Microscope for diatom identification work?

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Rorschach
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Microscope for diatom identification work?

#1 Post by Rorschach » Sun Mar 26, 2023 11:02 am

Hi people,

A friend of mine has asked for help in choosing a good microscope system for identifying diatoms (to species level). This would be for work and research purposes but will be paid by herself - so funds are not unlimited.

I do know that DIC would be perhaps the best method for diatom work but those systems tend to be prohibitively expensive, even used. Therefore, regular phase contrast and/or darkfield seem like the potential ways to go? However, I have never used either in a transmitted light microscopy setting. That's why I decided to ask here - there seems to be very knowledgeable folks on just about any topic.

I know that Olympus has very good and popular (and relatively recent) models, some with infinity optics but, alas, due to being so popular, prices seem to have gone quite high.

One option could be to build her a system around a spare Leitz Metalloplan frame that I have around as an extra. I think I may even have some phaco objectives around for it and an extra objective revolver, need to check. One of the benefits of the Orthoplan/Metalloplan approach, in addition to that being at hand, is the extreme modularity of it. Various options could quite easily be added later on if need arises. But which condenser should I get for the phaco setup? And is there something to look out for, like missing annuli etc.? I do know that for the higher power objectives, above and including 40x, Planapos would be preferable while lower power ones can be just plan or even achromats. And what about darkfield: which condenser and objectives to go for? Darkfield parts seem to be not that easy to find for the Orthoplan.

Other proposals are also welcome. But please, no chinese etc. models.

Best regards,
Riku

Hobbyst46
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Re: Microscope for diatom identification work?

#2 Post by Hobbyst46 » Sun Mar 26, 2023 12:12 pm

IMHO a light microscope cannot provide identification of all diatoms at species levels. Whatever illumination is used.

I would think of two alternatives for your friend.

1. Buy a second hand scanning electron microscope (SEM). I do not know about availability, prices and maintenance costs, but even a humble model will provide the necessary resolution in this case. However, since institutes tend to refresh their equipment arsenal from time to time, and relatively few amateurs are on the hunt for SEMs, there might be a chance of finding a SEM for a reasonable price - I believe.

2. If diatom identification is only needed infrequently, seek a service contract with a certified expert laboratory that carries a SEM and can at least provide the images for later identification.

The above are mentioned on the assumption that your friend has the diatoms as clean frustules. Otherwise, she will have to clean and mount them on the specimen carrier ("stub") for SEM. Cleaning diatoms for SEM does not differ much from cleaning them for optical microscopy - in fact, it might be even slightly easier. Moreover, mounting for SEM is easier than mounting for optical microscopy. IMHO.

At any rate, a good quality trinocular phase contrast microscope, even a 50-years old model, with 40X dry, and 60X or 100X oil immersion objectives, would be very useful for at least a primary classification of the diatom, hopefully at the genus level. Oblique illumination is easy to achieve, anyway, and is useful. Darkfield is nice to have. Yet I would consider phase contrast a must in this case. DIC might be an alternative, but it is very expensive and, again, not always sufficient for species ID.

PeteM
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Re: Microscope for diatom identification work?

#3 Post by PeteM » Sun Mar 26, 2023 1:54 pm

What is your friend's budget?

I don't know the requirements for diatom identification other than resolution and the desirability of some method of adding contrast. Something like an old AO 20 with its 100-watt lamp and a darkfield condenser might be around $500 for one complete and in good condition. It could also be configured for phase contrast at some added cost. It would be heavy to ship.

Good finite phase contrast microscopes (Olympus BH2, Nikon Labophot/Optiphot, or Zeiss Standard) might be around $1000. All three are excellent optically. A Reichert Microstar IV would likely be less, equivalent optically, and also not too hard to find darkfield condensers for it. Not knowing any better (about diatom ID), I wonder if phase contrast's halos and loss of resolution would be an issue.

Something like a Zeiss Standard equipped with DIC might be around $3000, and an Olympus BX or Nikon E600 / E800 $4000 up. I'd think the optical sectioning capability of DIC, along with its higher resolution compared to phase contrast, might be worth the stretch.

These are US prices, Riku. Probably harder to find equivalents in your area?

There is at least one intrepid SEM owner/user on this forum. It seems an expensive thing to repair and maintain ($40K??) and with very slow throughput in terms of specimen preparation. Superb resolution, though.

DNA analysis is getting cheaper all the time. I'm curious if anyone uses it to ID protists, algae, etc.??

Rorschach
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Re: Microscope for diatom identification work?

#4 Post by Rorschach » Sun Mar 26, 2023 3:24 pm

Hobbyst46 and PeteM thanks for the suggestions.

