Inverted vs Stereoscope or Compound for Pond water organisms

[einman]

Ok. I would like to open up a discussion and get individuals experiences and thoughts on the use of an inverted compared to a stereoscope for examining pond water organisms. Personally, I use both to examine water samples. Stereoscopes have a Larger FOV, a much larger working distance and can benefit from a continuous "zoom" function. However, the numeric aperture of most stereoscopes tends to be much lower than the objectives typically used on an inverted. Although an inverted does not have a "zoom" function, they do have multiple objectives which can be switched in and out.

Inverteds can, in some cases, exceed the performance of even standard compound scopes if the sample is placed in special culture dishes with a glass bottom having a thickness comparable to slightly thicker than a standard cover slip (0.19-0.22 mm) ie such as those sold by Nest Scientific. Others on the forum has made their own as well. Also today's technology allows some "plastic" culture dishes to have optics greater than or equal to standard glass without the cost. The benefit lies in the ability to view organisms without the sample drying out or restricting the organism to the point of causing physical damage. I am speaking of organisms that are not solely microscopic, such as insect larvae, nymphs etc. Limiting the depth of the water sample also improves your ability to utilize the better resolution of an inverted's objectives.

Inverted microscopes can also benefit from DIC, phase contrast, and oblique Technolgies. I am not taking a side as I use both.

Just some thoughts..any experiences, opinions etc?

[zzffnn]

Dear eiman, I thought about building a pond scope for a highly similar purpose:

https://www.microbehunter.com/microscop ... =5&t=17905

To summarize, there are 4 issues that interact with one another:

1) ability to see deeply;
2) pond debris / plants blocking viewing and lighting;
3) requirement for not disturbing pond organisms while viewing;
4) resolution.

I haven’t arrived at one universal solution yet. I am thinking to use 2-3 systems for my own “pond viewing” (which may not work well for others):

For a total magnification of less than 160x or NA of around 0.3: I would use the Wild M450 with or without 2x aux lens, under DIY side lighting (for darkfield / oblique effect).

For a total magnification of above 100x or NA of around 0.25, I would use an upright compound scope with inverted objectives with correction collar and a thin film of minced pond “soup” on a DIY pond slide.

That minced (with scissors) pond “soup” would reduce problem #2. Coupled with an inverted objective with correction collar, problems #1 to #4 would be alleviated.

The depth of that “film” of soup should be as thin as surface tension allows. The correction collar would allow viewing deeper in such a thin film.

It will still dry out, we just have to add water.

My reason is that I don’t want another scope to take up my space, if its advantage is not significant. An inverted scope hardware platform would not solve my issue #1 (of viewing deeply), if my pond organism is a vorticella that suspends in the middle of water column.

An inverted correction collar objective will solve that problem, when used on an upright scope for viewing through the thin “pond soup”.

For my application, I would rather not have cover slip or slide bottom between inverted objective and my subject of interest. I understand inverted objectives with correction collar is ideal for compensating glass thickness and not for compensating water column thickness. However, at low NA of 0.55-0.6 (for 30x-45x inverted objective), I believe that optical difference can be accepted as a compromise.

I did try it with a short Nikon 30x NA 0.55 with correction collar on an upright scope viewing into thin “pond soup” slide; image quality is decent and I was able to view deeply into water. Just play with the correction collar and use as less water as possible. There may be spherical aberration (fog) present; but then I think with a regular inverted scope used the correct way, that optical mismatch when viewing the vorticella still remains (and additional glass layer may exacerbate the fog).

If I want even higher resolution, I can use a water dipping objective with slender tip and long working distance (LOMO 40x NA 0.75) on an upright scope. My pond subject of interest and its environment will be disturbed by movement of the dipping objective though (my issue #3 would be exacerbate, as a compromise to trade for better resolution).

Also: those low NA inverted scope condensers seem very limiting to me. If I resort to using a high NA (~0.9 -1.25) condenser on an inverted scope, the true advantage of an inverted hardware platform quickly diminishes for my application.

That is why, for my own pond scope application , I would probably not use an inverted scope hardware paltform at all. Using inverted objectives on an upright scope may be a good compromise.

[Scarodactyl]

It's rather a shame that Nikon's AZ100 is the only macroscope to ever offer a DIC option. Still, techniques like rotterman contrast can give a very nice and tunable dic-like effect.

[MichaelG.]

Best of both worlds ?
https://bolioptics.com/8x-50x-wf-invert ... ing-light/

MichaelG.

