lens (menaing in between objectives BFP and eyepiece) though, because putting filters before objectives affects image quality much less.

Have you looked at a test diatom (for example, Frustulia) with and without Shinya mod and compared the image quality degradation, if there is any?

I should have some polarization filters left from years ago, if I can still find them.
Yeah I guess I should have put a disclaimer that I am unable to show the effect. The final minute or so was just some DIC footage I captured recently to fill time. In order to properly show it I’d need to record the left and right eyepieces simultaneously and produce a 3D video. And I might just do that some time!

This will certainly have an impact on image resolution because you’re reducing the effective aperture size but the images themselves are quite clear. As Bram says, try not to get chicken grease all over the filters and it’ll look excellent.

Believe me when I say the resolution hit will not matter much to you once you get it set up and start looking at an active sample. This isn’t really a mod for high end content creation or photography - it’s a mod for good old awe and wonder through the eyepieces :)

Here's a clarification video I just threw together which shows me toggling the view from left to right. Hopefully that helps!

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#9 Post by amphicyon » Fri Jan 28, 2022 10:31 am

Polish company PZO made same device for stereo phase contrast named KS. Dual rotated polarizing filters were inserted into the condenser (on phase ring) and in eyepieces tubes. Do not spoil the image of the objective by sticking the film on the back, insert them into the filter holder in condenser.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#10 Post by apochronaut » Fri Jan 28, 2022 1:58 pm

I have been doing DF for over 30 years now. Despite what one will read about DF reducing depth of field, my experience has been the opposite. It has been fairly common over the years , depending on sample depth, the condenser used, the objective used and some filtering to achieve a very deep plane of focus.

Visually as well but not photographically, this can be enhanced by some critical adjustment of the interpupillary distance. The depth of field can be altered, either positive or negative, especially with the parallax Jentsch heads used on AO microscopes up to the series 10/20 vintage. This imposed parallax, rather than the more common parallel eyetubes found on most microscopes has some advantage in creating a 3 dimensional effect but that is something additional and different than the innate depth of field associated with DF.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#11 Post by josmann » Fri Jan 28, 2022 7:19 pm

amphicyon wrote:
Fri Jan 28, 2022 10:31 am
Polish company PZO made same device for stereo phase contrast named KS. Dual rotated polarizing filters were inserted into the condenser (on phase ring) and in eyepieces tubes. Do not spoil the image of the objective by sticking the film on the back, insert them into the filter holder in condenser.
This doesn't work quite right for this method because you then introduce polarization dependence of the sample into the equation. I believe you will potentially create some odd effects but I will give it a shot. Moreover, at high numerical apertures, the polarization extinction effect will be diminished at certain azimuthal angles without the use of a compensator (another invention of Shinya Inoue). By placing the polarizers at the back of the objective, the only angles you have to contend with are the relatively modest projection angles from the rear aperture to the intermediate image.

Before you write this off, I encourage you to try it! It's only 10-15 dollars to implement, takes a few minutes of your time, and you can observe for yourself whether the image is ruined :)
apochronaut wrote:
Fri Jan 28, 2022 1:58 pm
I have been doing DF for over 30 years now. Despite what one will read about DF reducing depth of field, my experience has been the opposite. It has been fairly common over the years , depending on sample depth, the condenser used, the objective used and some filtering to achieve a very deep plane of focus.
Yes the enhanced depth of field from DF really makes this modification sing! The depth effect is primarily due to objects away from the image plane being projected at different angles. With darkfield, out of focus objects are always quite visible (sometimes to our frustration :lol:). Try this one out, Apo, I thought of you in particular when I found this mod out since you have talked at length about experiencing depth through darkfield before. I think you'll really enjoy this little twist on it :)
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#12 Post by Rossf » Sat Jan 29, 2022 12:04 am

Thanks josmann! VERY INTERESTING…I’ve had dreams about this being possible-I need to read that book you breezed through in the video-what is it called? Shinya sounds like he had an incredible knowledge and visionary appreciation of the possibilities of optics. Way cool-call out to MichealG-I think you need to see this!
Regards ross

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#13 Post by josmann » Sat Jan 29, 2022 1:06 am