SEM is not possible for her budget. Also, the maintenance of those older units is impossible for all but those who are trained on these machines. Furthermore, the large numbers of samples, each often with 500 valves/individuals identified, means that SEM isn't really a possibility.

DNA techniques are evolving (pun intended) quickly indeed, and we are already using eDNA in many of our more cutting edge research projects. Most recently last year in a stream ecology project that concentrated on microbial communities. However, for the kind of routine monitoring surveys that she is doing, they are not really an option either. These techniques also tend to only yield presence/absence data instead or abundance data and that is a major problem.

So that leaves the optical light microscopy way. This is how it has been done for decades in the environmental monitoring community, so it seems to be quite good enough in terms of species level identification.

DIC seems out of her budget. I am still waiting for a figure but will be surprised if it's above 1000-2000 euros for a complete system.

Pete, you are right: some of those like AO aren't available that often here on the old continent. And orders across the pond always carry a risk of breakage plus EU VAT & customs on top of the outrageous shipping costs. But Leitz is commonly available here :)

MichaelG.
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Re: Microscope for diatom identification work?

#5 Post by MichaelG. » Sun Mar 26, 2023 4:33 pm

Rorschach wrote:
Sun Mar 26, 2023 11:02 am
Hi people,

A friend of mine has asked for help in choosing a good microscope system for identifying diatoms (to species level). This would be for work and research purposes but will be paid by herself - so funds are not unlimited.

[…]
Please permit me a naive question, to the wider forum:

Is it really feasible to identify diatoms (to species level) using a light microscope ?

Having recently moved to North Wales, I was pleased to find this site:
https://naturalhistory.museumwales.ac.u ... s/Home.php

and I am gently browsing my way through it
… but that’s a very different proposition to actually identifying a random specimen as being of a particular species.

MichaelG.
Too many 'projects'

Rorschach
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Re: Microscope for diatom identification work?

#6 Post by Rorschach » Sun Mar 26, 2023 4:40 pm

MichaelG. wrote:
Sun Mar 26, 2023 4:33 pm
Rorschach wrote:
Sun Mar 26, 2023 11:02 am
Hi people,

A friend of mine has asked for help in choosing a good microscope system for identifying diatoms (to species level). This would be for work and research purposes but will be paid by herself - so funds are not unlimited.

[…]
Please permit me a naive question, to the wider forum:

Is it really feasible to identify diatoms (to species level) using a light microscope ?

Having recently moved to North Wales, I was pleased to find this site:
https://naturalhistory.museumwales.ac.u ... s/Home.php

and I am gently browsing my way through it
… but that’s a very different proposition to actually identifying a random specimen as being of a particular species.

MichaelG.
Hello Michael,

I'm no expert on diatoms, but as far as I know, it is possible and has been routinely done for a long time. Of course, one doesn't get to species level with all of the valves, some are left to genus level, but that is quite normal. Some frustules are positioned in a bad way or damaged etc.

It is the same situation for my darlings, the benthic macroinvertebrates, especially the larval ones: some of them are impossible to get beyond genus (or in rare cases: family) because of excessive damage (from sampling) or because the individual is simply too young a larva which hasn't yet developed the features that are needed to use the identification keys.

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zzffnn
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Re: Microscope for diatom identification work?

#7 Post by zzffnn » Sun Mar 26, 2023 4:52 pm

DIY circular oblique lighting or offset oblique lighting techniques are very useful for diatom identification too, and cost almost nothing.

Phase contrast and oblique are not difficult to find from European microscopes. You just need some patience and research to find a suitable darkfield condenser.

For diatom identification though, I don’t think darkfield is required. It is very nice to have though.

I personally would suggest getting an older 160mm tube binocular or trinocular scope that can focus with 33mm parfocal LOMO water immersion objectives and use water immersion (which is useful for live diatoms). Maybe also an immersion 40x or 50x objective.

MichaelG.
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Re: Microscope for diatom identification work?

#8 Post by MichaelG. » Sun Mar 26, 2023 5:32 pm

Thank you for responding, Rorschach
… I feel more comfortable with your ambitions now
Perhaps I read a little too much emphasis into your parenthetic ‘to species level’

Wise words from zzffnn

MichaelG.
Too many 'projects'

PeteM
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Location: N. California

Re: Microscope for diatom identification work?

