Poor man's polarized light microscopy

[Hobbyst46]

A demonstration with a microscope that has no polarized light gadgets.
A piece of linear polarizing plastic sheet inside the 10X barrel (Olympus objective on Zeiss scope - hence some CA).
Another piece of polarizing material (rotatable) on the field diaphragm.

The specimen is an anti-headache pill dissolved in ethanol. Filtration and pouring onto slide. A 10 minutes preparation.
Three different rotations of the polarizer.
Single images, resized, cropped, the contrast enhanced by 20% in software.
Alas, the actual view was much sharper and contrast.
Most fascinating is the drying of the solvent and disappearance of the black fjords among the crystals - apt for a movie clip.

[75RR]

Well it works but I think you will get a sharper and better image if you place the analyzer like so:

It does not block too much light (couple of stops perhaps) so you can just leave it in.

[Hobbyst46]

Well it works but I think you will get a sharper and better image if you place the analyzer like so...It does not block too much light (couple of stops perhaps) so you can just leave it in.

Thanks a lot for the idea!! Never crossed my mind.
I will give it a try. My LED light is so intense that I often use ND filters. Since POL is done with the low mag objectives it looks promising and become independent of a specific objective...

Incidentally, did you draw these nice figures ?

[75RR]

Incidentally, did you draw these nice figures ?

No credit due, just modified 'borrowed' drawings. Such is the basis of most graphic design ;)

[Hobbyst46]

Well it works but I think you will get a sharper and better image if you place the analyzer like so:...

Done! and it works fine.
The sharpness is the same as before. Through the eyepieces they look razor sharp, and nice colors, but my photos lack sharpness and contrast. Either the combination: (camera)+(15-45mm zoom lens)+(Zeiss 8X KPL as photo eyepiece) is not optimal, or camera settings are not optimal, or both...
Camera shutter shake ? the camera is mounted on the trino phototube with adapters. Disconnecting them and mounting the camera above the phototube, in air, is not possible in my workspace.

At least now, I have POL with several objectives.
Thanks again for the good, useful idea!

[75RR]

Camera shutter shake ? the camera is mounted on the trino phototube with adapters. Disconnecting them and mounting the camera above the phototube, in air, is not possible in my workspace.

Old image of what was my Standard RA at the time. Still use the same setup though with my WL. Vibration free!

[MicroBob]

Hi Doron,
thank you for showing!

How did you focus the camera? I can imagine that the polarized light might influence the auto focus.
These crystals are not flat, they have more height than can be photographed in one image.

Some (not all, different systems) 3D-cinema glasses have flat circular polarizers in them. They can be used for this purpose.

Bob

[Hobbyst46]

Hi Bob,

How did you focus the camera? I can imagine that the polarized light might influence the auto focus.

I always focus the camera manually. This camera only has its small screen, no optical viewfinder. The details in focus are suggested by the camera and are shown by thin red traces. For easier focusing I use the Camera Connect remote application so I can focus with a 10" tablet. I do not know if a linear polarizer, apart from being a sort of ND filter, influences the sensor of a mirrorless camera. Can check it with a stage micrometer.

These crystals are not flat, they have more height than can be photographed in one image.

Yes. That was just a preliminary test.

Some (not all, different systems) 3D-cinema glasses have flat circular polarizers in them. They can be used for this purpose.

Yes. I have a pair of these. If the linear polarizer affects focus I can try them instead.
Thanks!

[MicroBob]

Hi Doron,
some of these modern mirrorless system cameras have two AF systems, both based on the sensor data. One is contrast, precise but slow, the other is "phase", more like a DSLR. Maybe this influences this red trace focussing aid. Many cameras offer the enlargement of part of the screen to focus manually. It is very likely that yours offer this too. May be you have to configure it in the menu.

Linear or circular polarizer won't explain the somewhat unsharp images. AFAIK a circular polarizer is a linear one with a retarder added.
Photography polarizers are the most likely filters to adversely affect image quality because often they are made from several layers. My 3D glasses offered acceptable image quality. Maybe simple plastic foil is less critical because it is just one layer?
I have a small plastic bag with used 3D glasses because I instructed my sons to bring them home when they went to the cinema.