[BramHuntingNematodes]

AN inverted scope is best used to look at things that sink to the bottom so the can press up against the glass, or things you need to access while looking at them. You can get LWD condensers with NA of .5 readily-- they are pretty big and heavy and don't always fit. I use a .4 Nikon condenser when using phase in the inverted. If I wanted higher NA I would use a regular scope to sandwich the specimen between slide and slip to minimize the relief. zzffnn's use case is different from anything I would do, for instance. I think idflan was quite wrong in their assumption that the glass sandwich would not flatten protists, it most definitely does, avoiding which would be the point in looking at them in the middle of an aquarium. So what would you be looking at, is a good question?

[einman]

It's rather a shame that Nikon's AZ100 is the only macroscope to ever offer a DIC option. Still, techniques like rotterman contrast can give a very nice and tunable dic-like effect.

I have photos of that scope and it is quite impressive. I have never actually seen one though. There was a time I looked on E-bay and the web but after a time it eventually gave up.

[einman]

Best of both worlds ?
https://bolioptics.com/8x-50x-wf-invert ... ing-light/

MichaelG.

That is more or less a stereoscope and still restricted by resolution. Im not sure how that would be superior to using a stereoscope with a culture dish other than being a bit more efficient for organisms that tend to drift to the bottom. Shallow culture dishes tend to solve that issue, especially given a stereoscopes LWD. Here is a video without any real preparation other than popping the planaria into a culture dish and making the video. It was a speedy creature so there is no real detail as focusing was a challenge. Even so the Nikon only has a peak resolution of about 0.09 with a 1x objective and 0.18 with a 2x objective something a 10X inverted objective exceeds easily.

[Scarodactyl]

It's rather a shame that Nikon's AZ100 is the only macroscope to ever offer a DIC option. Still, techniques like rotterman contrast can give a very nice and tunable dic-like effect.

I have photos of that scope and it is quite impressive. I have never actually seen one though. There was a time I looked on E-bay and the web but after a time it eventually gave up.

I don't think it was that good a seller. It had amazing options but its optical specs were worse than the Leica Z6 and much worse than the olympus mvx10 or zeiss axiozoom. I've seen two come and go on eBay.

[einman]

AN inverted scope is best used to look at things that sink to the bottom so the can press up against the glass, or things you need to access while looking at them. You can get LWD condensers with NA of .5 readily-- they are pretty big and heavy and don't always fit. I use a .4 Nikon condenser when using phase in the inverted. If I wanted higher NA I would use a regular scope to sandwich the specimen between slide and slip to minimize the relief. zzffnn's use case is different from anything I would do, for instance. I think idflan was quite wrong in their assumption that the glass sandwich would not flatten protists, it most definitely does, avoiding which would be the point in looking at them in the middle of an aquarium. So what would you be looking at, is a good question?

I agree for the most part. If I am wanting to view a protozoan for example I would definitely use an upright and a concave slide. Maybe even a well slide made using washers. In this video I used a stereoscope to locate the specimens than moved it to an AO 120. The sample could have been prepped better and the camera settings optimized. It was taken quite sometime ago. I post it to show how I use the various scopes. Most of my youtube videos are quite old as I stopped making them..due to a housefire etc.



This one used an Olympus IM and the lack of resolution is obvious. But then once again the sample could have been prepped better.

[einman]

It's rather a shame that Nikon's AZ100 is the only macroscope to ever offer a DIC option. Still, techniques like rotterman contrast can give a very nice and tunable dic-like effect.

I have photos of that scope and it is quite impressive. I have never actually seen one though. There was a time I looked on E-bay and the web but after a time it eventually gave up.

I don't think it was that good a seller. It had amazing options but its optical specs were worse than the Leica Z6 and much worse than the olympus mvx10 or zeiss axiozoom. I've seen two come and go on eBay.

That seems to be the case for many of what I consider to be expensive stereoscopes. Leica scopes (MZ-series) have a tendency to be costly with relatively poor specs. You have to purchase a model MZ9.5 or higher to get a resolution higher than say an older Nikon SMZ-U or even a B&L Stereozoom 7.

[einman]

Sorry for the older videos! But it helps to use older videos as a baseline to assess my improvement over the years. I plan on posting again soon.

Anyone with a high end stereoscope such as a leica M165/205 or a comparable Olympus SZX16 or a Zeiss Discovery V12 that we could see images compared to an inverted? Apochronaut--anything with your Bestscope?

[einman]

I will be posting some useful equipment, slides, devices etc for use with inverteds and stereoscopes for pond sample examination. Please do the same. I will start another topic similar to this one but related to entomological studies.

[blekenbleu]

techniques like rotterman contrast

I yet to find a written description of Rotterman Contrast, for which Leica seemingly claims a trademark:

rotterman.jpg (49.69 KiB) Viewed 24184 times

.. from page 11/16 of http://biomarker.hu/sites/default/files ... ure_en.pdf;
Is it basically oblique illumination?