#9 Post by PeteM » Sun Mar 26, 2023 6:36 pm

Rorschach wrote:
Sun Mar 26, 2023 3:24 pm
. . . Pete, you are right: some of those like AO aren't available that often here on the old continent. And orders across the pond always carry a risk of breakage plus EU VAT & customs on top of the outrageous shipping costs. But Leitz is commonly available here :)
Riku, an older Leitz Dialux (gray paint, 170mm tube length, right before the change to 160mm) might prove an affordable solution. If your Metalloplan uses the same condensers and has full Kohler illumination, it might be a great choice as well - especially if you can adapt a mechanical stage to it. Darkfield and phase contrast condensers show up fairly frequently and affordably. The focus mechanism is a marvel of precision, should focus stacking be desired. Excellent Apo optics often show up at modest prices. The removable nosepieces are a plus, though spare ones can be hard to find. 100-watt halogen lamps are available and It's very easy to retrofit a Dialux (and maybe a Metalloplan) to LED.

Given that these could accommodate Leitz short barrel objectives as well as DIN 45mm - they might focus closely enough (an extra 4mm or so) to handle the Lomo water immersion objectives Fan suggests. A Zeiss WL with its stage on a dovetail would be another option.

The main negative in terms of future upgrades -- and a plus for someone on a limited budget -- is that transmitted DIC will be near impossible to find. At least here in the US. The newer Dialux 20 and 22 models are also excellent, but more expensive and less commonly with darkfield condensers - at least here.

The confusion between 170mm and 160mm tube lengths and the different eyepieces required, along with having four different head dovetail diameters over the years, puts off many buyers. However, once your friend understands the Leitz guidance on what can mix and match - a world of both 160mm and 170mm legacy optics opens up.

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Re: Microscope for diatom identification work?

#10 Post by Phill Brown » Sun Mar 26, 2023 7:32 pm

With respect,no Chinese excludes some good optics.
I have a Euromex iscope with PH/PL ∞ optics.
It's a long way from bad.
Unlikely it didn't originate from China.
The only thing I can fault is the relatively poor slide clamp, not the hardest job to change. it's also LED which isn't my preference.
I'm not saying buy one new but I wouldn't have been disappointed with it at the budget.

Sure Squintsalot
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Re: Microscope for diatom identification work?

#11 Post by Sure Squintsalot » Sun Mar 26, 2023 9:02 pm

I'd have thought that, by now, gross identification of diatom species by visible morphologies is fairly complete. I know that there are extensive efforts to refine species by DNA. A SEM would be invaluable for variant ID but probably overkill for species level ID. That being said, there are a lot of good reasons for wanting to identify diatom species that would influence a choice in microscope, never mind illumination technique.
  • I'm not sure DIC is all that critical for gross morphological ID. I can see some amazing sub-micron(?) detail with a simple darkfield setup that my DIC does not reveal.
  • Is the plankton ID for survey purposes? Do they have to be counted? Live samples fresh off the boat?
  • Is quantitative measurements of pattern angles required? What is the level of detail you need to see?
  • Does the plankton have to be manipulated? Sorted?
Just "looking at plankton" requires a different tool kit than doing real plankton work. If it were me, I'd be looking at an inverted scope with easy access to a rotatable stage and expandable capabilities to allow high end illumination techniques down the road.

apochronaut
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Re: Microscope for diatom identification work?

#12 Post by apochronaut » Sun Mar 26, 2023 9:48 pm

Having done a fair amount of water examination for many years with an eye to doing specific indicator species counts at target locations on target dates, I can recommend a few techniques and tools that ease the task without breaking the budget. Only some of the target species in this survey are diatoms.