Rossf wrote:
Sat Jan 29, 2022 12:04 am
Shinya sounds like he had an incredible knowledge and visionary appreciation of the possibilities of optics.
This, my friend, is an understatement! The more I read and learn of this man, the more I think it's a tremendous shame he is not more well known. I always have a ton of respect for people who can operate in what I call "crossover domains." Dr. Inoue operated in a crossover space of optical physics, materials science, and cellular biology. From biology, he could intuit what processes might lead to optically active matter arrangements (for instance, the tubulin fibers of the mitotic spindle aligning to create a birefringent medium) and then he could image them with the masterful and precise manipulation of polarized light - mostly with microscopes he assembled himself (the so-called "Shinya-Scopes"). These stain-free techniques allowed him to record dynamic cellular processes live with electronic video (new at the time) and implement nonlinear filters to boost contrast of weak birefringent effects. I suppose he never invented something so ubiquitously useful as phase contrast and so never garnered the acclaim of Zernike, but he was well known and respected in his field.

The mod I've described here is, frankly, a cute party trick compared to the sum total of what he accomplished through microscopy - he submitted the patent describing this technique before he was 20 years old.

The English translated patent is in "Collected Works of Shinya Inoue." Frankly, most of this 1000 page book is only going to be interesting to a Shinya/PLM enthusiast as it's mostly a sequential series of papers culminating in Dr. Inoue being awarded the International Prize in Biology in 2003. It does come with a DVD which has a video lecture and some powerpoints on it, but I haven't looked at the powperpoints yet since I don't have a DVD drive! "Video Microscopy" is available used for about 7 dollars and is an easy recommendation even if the middle 2/3s of the book is pretty outdated (an EXTENSIVE explanation of analog video and its applications for microscopy). The analog video stuff is bookended by an excellent initial few chapters covering practical microscopy and human vision and a final few chapters on polarized light microscopy for biology. There was a revised edition of video microscopy released after digital video soon took over, but I have not read it and it's more expensive. One thing that makes him an endearing figure is that he always sprinkles in little practical microscopy tips and tricks into his writings. In Video Microscopy, for instance, he describes using a drop of benzene and a kimwipe to clean high-end oil immersion lenses without touching the glass. In "Collected Works" he has an article describing a simple coverslip thickness gauge which doesn't require the use of a micrometer. Some folks recently improved and resurrected this gauge and made a pdf about it: https://www.researchgate.net/profile/Mi ... ckness.pdf

This video is a great introduction to his work:



Here is the appendix to "Collected Works" which includes descriptions of his ShinyaScopes: https://www.worldscientific.com/doi/pdf ... 66_bmatter
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#14 Post by Rossf » Sat Jan 29, 2022 2:46 am

Thanks again josmann -it appears I’ve got some reading and YouTube watching to do!
Cheers ross

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#15 Post by dtsh » Sat Jan 29, 2022 3:41 am

josmann wrote:
Sat Jan 29, 2022 1:06 am
In "Collected Works" he has an article describing a simple coverslip thickness gauge which doesn't require the use of a micrometer. Some folks recently improved and resurrected this gauge and made a pdf about it: https://www.researchgate.net/profile/Mi ... ckness.pdf
There's an elegance in the simple and effective way that operates.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#16 Post by apochronaut » Sat Jan 29, 2022 3:49 am

An astute observer he was. I will definiteley put some time into trying the technique out, Jason. Thanks for the thread!

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#17 Post by josmann » Sat Jan 29, 2022 4:49 am

dtsh wrote:
Sat Jan 29, 2022 3:41 am
There's an elegance in the simple and effective way that operates.
Yeah that's another compelling aspect about him - he was clearly a very practical and resourceful man.
apochronaut wrote:
Sat Jan 29, 2022 3:49 am
An astute observer he was. I will definiteley put some time into trying the technique out, Jason. Thanks for the thread!
If you have the choice, a sliding type head will perform the best for this as the internal optics will maintain polarization perfectly. The key to the clearest image is total extinction of the outer semicircle apertures and that comes naturally for sliding heads. To achieve the same on a folding head, you'll need to custom tailor the polarizer angles based on your IPD. 45 degrees definitely still works pretty darn well as an approximation in my experience, but still it's technically better and easier if you have a sliding head.
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#18 Post by tpruuden » Sun Jan 30, 2022 11:08 am