[Scarodactyl]

That seems to be the case for many of what I consider to be expensive stereoscopes. Leica scopes (MZ-series) have a tendency to be costly with relatively poor specs. You have to purchase a model MZ9.5 or higher to get a resolution higher than say an older Nikon SMZ-U or even a B&L Stereozoom 7.

To be fair the lower numbered m/mz series are not really research stereos while the smz-u definitely is and the sz7 is at least a swipe in that direction. The smz-u would have been meant more to go up against the m10 or mz apo/mz12 at the time.

Rotterman is just a tunable oblique technique. It's nice but can be achieved more affordably.

[Scoper]

For pond viewing, what magnifications are most useful?

I would love to see the setups others are using..what works, what doesn’t

[einman]

That seems to be the case for many of what I consider to be expensive stereoscopes. Leica scopes (MZ-series) have a tendency to be costly with relatively poor specs. You have to purchase a model MZ9.5 or higher to get a resolution higher than say an older Nikon SMZ-U or even a B&L Stereozoom 7.

To be fair the lower numbered m/mz series are not really research stereos while the smz-u definitely is and the sz7 is at least a swipe in that direction. The smz-u would have been meant more to go up against the m10 or mz apo/mz12 at the time.

Rotterman is just a tunable oblique technique. It's nice but can be achieved more affordably.

When comparing the SMZ-U to the MZ series ( I own a Leica M80 which I love, just a disclosure that I am not biased against Leica) I was primarily comparing price per performance. A good used SMZ-U can be had for significantly less than even a Leica MZ7.5. I believe the MZ12 came out round about ~1999 maybe a little sooner, whereas the SMZ-U came out in 1990 so I don't think it was meant to compete against the MZ12. The MZ12 did not exist yet at the time. Do you have any literature on the MZ12? I would love to add it to my collection and table of specs I have been creating for Stereoscopes. Perhaps the M10 would have been a target, although I have never seen actual specs, but was told at one time the na was around 0.125 with a planapo objective. I have never been able to confirm that via any Leica/Wild literature. I am suspicious of those numbers because that is the same spec as the MZ12.5 which came along much later. Feel free to correct any of my comments with literature. I am writing a paper on stereoscopes and collecting all the data I can get.

Don't get me wrong I have invested a great deal in Leica stands lenses etc for my Leica M80 and love Leica's designs. They are just overpriced with respect to the "used" market. When comparing utility of an inverted vs a stereoscope I think price should be considered as well.

[einman]

For pond viewing, what magnifications are most useful?

I would love to see the setups others are using..what works, what doesn’t

I generally use the lower magnifications with 20X being my sweet spot. Primarily because I view live specimens and the higher magnifications tend to require greater restrictions on the organism to prevent them from leaving the FOV. This is especially true if using culture dishes. Concave slides are not as difficult, but then you start to wonder how the restricting the environment impacts their behavior. "“What we observe is not nature itself, but nature exposed to our method of questioning.” ― Werner Heisenberg. Quite another topic. I will post some photos of my Inverted scopes and Stereoscopes once I finish putting my lab back together.

[charlie g]

Hi all, the foot print at your microscopy bench is huge...for an inverted microscope, and the use of a stereo scope has a strict 'sweet spot' of magnification which is useless for most protozoa and protist algae.

A good Plan: 1X, Plan 2X, Plan 4X objective on a trinoc-cmpd scope , and 6X oculars (as well as your standard 10X, 12X oculars) are excellent for Meiofauna such as flatworms, water fleas, insect aquatics, water mites...finger nail clams....etc., etc....meiofauna.


It is simply a tease to delve into pond water biology observations with a stereo scope...and the inverted scope has a strict mission...culture vessel studies.. you can not offer pond life the freedom of vast volumes of sample water, and expect good observations of the variety of phyla , as they react with irritation to being sampled.

The depth of waters under a coverslip does not distort meiofauna to the point of abnormal morphology for hydras, flat worms, water mites, microcrusteaceans, tuberflix worms...an inverted stand offers so many obstacles to crisp meiofauna observations, to image captures to share...too much sample volume for live and irritated organisms to frantically move about in..as you attempt good observations of pond life.

Think about low-magnification Plan objectives...think about methylcellulose viscosity tool mixed with your pond life sample: 50/50 by volume.

The water thickness under a coverslip can be huge...learn how to 'feed waters to a wetmount slide' with a fine point dropper...to enjoy observing for hours.

I have an Olympus CKA inverted microscope which I am willing to part with...very low...very low cost...but with it's foot print..shipping even con-US would be high.

Stereo microscopes have a 'sweet spot magnification range ' which is simply a tease for comfortable real world appreciation of what is going on in our surrounding: 'microscopy world views'.

all the best, charlie g/ fingerlakes/US

[einman]

All excellent comments Charlie and, in fact, where most of my thoughts lie. The bench space is definitely something to be considered. Slowing down specimens via CMC etc is great for the organism's anatomy but still interferes with its natural behavior. You can anesthetize them as well if behavior is not a consideration. I think the size of the organism you wish to observe is definitely a variable in choosing the stand. I have found compound plan 1x objectives to be a bit pricey if from any of the big 4.