1) The age or brand of the microscope is not important. The condition and accessory complement are the most important things and the microscope must be a system. Looking for an acceptable system that can be made as complete as possible, as cheaply as possible is the challenge.
2) The most important specification of the optical system is it's colour correction. An apochromat system, or fluorite system is essential. An old apochromat system is preferable to a new achromat system. To begin, an achromat system could be started with if the system was in excellent condition at a good price and the possibility to improve it with superior optics at a good price in future was guaranteed.
3) The objectives with an N.A. above .75 should have some method of stopping the N.A. down. Iris diaphragms preferred.
4) There should be a minimum of 4 objectives. 4-5X, 10X, 40X, and 100X but 5 would be better adding in a 20X. or 6.
5) 15X eyepieces should be easily availalable for the system. These are useful, for providing intermediate magnifications and a useful 60X.
6) Objectives must be parfocal and parcentered. No, ifs ands or buts.
7) There must be a trinocular head and economical photo options secure.
8) There must be 3 easily available flexible condenser options. High N.A. achromat aplanat with some method of achieving a wider field coverage, for the low magnification, oil DF and oblique at a minimum.
9) Phase contrast should be an available option too. I would consider positive and negative phase.
10) DIC is another possible option but usually throws the economy out the window. Slit condenser interference contrast is possible and several condenser options are available there, PZO and Goerz, both available at economical prices but there is a lot of diy necessary to get them much above the capability of circular oblique.
11) There must be a 100 watt halogen illumination source. This is essential for DF. Led retrofits that claim to perform as well as 100 watt systems are abundant but I would want proof that that is the case. Many work o.k. but don't provide the required lumens necessary to duplicate an original 100 watt system plus they shift the colour temp. to an excessive blue. Lightly filtered halogen is best.
12) If possible a swappable nosepiece design is advisable, especially if phase contrast is to be included as a tool. It isn't a dealbreaker, though. With many older systems, the stand itself is the cheapest component, so setting up a BF, DF, COL even DIC 100 watt stand with one set og objectives, alongside a similar or even a different phase stand with 30 or so watts or led illumination is possibly a better option if an extra 4 sq. feet of bench space is available. Phase exists outside the rule of using highly colour corrected objectives only. That exists for some systems but many phase systems fell short of that target because filtering the image removed extraneous colour artifacts caused by chromatism and making achromstic phase objectives. Utilizing that tool helps with i.d.'ing and conventional achromatic phase objectives work well. In my work I use 8 different phase systems but all achromat. I filter the image but also have a partial dark phase planapo system too for the other BF, DF, oblique stand. Granted, that is a pretty extensive phase capability but it gets used.
13) Field of view. Wide fields of view became popular as the science of pathology evolved. Super wide fields of view are very useful on stereo microscopes and for special applications on diascopic microscopes but the benefit usually outweighs the extreme cost. Currently there is an amateur obsession over the field of view, just like with DIC. People will fill their nosepiece with cheap planachros then want a 26mm f.o.v. and DIC??? You have to contort your eyesockets to use that f.o.v.
It isn't necessary for identification, just for scanning a pretty panorama and pretending to be a microscopist, unless you have a defined need for it. Whatever the f.o.v. is for the complete system that you have chosen is adequate. At one time , there was no conformed standard, then it became 18mm, then 20. Now it seems to be heading to 22mm as more Chinese systems use a 30mm tube. It might be of some consequence with a very large species but usually you can reduce the magnification and get as good or possibly better detail.

Rorschach
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Re: Microscope for diatom identification work?

#13 Post by Rorschach » Tue Mar 28, 2023 4:48 am

PeteM wrote:
Sun Mar 26, 2023 6:36 pm
Rorschach wrote:
Sun Mar 26, 2023 3:24 pm
. . . Pete, you are right: some of those like AO aren't available that often here on the old continent. And orders across the pond always carry a risk of breakage plus EU VAT & customs on top of the outrageous shipping costs. But Leitz is commonly available here :)
Riku, an older Leitz Dialux (gray paint, 170mm tube length, right before the change to 160mm) might prove an affordable solution. If your Metalloplan uses the same condensers and has full Kohler illumination, it might be a great choice as well - especially if you can adapt a mechanical stage to it. Darkfield and phase contrast condensers show up fairly frequently and affordably. The focus mechanism is a marvel of precision, should focus stacking be desired. Excellent Apo optics often show up at modest prices. The removable nosepieces are a plus, though spare ones can be hard to find. 100-watt halogen lamps are available and It's very easy to retrofit a Dialux (and maybe a Metalloplan) to LED.

Given that these could accommodate Leitz short barrel objectives as well as DIN 45mm - they might focus closely enough (an extra 4mm or so) to handle the Lomo water immersion objectives Fan suggests. A Zeiss WL with its stage on a dovetail would be another option.

The main negative in terms of future upgrades -- and a plus for someone on a limited budget -- is that transmitted DIC will be near impossible to find. At least here in the US. The newer Dialux 20 and 22 models are also excellent, but more expensive and less commonly with darkfield condensers - at least here.

The confusion between 170mm and 160mm tube lengths and the different eyepieces required, along with having four different head dovetail diameters over the years, puts off many buyers. However, once your friend understands the Leitz guidance on what can mix and match - a world of both 160mm and 170mm legacy optics opens up.
The Dialux is a good candidate, thanks! I need to check the Metalloplan base that I have for lower light path stuff. It may have the mirror and iris etc. Yes, the focus system of an Orthoplan/Metalloplan is second to none! Indeed, DIC seems super scarce for these scopes.