This may have interesting application for stereo modified DIC - slightly shifted polarisation for left/right eye with active shutter, different/multiangle DIC gradient for left/right eye etc.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#19 Post by josmann » Sun Jan 30, 2022 7:33 pm

tpruuden wrote:
Sun Jan 30, 2022 11:08 am
This may have interesting application for stereo modified DIC - slightly shifted polarisation for left/right eye with active shutter, different/multiangle DIC gradient for left/right eye etc.
Yeah I've had similar thoughts! I do have a DIC system so I'll have to experiment some time. I'm also curious what'll happen if I implement this technique with the semicircle filters behind the DIC analyzer. I suspect the razor thin DoF may yield somewhat unimpressive effects but we'll see!
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#20 Post by tpruuden » Sun Jan 30, 2022 11:43 pm

After DIC analyzer it will not work directly as passive method - the analyzer itself is polarisation filter. It may be possible have additional stereo depth effect if the polarisation angle is slightly rotated, with shuttering periodically one or other eye, offering each eye a little different viewpoint.

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#21 Post by josmann » Mon Jan 31, 2022 1:19 am

tpruuden wrote:
Sun Jan 30, 2022 11:43 pm
After DIC analyzer it will not work directly as passive method - the analyzer itself is polarisation filter. It may be possible have additional stereo depth effect if the polarisation angle is slightly rotated, with shuttering periodically one or other eye, offering each eye a little different viewpoint.
I think the polarizer on the BH2 intermediate tube is oriented at 45 degrees so using horizontal and vertical polarizers should work. That said, you can always use a quarter wave plate to circularize linearly polarized light if necessary.

Edit: Oh actually I forgot one important thing! I believe the last window on the intermediate tube may be a wave plate already. That’s because I’ve not been able to implement this mod on my BH2 with the intermediate tube in place. The eyepiece filters do not extinguish the light at all when this tube is in position :) Haven’t confirmed it yet but it would make sense considering other things I’ve discovered about how polarized light behaves in a binocular viewer.

Edit 2: just confirmed it. If I hold the empty tube in front of my polarized LCD display, there is an orientation I can hold the tube where a linear polarizer does not extinguish the light no matter how you turn it. That means it’s a quarter wave plate.
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#22 Post by patta » Sat Feb 05, 2022 12:43 pm

Inoue' and this mod are great!
But, digging today in Payne's book "Microscope design and construction", A.D. 1957, there is a paragraph (pag. 87) about those "Aperture-splitting binocular system"; and he cites a J.J. Jackson "Stereoscopic vision with the high-power binocular microscope", A.D 1948, for presenting this trick with polarizers. I couldn't find this last paper, it is available only by ordering an USB archive from the youngsters of the Quekett club. Somebody has it?

If we dig deeeper, maybe this method was described by Rayleigh.. or by some Dutch in the 16th century.....

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#23 Post by Chas » Sat Feb 05, 2022 3:26 pm

This is a pretty good overview of various approaches and some other polarising variants:
https://www.quekett.org/wp-content/uplo ... ocular.pdf

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#24 Post by josmann » Sat Feb 05, 2022 5:17 pm

Thanks so much for finding this! If I understand the description of Schulman’s technique (as presented by JJ to the QMC), it’s what amphicyon described - stick the polarizers in the condenser FFP. Still need to try this out myself! The advantages of Shinya’s version as I see it is there is no polarization dependence at the sample and its effectiveness shouldn’t change at higher numerical apertures. Reading that paper Chas posted - it sounds like they encountered related issues with DF/Rheinberg (scattered light doesn’t maintain polarization) which ShinyaVision circumvents. They do, however, mention the adjustment that needs to be made for the different head styles so I guess I can’t claim that as a novel thought :lol:

Polaroid introduced their polarizing film in the early 1930s so I think you wouldn’t see much exactly like this before then considering how much more cumbersome it was to polarize light. There were a lot of subsequent inventions and patents, including many related to implementing 3D vision of various sorts. One other paper Inoue mentions in Video Microscopy is Gaunt and Gaunt 1978 which is supposed to give a rundown on the history of stereoscopic microscopy techniques.
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#25 Post by crb5 » Sat Feb 05, 2022 8:19 pm

