Partially blocked Leica objective

[snik]

I got this objective for a reasonable $300: Leica N Plan L 40X/0.55 CORR

When I look throught the objective, there are two dark areas, one darker than the other as seen here

20240416-AA1_7323.jpg (78.61 KiB) Viewed 890 times

Is this a feature, or something inside the assembly that has been dislodged? I can get a sharp image, although the contrast is a bit lower than my HI Plan 40X. Not sure if that's expected due to the more complex optical design, or if the dark obstruction is lowering the contrast. Should I return it to the seller?

[Alexander]

Turn the correction collar and check what happens.

[apochronaut]

Looks like a modulation contrast gradient.

[snik]

It doesn't move with the correction collar.

I think you're right apochronaut, this looks like it:
https://www.leica-microsystems.com/scie ... trast-imc/
https://www.mikroskopie-forum.de/index. ... ic=41806.0

I wonder if I can diy a slit/polarizer to take advantege of this. I have an upright DMLS with a slider/single disc holder in the condenser.

[apochronaut]

I would think so. Having the modulater in the objective is probably covering 80% of the engineering complexity of the mechanism. The rest is kind of plug and play, not dissimilar to the related components in phase contrast.
The reduction in contrast and likely brightness you noted in BF would be a normal consequence of having such a barrier at the rear focal plane.

[wabutter]

do you have the catalog number for the objective. As pointed out, likely a Modulation contrast objective. also called LMC. Leica Modulcation Contrast. ala Hoffman.

[PeteM]

+3 It's a Modulation Contrast objective, especially useful to add contrast to live specimens on an inverted microscope. Someone will likely be willing to swap it for one without the added bits of polarizing film. You might also hope to acquire a matching condenser and a couple more objectives - at a significant additional cost.

If you plan to use this on an upright scope, you might better swap for a higher numerical aperture 40x N Plan or even phase contrast equivalent.

[zzffnn]

+4 That is a HMC (Hoffman Modulcation Contrast) objective. At 40x NA 0.55 and HMC, it is very likely an inverted objective designed for ~ 1mm cover.

If it does have a correction collar and you did not set it for the correct cover glass thickness, then contrast will be low due to spherical aberrations.

[snik]

Thanks all for the input. The catalog number is 506135. I've had a hard time finding info on older Leica parts, and even the current Leica website is sparse with details on their current offerings.

I bought this for looking through agar petri dishes, so upright scope should be fine I thought. I didn't think about the condenser working distance also being a factor.. Still, I'm getting more detail using this with 10x oculars compared to 10x/0.25 and 25x oculars. And Hoffman contrast looks cool, so I think I'm keeping it.

[MichaelG.]

Maybe not a lot of help, but … this appears to feature one in the appropriate setting
https://americanlaboratorytrading.com/l ... cope-14736

MichaelG.
.
.
Edit: __ __ Which quickly led me to find this
https://mmcri.org/wp-content/uploads/2 ... Manual.pdf

Diving-in at page 97 would probably be your best bet !

[apochronaut]

Thanks all for the input. The catalog number is 506135. I've had a hard time finding info on older Leica parts, and even the current Leica website is sparse with details on their current offerings.

I bought this for looking through agar petri dishes, so upright scope should be fine I thought. I didn't think about the condenser working distance also being a factor.. Still, I'm getting more detail using this with 10x oculars compared to 10x/0.25 and 25x oculars. And Hoffman contrast looks cool, so I think I'm keeping it.

Using the prescribed condenser from something like a DMIl would be nice but it isn't what is required in this case.
The condenser is part of the stand because it is designed to focus on the illumination source and then focus that light in a predictable way at the object. It only is part of the objective when that objective is used in it's original design framework as part of the system. Another condenser and it's correct stand configuration can theoretically provide the required illuminating conditions too , as long as it provides an adequate N.A. to the objective and fills the field with even light. In this case as well though, you have to provide a properly overlapping illuminating slit with polarized light, as well as have an analyzer above the objective. As mentioned before, a certain high percentage of the design/engineering of your intended system is already in the objective. The rest is just manipulation of slit width and vertical distance using the existing condenser in your stand but before any of that is fabricated, sort out your sample prep. set up.
The condenser's extra working distance in that objective's native stand is of importance when using that objective in an inverted stand only. Under such conditions , the condenser is designed to be well away from the stage in order to allow access as well as focus deeper into the sample. When that objective is used in an upright stand , there is no particular need for the specified condenser or specified condenser working distance because the distance of the condenser to object plane needs to be the same in your stand, irregardless of the objective used. Using a chamber or dish with exactly the same bottom thickness as a standard slide will keep your existing condenser comfy. Where you will run into problems is with whatever you use as a facsimile for a cover slip. I don't know what your collar adjustment range is but if it is 0-2mm for instance, that makes a good case for using a well slide for a chamber and a standard high grade polished slide as a cover slip. Measure it and adjust the collar accordingly. You could also use a thick .4 or .5 mm coverslip such as is used on certain blood counting chambers. You will have difficulty adjusting for and using standard cover slips. Whatever system you evolve, it needs to be kept fairly consistent because it will affect the slit width and focus.

You have to decide on a sample container/cover protocol first , test it to perfection in BF, then d.i.y. a polarizing/slit mechanism to suit. Fan is right, you probably didn't have the collar properly adjusted, thus some spherical aberration but it is typical for objectives with optical barriers or diversions placed internally to display lower contrast and brightness when used in BF, or outside of their design intention.