The different dovetail diameters are a pain, yes. But the 170mm and 160mm tube lengths less so: at least on the Orthoplan, at the higher powers you can use both, even mix them: https://www.microbehunter.com/microscop ... php?t=8936

What you say about legacy 160mm and 170mm optics is really one of the benefits of the Orthoplan and it's brethren. There is much less demand for these than the newer infinity types, which leads to a superb quality/price ratio. Sometimes it really pays off to stay off the trodden path :)

Rorschach
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Re: Microscope for diatom identification work?

#14 Post by Rorschach » Tue Mar 28, 2023 5:03 am

Phill Brown wrote:
Sun Mar 26, 2023 7:32 pm
With respect,no Chinese excludes some good optics.
I have a Euromex iscope with PH/PL ∞ optics.
It's a long way from bad.
Unlikely it didn't originate from China.
The only thing I can fault is the relatively poor slide clamp, not the hardest job to change. it's also LED which isn't my preference.
I'm not saying buy one new but I wouldn't have been disappointed with it at the budget.
Perhaps it does exclude also some good optics. However, I have personal bad experiences of chinese products, both microscopes and other products.

The following is not aimed at you or anyone else but illustrates my stance. There are several other very good reasons and these weigh even more than the quality issue; the Uigur genocide, the environment , reverse engineering, extremely systematic and vulgar theft of intellectual property, industrial espionage, being allied with putler, the list goes on... So no, I will not be buying products from there as long as I live. Not even rice. Everyone makes their own choices and those are mine.

Rorschach
Posts: 334
Joined: Sun Mar 10, 2019 11:44 am

Re: Microscope for diatom identification work?

#15 Post by Rorschach » Tue Mar 28, 2023 5:21 am

Sure Squintsalot wrote:
Sun Mar 26, 2023 9:02 pm
I'd have thought that, by now, gross identification of diatom species by visible morphologies is fairly complete. I know that there are extensive efforts to refine species by DNA. A SEM would be invaluable for variant ID but probably overkill for species level ID. That being said, there are a lot of good reasons for wanting to identify diatom species that would influence a choice in microscope, never mind illumination technique.
  • I'm not sure DIC is all that critical for gross morphological ID. I can see some amazing sub-micron(?) detail with a simple darkfield setup that my DIC does not reveal.
  • Is the plankton ID for survey purposes? Do they have to be counted? Live samples fresh off the boat?
  • Is quantitative measurements of pattern angles required? What is the level of detail you need to see?
  • Does the plankton have to be manipulated? Sorted?
Just "looking at plankton" requires a different tool kit than doing real plankton work. If it were me, I'd be looking at an inverted scope with easy access to a rotatable stage and expandable capabilities to allow high end illumination techniques down the road.
Yes, SEM is not a practical or financial possibility for something like this. Optical it is :)

It's reassuring to hear now from several sources that DIC is not absolutely critical! The aim now is for darkfield and phase contrast, using PlanApo optics at higher powers.

It's not just plankton, it's also a lot of diatom samples from stream bottoms and some from rocky lake shores. In other words the periphyton communities. Yes, they have to be counted, up to 500 but at least 400. No live samples, all will be preserved samples according to environmental monitoring protocols.

I'm not sure what 'pattern angle' means here?

No manipulation involved. The diatoms will go through an acid bath before mounting on slides for id and count.

This is real work and has to adhere to the protocols. And like I said, not just planktic diatoms, a lot will be periphytic. But the tip about inverted scopes is an interesting one, thanks!

Rorschach
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Re: Microscope for diatom identification work?

#16 Post by Rorschach » Tue Mar 28, 2023 5:53 am

apochronaut wrote:
Sun Mar 26, 2023 9:48 pm
Having done a fair amount of water examination for many years with an eye to doing specific indicator species counts at target locations on target dates, I can recommend a few techniques and tools that ease the task without breaking the budget. Only some of the target species in this survey are diatoms.