Thanks Josmann for your very clear video at https://www.youtube.com/watch?v=z6TUnvAD-Pg. I was wondering how convincing the effect might be given it depends on having a reasonable depth-of-field to get a 3D effect, so I gave it a go with my Amscope 120 with a Siedentopf head using the 4x objective and a marine plankton sample. I could see a 3D effect but it barely covered the thickness of the larger plankton (those > 50 um). The out-of-focus material also contributed to the 3D effect, as commented on by the article posted by Chas https://www.quekett.org/wp-content/uplo ... ocular.pdf. "Moreover, it allows the out-of-focus regions above and below the depth of field to be perceived more clearly such that their presence gives rise to less confusion and adds to the appreciation of three-dimensional structure"

I concur that using darkfield illumination adds a kind of magic, like you are swimming amongst the plankton. However, for this purpose I found using a darkfield condenser on a stereo scope (Amscope 305) at 10x and 30x magnification gave a more impressive effect. Such a condenser is available from Amscope https://amscope.com/products/dk-s but it is easy to make your own from electrical tape and recycled food containers https://www.youtube.com/watch?v=TIXAr5cCDOc&t=18s. Since making this video, I have added a ring of LEDs (50 mm Angel Eyes from Amazon) to supplement the existing light source which gives an even brighter darkfield image. As Josmann explains, it is not possible to illustrate the 3D effect without a fancy stereo camera system, so just try it and see!

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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#26 Post by josmann » Sat Feb 05, 2022 9:35 pm

Glad to hear you tried it out, crb! Did you try with the 10x or higher at all? I had similar concerns about depth and NA but I find the view through a .40 10x no less compelling than a .25 10x (and the enhanced detail makes it better overall I’d say). I think you’re definitely right that a stereoscope will always be better if you can match mag and illumination but the compound scope affords some benefits as well (like 3-axis sample manipulation).

I want to say that the stereo depth is independent of or at least only weakly tied to focus depth since it’s due to the geometric projection from the rear of the objective to the intermediate image. In other words, the depth of focus is reduced at higher NAs but the view doesn't feel flatter. The projection to the intermediate image has a lot less angular variability from objective to objective than the collection angle at the lens. It’s something I need to think about a little more…
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#27 Post by crb5 » Sat Feb 05, 2022 10:42 pm

Josmann
- my first attempt was with a 10X objective but that is before I got hold of some double-sided tape and was using electrical tape to hold the edge of the polaroid. This was not very satisfactory as it tended to snag on the turret when the objective was screwed in, so the polaroids may have moved. However, I could see plankton swimming in and out of focus, so thought 4x would be a better option with my sample when I retried with double-sided tape.

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josmann
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Re: 3D Vision on Compound Microscopes with Shinya Inoue's Forgotten Mod

#28 Post by josmann » Sat Feb 05, 2022 11:17 pm

crb5 wrote:
Sat Feb 05, 2022 10:42 pm
Josmann
- my first attempt was with a 10X objective but that is before I got hold of some double-sided tape and was using electrical tape to hold the edge of the polaroid. This was not very satisfactory as it tended to snag on the turret when the objective was screwed in, so the polaroids may have moved. However, I could see plankton swimming in and out of focus, so thought 4x would be a better option with my sample when I retried with double-sided tape.
Ah I see. Well I think you’ll find the 10x to be pretty unique and mesmerizing if you spend some more time with it. It will definitely test your skill with stage manipulation to follow around speedy critters but things like feeding bdelloid rotifers or heliozoans ominously floating in the void are easy to observe and take in. As I say in the video, make sure your sample is real thick on the slide!

Keep in mind that, when we look at something up close (an ant crawling on your hand, maybe), we also have a pretty sharp depth of focus and it is only by refocusing that we can view something a little closer or farther away. In this case, your fingers have to carry some of the burden that your ciliary muscle would normally shoulder!

By the way thank you for the video about the DF stereo illuminator - I have a B&L stereozoom that I will try that out on some time!
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