Bob

[Hobbyst46]

Many cameras offer the enlargement of part of the screen to focus manually. It is very likely that yours offer this too. May be you have to configure it in the menu

Thanks a lot for the tip!!
there is no need to configure it in the menu. It works, although cumbersome, because it involves several key strokes at a certain order, but it does improve focusing when using the camera without tablet. It provides up to a 10X magnification "sight". It cannot be used with the tablet though. With the tablet is easier to focus, but magnification is only by a factor of 6-7. So now I have two options. (there is actually a third option - using an HDMI monitor, but I do not have one).

Maybe simple plastic foil is less critical because it is just one layer?

This is exactly what I am using - the simple linear polarizing foil. Rather than the circular. Which is better - remains to be seen...

Linear or circular polarizer won't explain the somewhat unsharp images.

Yes, this is true for my setup. Just verified that the linear has no effect on the focusing. It only decreases the brightness by a small fraction, but it is not a problem since the illumination source is very bright.

[75RR]

Not sure if vibration is part of the problem (unsharp images) but in Afocal method (I assume you are using the afocal Method?) the camera should be focused at infinity.
This is an extract from Charles Krebs' Basic Considerations When Mounting a Camera on a Trinocular Tube

Afocal:
"A 'regular' eyepiece is inserted into the trinocular tube. In this case it is not
“raised”, and it should be in focus at the same time as the binocular viewing
eyepieces. Then, a camera with an attached lens (focused at "infinity") is located
very close and directly over the trinocular eyepiece. This can provide excellent
images, but it can be tricky to get a camera lens that can be positioned close
enough to the eyepiece to get a full image with no vignetting. Most lenses are
too large, and the entrance pupil is set back too far into the lens to "couple" well
with virtual image created by the eyepiece. Eyepieces with a "high eyepoint"
design can help."

http://www.krebsmicro.com/pdf/trinoc_a3.pdf

[Hobbyst46]

Not sure if vibration is part of the problem (unsharp images) but in Afocal method (I assume you are using the afocal Method?) the camera should be focused at infinity.

Hi 75RR and thanks for the comments,

Yes, I am doing afocal. Tried focal in the past, but CA as well as coverage of the FOV by the camera sensor were worse.

Focusing the camera at infinity: This camera has only electronic focusing, nothing mechanical. I have combed the web and other sources to find out how to focus at infinity with such camera, but found nothing. I assume, that the default setting of the camera in focus at infinity.

...Charles Krebs' Basic Considerations When Mounting a Camera on a Trinocular Tube
Afocal:
"A 'regular' eyepiece is inserted into the trinocular tube. In this case it is not
“raised”, and it should be in focus at the same time as the binocular viewing
eyepieces. Then, a camera with an attached lens (focused at "infinity") is located
very close and directly over the trinocular eyepiece. This can provide excellent
images, but it can be tricky to get a camera lens that can be positioned close
enough to the eyepiece to get a full image with no vignetting. Most lenses are
too large, and the entrance pupil is set back too far into the lens to "couple" well
with virtual image created by the eyepiece. Eyepieces with a "high eyepoint"
design can help."

This is the arrangement that I adopted when I first mounted the camera. The zoom lens of my camera is only slightly set back. So indeed, I have located the camera very close and directly over the trinocular eyepiece. Most (I think close to 90%) of the FOV is seen by the sensor.

Parfocality between the eyepieces and the camera screen: Is not perfect, but I think it is acceptable. For example:
When I focus (very carefully) on a target by eyepiece view, then modify it to get exactly the same focus on the camera screen, the difference is 56 micrometer for the 10X0.30 objective, and 28 micrometer for the 16X0.40 objective. Do you think it is a problem?

Camera vibration: always a concern. your setup with the tripod looks good, unfortunately it is not practical at my facility. That is one reason why I bought the mirrorless camera.

[75RR]

What camera do you have?
Does it not have a manual setting?

[Hobbyst46]

What camera do you have?
Does it not have a manual setting?

Canon EOS M10 camera. It has manual focus, which I practice exclusively, and also a manual exposure mode, which I do not use. Rather, I use shutter priority mode. To avoid interference from the LED light, I shoot at shutter speeds <250 (longer than 1/250).