1) The age or brand of the microscope is not important. The condition and accessory complement are the most important things and the microscope must be a system. Looking for an acceptable system that can be made as complete as possible, as cheaply as possible is the challenge.
2) The most important specification of the optical system is it's colour correction. An apochromat system, or fluorite system is essential. An old apochromat system is preferable to a new achromat system. To begin, an achromat system could be started with if the system was in excellent condition at a good price and the possibility to improve it with superior optics at a good price in future was guaranteed.
3) The objectives with an N.A. above .75 should have some method of stopping the N.A. down. Iris diaphragms preferred.
4) There should be a minimum of 4 objectives. 4-5X, 10X, 40X, and 100X but 5 would be better adding in a 20X. or 6.
5) 15X eyepieces should be easily availalable for the system. These are useful, for providing intermediate magnifications and a useful 60X.
6) Objectives must be parfocal and parcentered. No, ifs ands or buts.
7) There must be a trinocular head and economical photo options secure.
8) There must be 3 easily available flexible condenser options. High N.A. achromat aplanat with some method of achieving a wider field coverage, for the low magnification, oil DF and oblique at a minimum.
9) Phase contrast should be an available option too. I would consider positive and negative phase.
10) DIC is another possible option but usually throws the economy out the window. Slit condenser interference contrast is possible and several condenser options are available there, PZO and Goerz, both available at economical prices but there is a lot of diy necessary to get them much above the capability of circular oblique.
11) There must be a 100 watt halogen illumination source. This is essential for DF. Led retrofits that claim to perform as well as 100 watt systems are abundant but I would want proof that that is the case. Many work o.k. but don't provide the required lumens necessary to duplicate an original 100 watt system plus they shift the colour temp. to an excessive blue. Lightly filtered halogen is best.
12) If possible a swappable nosepiece design is advisable, especially if phase contrast is to be included as a tool. It isn't a dealbreaker, though. With many older systems, the stand itself is the cheapest component, so setting up a BF, DF, COL even DIC 100 watt stand with one set og objectives, alongside a similar or even a different phase stand with 30 or so watts or led illumination is possibly a better option if an extra 4 sq. feet of bench space is available. Phase exists outside the rule of using highly colour corrected objectives only. That exists for some systems but many phase systems fell short of that target because filtering the image removed extraneous colour artifacts caused by chromatism and making achromstic phase objectives. Utilizing that tool helps with i.d.'ing and conventional achromatic phase objectives work well. In my work I use 8 different phase systems but all achromat. I filter the image but also have a partial dark phase planapo system too for the other BF, DF, oblique stand. Granted, that is a pretty extensive phase capability but it gets used.
13) Field of view. Wide fields of view became popular as the science of pathology evolved. Super wide fields of view are very useful on stereo microscopes and for special applications on diascopic microscopes but the benefit usually outweighs the extreme cost. Currently there is an amateur obsession over the field of view, just like with DIC. People will fill their nosepiece with cheap planachros then want a 26mm f.o.v. and DIC??? You have to contort your eyesockets to use that f.o.v.
It isn't necessary for identification, just for scanning a pretty panorama and pretending to be a microscopist, unless you have a defined need for it. Whatever the f.o.v. is for the complete system that you have chosen is adequate. At one time , there was no conformed standard, then it became 18mm, then 20. Now it seems to be heading to 22mm as more Chinese systems use a 30mm tube. It might be of some consequence with a very large species but usually you can reduce the magnification and get as good or possibly better detail.
First off: thanks for a very comprehensive reply, apochronaut!

1) This was my gut feeling as well and is music to my ears. The approach has the additional, big benefit of being firmly in the field of recycling and circular economy, as opposed to the destructive way of linear economy of always buying new stuff (with often vastly inferior quality/price). So it is both ecological and economical - not to mention being also a lot more fun fishing the used market than just coldly buying a new product somewhere!
2) This is another one that confirms my suspicions. Better to have an older 160mm/170mm system with PlanApos than a new infinity system with achros. I suppose you wrote this one with DF in mind as at point 12) it seems that a phase contrast system is fine with just basic achros?
3) In the Orthoplan, there is the field iris/diaphragm in the lower light path plus there are iris diaphragms in many condensers (not sure about the phase contrast condenser, the 402a, though)
4) I agree. I think the plan is to go for a five-objective turret, unless it happens to be a four-turret one that I have lying around as an extra.
5) Ok, this one I did not think about beforehand. However, with the 23mm adapter, those eyepieces are easily available for the Orthplan as well. Might even have a pair somewhere.
6) This may require some planning. Probably means that all should be phacos. Not sure about parfocality of Leitz objectives accross types of objectives (staying strictly within the 170mm tube length objectives).
7) I agree. Although this can be swapped later on if a reasonably priced can't be sourced right away.
9) Thanks for the tip. This is probably easily fulfilled with many of the legacy systems, at least the modular ones. Shouldn't be a problem for an Orthoplan-based setup, I would think?
10) Yes, DIC is probably not worth considering and hunting for. This DIY options sounds quite interesting, though. Not sure which Orthoplan condensers would suit this approach and whether this would be within my capabilities.
11) Yes, this is what I also thought. Having the experience already from the world of DF stereo microscopes, I know that more is better! The LED options does interest me more and more, though. Would be great to know if there's a provider on eevilbay or elsewhere whose conversion does have the lumens, without the blue-shift?
12) I am a strong proponent of modular systems in general, so a swappable nosepiece is a no-brainer to me. However, what you write about using mainly achromats for phase work is certainly intriguing. I had thought that Apos should be the target, especially for the higher powers.
13) Agreed: this is not an important consideration here. Especially not for phaco as the system even for Orthoplan does not use it's WF capabilities or eyepieces. However, for future other purposes it may be very useful to have the possibility - benefits of a super-modular system.