[Scarodactyl]

Oh yeah, I just wasn't sure which of the m series would be contemporary. No question the leicas are priced higher and I like the smz-u quite a bit. I've never done a real head to head, and maybe I should since I somehow still have an smz-u on hand. The m10 having the same specs as the mz12 would definitely track, I think I read literature somewhere but don't remember if I still have it.
I will say the Leica rotterman system easily beats the Nikon OCC base I tried.

A higher end stereo with a higher mag objective shouldn't struggle too badly to reach 20x equivalent but it wouldn't be cheap.

[einman]

Any experience with using inverted LWD objectives on an upright stand? Any benefits? I know most of us have probably tried it at one time or another. I have done it for photography where I wanted higher resolution on an entomological specimen such as an ant. Although I eventually eliminated the stand altogether and went with objectives mounted on this aparatus:






Of course the camera has to be tethered to a computer to "see", taking the place of the eyepieces of an upright stand.

[Hobbyst46]

Any experience with using inverted LWD objectives on an upright stand? Any benefits? I know most of us have probably tried it at one time or another.

I happened to test an Olympus BH2 that had been fitted with a 10X DPlan and 60X Planapo objectives as well as a 40X0.55 LWD. The image from the LWD was OK, although somewhat lacking contrast. The big issue was the (expected) lack of parfocality with the other objectives.

[macnmotion]

For pond viewing, what magnifications are most useful?

I would love to see the setups others are using..what works, what doesn’t

I've been shooting fresh and marine water organisms with an inverted scope using 10x and 40x plan phase contrast, and 40x APO oil. Because that's what I have I'd love a 20x, will be getting one. I'm pretty new at this all, you're welcome to take a look at my results on my site: thaimicrocosmos.com

[MichaelG.]

Best of both worlds ?
https://bolioptics.com/8x-50x-wf-invert ... ing-light/

MichaelG.

That is more or less a stereoscope and still restricted by resolution. […]

Apologies for my misunderstanding of your priorities … I had presumed that the stereoscopic view would offer a significant advantage over the usual inverted, which is simply binocular.

MichaelG.

[einman]

MichaelG- No apologies necessary. We are simply exploring the plus and minuses of both instruments. My comment regarding the instrument you mentioned was intended to say I saw no added advantage of verse a regular stereoscope other than perhaps being able to see organisms at the bottom of a container a bit better which for me has not been an issue due to their huge working distance. There is no wrong, right, better or worse comment. Just sharing ideas and opinions.

[einman]

Any experience with using inverted LWD objectives on an upright stand? Any benefits? I know most of us have probably tried it at one time or another.

I happened to test an Olympus BH2 that had been fitted with a 10X DPlan and 60X Planapo objectives as well as a 40X0.55 LWD. The image from the LWD was OK, although somewhat lacking contrast. The big issue was the (expected) lack of parfocality with the other objectives.

ahhh..lack of contrast. I have noticed that as well with the objectives on my Olympus IM and AO Biostar. I recently noticed a lack of contrast on a Nikon LWD objective, which looks like new, when mounted on a Leitz. At times I wondered if it was just my specific objectives (age related etc) or a property of LWD in general. That was until I received a Zeiss invertoskop which has incredible contrast. in regards to teh parfocality I was thinking of loading an upright stand with all LWD objectives for just that reason. Try it for a while.

[zzffnn]

Lack of contrast (what I referred to as “fog” above) may be caused by spherical aberrations (for example, optical mismatch between glass / water depth). Correct collar and controlling water depth will help.

[einman]

Lack of contrast (what I referred to as “fog” above) may be caused by spherical aberrations (for example, optical mismatch between glass / water depth). Correct collar and controlling water depth will help.

Thanks Fan! I had read that the "fog" could be related to an optical mismatch. It is for this reason I never gave up on using inverteds or LWD objectives. How are those Nikon Eyepieces working for you?

[zzffnn]

They work very well (and almost perfectly parfocal) on my Wild M450. Thank you!

[MichaelG.]

Rotterman is just a tunable oblique technique.

It’s new to me … but I’m intrigued
I have just found this crumb of information on the Leica site:
https://www.leica-microsystems.com/prod ... tl4000-rc/

MichaelG.
.

Edit: __ I don’t use Facebook, but this is worth a look:
https://www.facebook.com/nuhsbaum/photo ... 179755767/

as is this, from Leica: https://youtu.be/qD5ew109KZs
… to me, this is barely recognisable as a microscope