[75RR]

I have an olympus m4/3 camera which I use in manual mode with a legacy lens (Olympus OM) via an adapter, which I focus manually on infinity.

https://www.amazon.com/Fotasy-Canon-Mir ... FD+adapter

You would need to figure out which manual lens to get that would go best with it. (Area of Field of view coverage)

[Hobbyst46]

I have an olympus m4/3 camera which I use in manual mode with a legacy lens (Olympus OM) via an adapter, which I focus manually on infinity.

https://www.amazon.com/Fotasy-Canon-Mir ... FD+adapter

You would need to figure out which manual lens to get that would go best with it. (Area of Field of view coverage)

Thanks for the link.
In fact, having read in the past that a prime camera lens might be best, I have already tried the option: A Canon EOS-Minolta MD lens adapter, onto which goes a Minolta lens: The prime MD Minolta, 50mm 1:1.7. It was rather difficult to attach it afocally, precisely because the front glass surface of the MD lens is deeply set back in the barrel, but I managed. I manually focused the camera at infinity and shot test photos in the manual exposure mode. However, the results were disappointing - not as good field of view coverage and intense CA. So I gave it up.

I have also read that in two or three different afocal setups of this sort, an Olympus Zuiko prime 50mm lens performed well.

Based on other poeple's experience, I feel that finding the appropriate old mechanical-focus, manual-mode SLR prime lens for afocal photomicrography is a hit-or-miss project. However, I will have to buy each lens for trying...perhaps I should try and locate an inexpensive manual Olympus prime lens locally.

[75RR]

I manually focused the camera at infinity and shot test photos in the manual exposure mode. However, the results were disappointing - not as good field of view coverage and intense CA. So I gave it up.

Focusing on the sharpness first (pun) you need some way to adjust the height of the camera - fine tuning the height is critical to a sharp image.

[Hobbyst46]

I manually focused the camera at infinity and shot test photos in the manual exposure mode. However, the results were disappointing - not as good field of view coverage and intense CA. So I gave it up.

Focusing on the sharpness first (pun) you need some way to adjust the height of the camera - fine tuning the height is critical to a sharp image.

OK, thanks again. I will draw a scheme of the optical path of my setup and post it, to see what should be modified.

BUT I am afraid that my description of the focusing procedure was somewhat misleading, so let me clarify:
1. The camera and eyepieces are set to optimal parfocality as far as I can. This includes:
setting the distance (a few mm) between the photo eyepiece (an ordinary eyepiece inserted into the phototube) and the top end of the
phototube,
setting the distance (a few mm) between the front glass surface of the camera lens and photo eyepiece,
setting the interpupilary distance and the corresponding angular position of the viewing eyepieces, this is done because it is the old type
black 45 degrees trinocular head.

The result is parfocality within 56 microns with the 10X objective, 28 microns with the 16X objective.

2. The camera is configured to Manual Focus.

3. With a specimen under the objective, I focus first by viewing.

4. I switch to camera view (it is a 0/100, 100/0 head) and tweak the focus with the microscope according to the camera screen. I never focus with the camera (so I believe that, from the viewpoint of the camera processor, the focus never changes).

[Hobbyst46]

Hi Doron,
Many cameras offer the enlargement of part of the screen to focus manually. It is very likely that yours offer this too.

Thanks for this reminder!! I never cared for this feature before ( )...since yesterday I have adopted this method and it already improved the sharpness. Hopefully will demonstrate it in a post soon!

[75RR]

I will draw a scheme of the optical path of my setup and post it, to see what should be modified.

That sounds like the best approach.

[Hobbyst46]

...That sounds like the best approach.

Here is a scheme and photo of my gear.
Starting from the camera, a Canon EOS-M10 with zoom lens, filter thread is female M49. Then, 1.25" to 49(male)x0.75mm telescope adapter. It sits on the photo tube, stabilized by means of the screws but indirectly, over a PVC spacer sleeve (grey-silver color in the photo).
The photo eyepiece is a Zeiss 8x18KPL eyepiece, of ~7mm eyepoint, inside the adapter and raised somewhat relative to the photo tube.
So the only optics is the photo eyepiece and the camera zoom lens. The zoom lens is fixed at a focal length of 45 mm, with piece of adhesive tape. The photo eyepiece is very close to the front glass end of the camera lens, so a soft rubber annular spacer is placed in between, to protect.