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Re: Microscope for diatom identification work?

#17 Post by apochronaut » Tue Mar 28, 2023 11:22 pm

I just noticed a few typos in my text all courtesy of my clumsiness coupled to the stupid hover sensitivity on my tablet.
An Orthoplan is a good choice. Lots of potential options and it's general current lack of sexiness means that they are way undervalued, so many accessories are cheap, if you can find them. That is always the problem with good oldies. They continue to get used in their existing life and accessories are coveted.
The O.M.N.R. here, colloquially known as the Ontario Ministry of No Results has a fisheries research station not too far from me and they bought an Aristoplan just about the time they first came out, a later Orthoplan. The last time I was over there it was still a prime tool in their arsenal.

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Re: Microscope for diatom identification work?

#18 Post by Rorschach » Wed Mar 29, 2023 7:19 am

Yes, the Orthoplan is not as commonly coveted than, say the Olympus BH-2 or some others. Very good bang for buck. I also like the fact that it has virtually zero electronics in it's entire system - this goes a very long way towards reliability, avoiding a lot of potential and expensive pitfalls. I've heard of many grievous problems with electronics with the next generation of scopes. Despite those risks, I couldn't say no to an Aristoplan if an superb opportunity arose, that's for sure :)

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Re: Microscope for diatom identification work?

#19 Post by woyjwjl » Wed Mar 29, 2023 9:34 am

In my humble opinion, due to the fact that the diatom shell is composed of non birefringent materials, DIC can increase contrast while exerting the maximum numerical aperture of the objective lens. This is impossible in phase difference and dark fields. Considering the price, I prefer oblique light.



You can take a look here:

https://www.microbehunter.com/microscop ... =6&t=16055

https://www.microbehunter.com/microscop ... =6&t=17998

https://www.microscopy-uk.org.uk/mag/ar ... trast.html

https://www.microbehunter.com/microscop ... 24&t=17919

Finally, I fully agree with Hobby 46's opinion on second-hand SEM. Sometimes learning a little new knowledge can greatly broaden our horizons.


Best regards.
Micrographers from China, thanks to the forum for providing a platform for exchange

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Re: Microscope for diatom identification work?

#20 Post by Rorschach » Thu Mar 30, 2023 11:21 am

woyjwjl wrote:
Wed Mar 29, 2023 9:34 am
In my humble opinion, due to the fact that the diatom shell is composed of non birefringent materials, DIC can increase contrast while exerting the maximum numerical aperture of the objective lens. This is impossible in phase difference and dark fields. Considering the price, I prefer oblique light.



You can take a look here:

https://www.microbehunter.com/microscop ... =6&t=16055

https://www.microbehunter.com/microscop ... =6&t=17998

https://www.microscopy-uk.org.uk/mag/ar ... trast.html

https://www.microbehunter.com/microscop ... 24&t=17919

Finally, I fully agree with Hobby 46's opinion on second-hand SEM. Sometimes learning a little new knowledge can greatly broaden our horizons.


Best regards.
Great photo, thanks for sharing!

Yes, DIC would be great but looks like that has to left for possible future endeavors.

SEM isn't practical when one has to rapidly id a large number of samples and 400-500 individual diatoms in each sample. Plus maintaining an old SEM machine is quite impossible for most of us. I do agree that SEM would be the best for eeking out details of morphology.

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Re: Microscope for diatom identification work?

#21 Post by Rorschach » Thu Mar 30, 2023 11:25 am

I would like to ask whether there are major cons in mixing phaco objectives with regular transmitted light objectives in the same turret? As long as they are all the same tube length (170) and from the same manufacturer. I may be wrong here but I am guessing that having phase capabilities on the low power objectives (4/5x and 10x) isn't likely to be yielding much so regular good quality lenses could be used?

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Re: Microscope for diatom identification work?

#22 Post by apochronaut » Thu Mar 30, 2023 12:49 pm

With many systems the low power objectives will function in DF with one or more of the phase diaphragms, as well as the 10X and often the 20X phase objectives too. The DF is usually very good because the phase diaphragm/condenser relationship is a very controlled precise set up, with pin point centering capability and often of a corrected type too. For instance in one of my 6 place nosepieces for a Diastar I have 2.5X .06, 4X .12 , 10X .25 med. dark phase, 20X .50 med. dark phase, 40X .66 med. dark phase and 100X 1.25 med. dark phase, all as planachros. This yields 2.5X BF and DF, 4X BF and DF, 10X DF and PH., 20X DF and PH, 40X PH, 100X PH, so 10 different contrast/magnification options from 6 objectives and 5 condenser settings. The condenser is a .90 dry achromat aplanat.

There is some belief out there that phase objectives are useable for BF too , which is only true if you are willing to accept a resolution and contrast loss in each objective due to spherical aberration. It is noticeable but the objectives do work in BF.

Preferable to that I have another nosepiece fitted with 6 high N.A. objectives for a further high resolution, finely colour corrected 10 contrast/magnification options in DF and BF using two quickly interchangeable condensers, a 1.4 N.A. achromat aplanat and a toric cardioid oil DF. However for most BF sessions, I just rotate the open port in the phase condenser under the .90 achromat aplanat and use it dry. It is really only the 100X 1.32 planapo that needs the 1.4 N.A. condenser at full throttle but it works very well with a .90 achromat aplanat condenser too with some small resolution loss. In your case with diatoms, in some cases every sub micron bit of resolution will be useful, so I can see you oiling your condenser frequently.
All the condensers have the capacity for oblique too, since it can be created in the filter pack situated in the illumination pathway.

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Re: Microscope for diatom identification work?

#23 Post by Hobbyst46 » Thu Mar 30, 2023 4:51 pm

May I note that for counting hundreds of diatoms in an image, software might be fairly important. Years ago I searched for an image analysis software that automatically identifies frustules. I think that such software has evoved in recent years, though perhaps not as a finished product.

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Re: Microscope for diatom identification work?

#24 Post by apochronaut » Thu Mar 30, 2023 5:26 pm

Or you might develop the rare medical condition of Frustulaphobia.

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Re: Microscope for diatom identification work?

#25 Post by zzffnn » Thu Mar 30, 2023 7:09 pm

Rorschach wrote:
Thu Mar 30, 2023 11:25 am
I would like to ask whether there are major cons in mixing phaco objectives with regular transmitted light objectives in the same turret? As long as they are all the same tube length (170) and from the same manufacturer. I may be wrong here but I am guessing that having phase capabilities on the low power objectives (4/5x and 10x) isn't likely to be yielding much so regular good quality lenses could be used?
In theory, phase objectives would not work as well as brightfield objectives for all non-phase applications. In practice, I heard that some Phaco objectives are still good performers in non-phase (a microscopist at the other forum did direct comparisons). I don’t know if Phaco offers low contrast, if so, they tend to perform better than medium or high contrast, in non-phase lighting.

I personally don’t think phase is that much useful for 10x and lower objectives. Darkfield and oblique work very well at those powers. Personally, I don’t even care for 20x phase objectives, but I do keep 40-45x and 90-100x phase objectives at hand.

My turret phase condenser has 5 slots. I keep 45x and 90x phase annulus there (90x annulus can provide good darkfield for low power objectives 4x-20x). I removed annulus for 4x, 10x and 20x and sold them as sets with their matching phase objectives (because I don’t have use for them; I prefer DF and oblique). With the 3 empty slots, I put in DIY (removable) circular oblique masks that fits my favorite objectives and their respective NAs, to obtain COL and DF. Same idea goes for objective turret.

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Re: Microscope for diatom identification work?

#26 Post by Hobbyst46 » Thu Mar 30, 2023 8:28 pm

zzffnn wrote:
Thu Mar 30, 2023 7:09 pm
In theory, phase objectives would not work as well as brightfield objectives for all non-phase applications. In practice, I heard that some Phaco objectives are still good performers in non-phase (a microscopist at the other forum did direct comparisons).
zzffnn, please, would you provide the link to that comparison ?
I am specifically interested because a couple of years ago I compared a (Zeiss) 40X phase contrast objective with its non-phase brother, and posted the results on this forum. To my eyes the results in BF, DF and oblique were not significantly affected by the phase contrast, but maybe image quality was below optimum anyway..

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Re: Microscope for diatom identification work?

#27 Post by apochronaut » Thu Mar 30, 2023 9:09 pm

Spherical aberration due to the phase annulus can be very subtle and sample dependent. It is not seen as easily with high contrast subjects. I 'd like to see a valid comparison too. It doesn't tally with me. Granted , the effect is subtle but who needs it or would want it, when for most amateurs used BF objectives aren't that expensive.
There has to be a negative effect of an obstruction in the middle of the aperture.

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