[75RR]

Nice and detailed drawing!

I suppose that the first thing to look at is camera vibration.
This is because if it exists then it will have to be solved first as it will negate all the other improvements.

My camera, an Olympus EP2 is also mirrorless and yet it kicks like a mule!

Easiest way to tell is to place a wet mount slide (cover slip with the ‘normal’ amount of water under it) on the stage (trinocular switch set to let light to the binocular head) and activate the camera shutter.
That way you can actually see if there is a visible wobble.
The sort of wobble you are looking for is similar to what you get if you tap the microscope with your finger.
If there is wobble you can try with a mount with less water and see if that helps.
If there is still a visible wobble then you will need to consider ways to isolate the camera from the microscope.

I used to have the camera permanently set up on the tripod but I don’t have the space at the moment for that as both my computer and the microscope have to take turns on the same desk.
Nevertheless, moving the monitor and setting up the microscope and tripod for a session does not take long.

Alternatives to a tripod include an enlarger copy-board or mounting a bracket on a shelf or on a wall that can hold the camera.
The next stage assuming there was no vibration, or if there was it has been dealt with, is setting the camera at infinity and camera positioning.

To set the camera at infinity it is best not to trust the focusing dial readings too much as they are not precise enough.
Much better to place the camera (provisionally) on a tripod and focus manually at an object in the distance and then tape the focusing dial down so that it remains fixed at this position.

Assuming that the trinocular port is parfocal with the binoculars (if not it should be so arranged), then the camera focusing is achieved by fine vertical movement of the camera, usually positioned just a few mm above the eyepiece.

[Hobbyst46]

Many thanks 75RR for the detailed methodical layout!

an Olympus EP2 is also mirrorless and yet it kicks like a mule!

Important info - I did not know that a mirrorless behaves such.

Easiest way to tell is to place a wet mount slide (cover slip with the ‘normal’ amount of water under it) on the stage (trinocular switch set to let light to the binocular head) and activate the camera shutter.

Done. There are no shutter-induced vibrations. In addition, the camera trigger is extremely sensitive: just moving my fingertip very close to the camera screen, without actually touching it, activates the shutter.

I used to have the camera permanently set up on the tripod...moving the monitor and setting up the microscope...
Alternatives to a tripod include an enlarger copy-board or mounting a bracket on a shelf or on a wall that can hold the camera.

My Microscope and computer and monitor are all on the same desk. This is a must.
If I tap the desk, there are visible vibrations, but I take care to work without such... anyway, as an additional remedy, I have now placed a 7mm thick flat rubber foam pieces under the microscope. These visibly dampen desk-induced vibrations. The shelf bracket is another option, if needed.

The next stage...is setting the camera at infinity and camera positioning. To set the camera at infinity it is best not to trust the focusing dial readings too much as they are not precise enough. Much better to place the camera (provisionally) on a tripod and focus manually at an object in the distance and then tape the focusing dial down so that it remains fixed at this position.

That answers my previous question about the infinity focus. Will try it.

Assuming that the trinocular port is parfocal with the binoculars (if not it should be so arranged),

yes, it is.

then the camera focusing is achieved by fine vertical movement of the camera, usually positioned just a few mm above the eyepiece.

I assume that this recommendation is related to the question added in green fonts on the drawing - can the distance be changed? I have fixed it once, at what seemed to be the correct height; namely, both the camera and eyeview were in focus. This I should perhaps re-check, because the adapters on the photo tube are not rock-stable. But the space cannot be easily adjusted: there is no focusing aid, like a helical tube or telescopic tube, to provide smooth easy movement of the camera.

[wporter]

Greetings Hobbyst46,

Looking at your images, they seem to have a very slight out-of-roundness to spots, indicative of motion during exposure, rather than poor focusing.

Apparatus vibration can be a perplexing issue. It dogged me for a while until I upped the shutter speed until it went away. And this was with a mirrorless camera (Oly OM-D EM5II) set on it's anti-vibe settings (this helped), the use of a remote shutter release cable (also helpful), and coupled solidly to the chassis of a 120-pound microscope (my Univar; so the scope whipping around presumably was not an issue). The thing is, all cameras will vibrate internally somewhat; in my case I think it was significant.

Shooting at a higher shutter speed solves many problems; all you need is more light (a flash would do very well).

The issue of inadequate focusing seems to be solved by your using a monitor w/ magnification.

I would also encourage you to get a real photo eyepiece for your Zeiss, such as a FK 10x. They are made to flatten out the field for projection onto a flat film surface; regular eyepieces are made for planarity for curved retinas. (I realize you are shooting afocally, but if you used a photo eyepiece, you could get rid of the camera lens from your optical train.)

Keep up the good work.

[Hobbyst46]

Thanks wporter,

Looking at your images, they seem to have a very slight out-of-roundness to spots, indicative of motion during exposure, rather than poor focusing.

Please, can you lead me to a point where this effect is visible? I do not recognize it, probably do not know how to find it..

Apparatus vibration can be a perplexing issue. It dogged me for a while until I upped the shutter speed until it went away. And this was with a mirrorless camera (Oly OM-D EM5II) set on it's anti-vibe settings (this helped)

I presume you do not mean the camera picture stabilizer? because it exists within the lens, is continuously ON, and I doubt that it matters. But yours and 75RR's previous message about vibrations with mirrorless cameras - WOW. I never expected it.

...the use of a remote shutter release cable (also helpful),

I guess that remote triggering by a WiFi device is better than a mechanical cable.

and coupled solidly to the chassis of a 120-pound microscope (my Univar; so the scope whipping around presumably was not an issue).

Important note! I googled Univar, to learn that my years-ago Reichert Zetopan, whom I thought was heavy weight, was a toy scope by comparison. Owning a heavyweight scope on my desk is beyond my means. Side note - I became aware of your nice website (under construction, right? no pictures yet?).

Shooting at a higher shutter speed solves many problems; all you need is more light (a flash would do very well).

The subject of shutter speed on the microscope has been confusing to me. Intuitively, I would always strive to shoot at highest speeds. But, there are comments in forums that slow speeds are advantageous, because then, triggering occurs after a delay, by virtue of which, the scope's vibrations have faded away.
So which is better (assuming that light and lens apertures permit)? 1/250 or 1/15 (say)? Adding a flash will be an interesting project...

I would also encourage you to get a real photo eyepiece for your Zeiss, such as a FK 10x. They are made to flatten out the field for projection onto a flat film surface; regular eyepieces are made for planarity for curved retinas. (I realize you are shooting afocally, but if you used a photo eyepiece, you could get rid of the camera lens from your optical train.)

I have often seen Zeiss photography adapters, like 0,63 (or some like) but they are rare and expensive and maybe won't fit my trino head. I do not find an FK10X eyepiece in the Zeiss catalogues for the GFL, WL models, I guess there were fil;m camera adapters with 0.6X relay lenses rather than photo eyepieces. Dave Walker has recommended 10X KPL or 8X KPL and I followed suit. I will try to locate an FK 10X photo eyepiece.
In fact, this whole photography setup yields some CA as well, which is a further reason to look for a modification.
thanks again.

[wporter]

Below is an enlarged screenshot of an area in the first image. Notice how it seems smeared in one direction. Could indicate camera movement, or just the structure of the crystals at that area, so I may be wrong.

The Oly camera I use has settings one can set, to provide a delay after the shutter is tripped until an electronic front-curtain shutter actually trips, so as to let vibration die down. I use 1/2 sec. It is called "Anti-shock" and is made for microscopes and telescopes. It is separate from the image stabilization feature of the camera or its lens.

My shutter release cord is a simple electrical one, no wi-fi.

No, my website is lagging far behind reality. Maybe soon some pix.

I don't buy it that a slow shutter-speed is better. Maybe if the light is so dim, and the exposure is a 'time' exposure of many seconds, so that most of the exposure is captured after the vibes die down, but in my humble experience I've gotten nothing but crisper pictures shooting faster (> 1/200 sec).

I have a FK10x eyepiece (called by Zeiss a "photocompensating" eyepiece), but haven't had it long and haven't shot any images through it yet. So I better shut up about it's glories. Maybe I'll do some trials for the forum to see it it is really better than a KPL10x.

The eyepiece is mentioned on page 24 of the GFL manual referenced at this site:

http://www.micromagus.net/microscopes/zeiss_gfl.html

It would go into the top of the trino head tube, and the lensless camera would be placed over it (hard-coupled or not to the scope).

[Hobbyst46]

Thanks wporter for all answers. I appreciate them a lot.
An "anti shock" delay is an option on my camera too.

[Hobbyst46]

Hello all,
I am looking for a photo compensating ocular from Zeiss, FK10X, mentioned in a catalogue, but it seems to be between rare and extinct (?).
On the other hand, I occasionally see listings of LOMO eyepieces, like this for example:
"LOMO Compensating eyepiece K 6,3x /inf. microscope ZEISS..." and similar ones. They are specified to be 23.2mm diamater, so will mechanically fit.

Does anyone know if such eyepieces are worth trying on the Zeiss? as photo eyepieces in the trino head, for afocal/focal shooting? I am using KPL's right now.

[75RR]

Hello all,
I am looking for a photo compensating ocular from Zeiss, FK10X, mentioned in a catalogue, but it seems to be between rare and extinct (?).
On the other hand, I occasionally see listings of LOMO eyepieces, like this for example:
"LOMO Compensating eyepiece K 6,3x /inf. microscope ZEISS..." and similar ones. They are specified to be 23.2mm diamater, so will mechanically fit.

Does anyone know if such eyepieces are worth trying on the Zeiss? as photo eyepieces in the trino head, for afocal/focal shooting? I am using KPL's right now.

Nothing wrong with wanting to try out other eyepieces.
Would just like to say however that the problem with your pol images is not related to the Zeiss 8x18KPL eyepiece you are using now.
I have used a 'normal' 12,5x/18 KPL eyepiece for both afocal and projected imaging until recently with what I consider to be very good results.
Now using a Mipro/Projekiv f=63mm
See image for both:

[Hobbyst46]

... the problem with your pol images is not related to the Zeiss 8x18KPL eyepiece you are using now.
I have used a 'normal' 12,5x/18 KPL eyepiece for both afocal and projected imaging until recently with what I consider to be very good results.
Now using a Mipro/Projekiv f=63mm
See image for both:

I think that at least some of the CA in results - not the POL maybe, but others - might have been caused by the photo eyepiece, although other factors might have contributed as well. And I hope to succeed with direct projection in the future, so...
Please tell if you see any visual advantage of the Mipro. These lenses I have seen here and there on eBay, but ignored them for the time being (prices being the main reason).

Incidentally, I have just finished a major overall of the photo setup, as follows:

1. Took apart all items installed on the head
2. Replaced the free-floating, curving piece of polarizer sheet with a more sturdy polarizer - the same piece but fitted inside a thin aluminum ring which, in turn, is forcefully fixed into the dovetail of the head - so the polarizer is flat and perpendicular to the optical axis
3. Set the camera to focus at infinity, using the autofocus feature and pointing at a remote road sign
4. Taped the focusing ring on the camera, to fix it in place, like I had done to the zoom ring
5. Seated the 8X KPL photo eyepiece into the head tube and verified parfocality between the photo eyepiece and viewing eyepieces
6. Improved the contact between the PVC spacer sleeve and the 1.25"->49mm telescope adapter, by using steel shims (~0.1mm thick) instead of the cellotape I had used before
7. Verified the height of the eyepoint above the photo eyepiece, by locating the smallest image
8. Attached the camera back to the adapter --- and was rewarded with exact parfocality between the camera and viewing eyepieces, better than I had before. This I check with a stage micrometer.
I added thick rubber foam stand-offs beneath the scope, to better eliminate desk-induced vibrations.
Will proceed to real-life tests...

P.S. what are the greyish spacer (?) rings in your photo used for? height limiters on a photo tube? I see that their heights are according to a series 1,2,4,...etc, to enable all height combinations?