A 25X eyepiece is pushing it with an achromat objective, even if the N.A. theoretically limits the level of empty magnification., As the colour correction of the objective increases, it seems that limit is more approachable and in fact exceedable. Thus for instance, 15X eyepieces with their typically wider apparent field were used as the default eyepiece in some older apochromatic systems. The 90X 1.30 oil immersion objective of those systems was purposeful, allowing for 1350X magnification with 15X eyepieces, even though the predictable limit was 1300X. You may find 15X eyepieces to be acceptable at 600X with a plan fluotar, even if the N.A. is .55.

[snik]

The problem with condenser WD I was thinking of is that standard petri dishes are 16mm high. When I flip a dish upside down, the condenser focus is something like 12-13 mm away from the bottom of the dish. Here's how it looks when I look through the bottom:

IMG_3447.jpg (135.47 KiB) Viewed 660 times

This 10x has a WD of 18mm, so I can also look through the lid of the dish. That way I can get phase contrast if I want, although I get better images through the bottom. The 10x doesn't seem to mind much that the condenser is way out of focus.

The 40x has a 0-2mm correction, and I get really poor images through the bottom. I can see a bit more detail than the 10x, but not much. Going further with the 40x gets complicated as I understand from your explanation. Dishes with coverslip wells and whatnot are difficult to source and I'm guessing quite expensive.

I got a pair of Wild 25x at a reasonable cost. Leica 15x have been hard to find and/or expensive, so I just went with it. Sure there's some empty magnification, but man it's so much easier to spot clamp connections using 25x.

[wabutter]

Thanks all for the input. The catalog number is 506135. I've had a hard time finding info on older Leica parts, and even the current Leica website is sparse with details on their current offerings.

As a reference point. The 506135 is a LMC objective. possessing the following specs. N Plan L 40x/0.55 CORR 0-2, FWD 3.9 -1.9, with M25 threads

All CORR objectives from Leica are referred to as Contrast Automatic. This means you can adjust the correction collar until the contrast is best acheived and the objective will be set for the coverglass that is in use. Of course having Koehler Illumination set on the scope is necessary to make this work as contrast and resolution are dependant on maintaining conjugate planes for image and illumination.

[PeteM]

Wayne - that "contrast automatic" feature is new to me. Thanks - it should be much easier to use than fiddling with perceived resolution and refocusing. My question - do you know if Nikon and Olympus infinite objectives behave in much the same way?

[apochronaut]

The problem with condenser WD I was thinking of is that standard petri dishes are 16mm high. When I flip a dish upside down, the condenser focus is something like 12-13 mm away from the bottom of the dish. Here's how it looks when I look through the bottom:
IMG_3447.jpg

This 10x has a WD of 18mm, so I can also look through the lid of the dish. That way I can get phase contrast if I want, although I get better images through the bottom. The 10x doesn't seem to mind much that the condenser is way out of focus.

The 40x has a 0-2mm correction, and I get really poor images through the bottom. I can see a bit more detail than the 10x, but not much. Going further with the 40x gets complicated as I understand from your explanation. Dishes with coverslip wells and whatnot are difficult to source and I'm guessing quite expensive.

I got a pair of Wild 25x at a reasonable cost. Leica 15x have been hard to find and/or expensive, so I just went with it. Sure there's some empty magnification, but man it's so much easier to spot clamp connections using 25x.

I wasn't aware that you were lifting the sample so far above the stage.
So, just to be clear on what I am seeing : you are looking at a sample that is adherent to the inverted bottom and the bottom is just too thick ( more than 2mm?) or is it that the condenser is not capable of focusing that far above the stage? I'm not perfectly clear what is affecting the image quality so negatively.

I was thinking in terms of using a well slide not a dish, and then using a 1mm slide as a cover slip, so it would be a standard slide set up with a well, using a thick cover. The sample space is somewhat capacious. The wells are about 8-10mm in diameter. You could correct accurately and be looking into the sample with Köhler illumination. I don't think the well slides are that expensive.

An option could be a flip top condenser, if you need or want to stay with the current petri dish set up. I have one here that has the same dovetail as the later AO and Reichert scopes but a Leica part # I think. I began to think it must be for some early Leica DM. It fits an AO series 100 or 400 but you can't flip the top lens down, so it isn't for one of those. In your application, if it fits your dovetail , in flipped down mode it would still have enough N.A. for your .55 and could likely focus deep enough above the stage to illuminate the sample at .55 . I am pretty sure it is new.
If you are interested, post a picture of your dovetail. This one has a double angle, not just a straight slope from top to bottom. It might be a helpful addition for that objective.

[snik]

As a reference point. The 506135 is a LMC objective. possessing the following specs. N Plan L 40x/0.55 CORR 0-2, FWD 3.9 -1.9, with M25 threads

All CORR objectives from Leica are referred to as Contrast Automatic. This means you can adjust the correction collar until the contrast is best acheived and the objective will be set for the coverglass that is in use. Of course having Koehler Illumination set on the scope is necessary to make this work as contrast and resolution are dependant on maintaining conjugate planes for image and illumination.

Interesting, thanks. Where do you find information like this? I tried to adjust the collar for best resolution. It takes some fiddling, and with the petri dishes it's difficult to say what's the best setting, since the image is not sharp at any setting really.

I wasn't aware that you were lifting the sample so far above the stage.
So, just to be clear on what I am seeing : you are looking at a sample that is adherent to the inverted bottom and the bottom is just too thick ( more than 2mm?) or is it that the condenser is not capable of focusing that far above the stage?

The plastic bottom is around 1mm thick, agar is around 2mm. I've cut slices out of the agar so mycelium can grow on the plastic, lowering the WD. I'm guessing poor image is due to inadquate NA from the condenser at this distance. I can close the condenser all the way down without much change in the image. This is as good as I get it with 40x through the bottom:

pclamp.jpg (170.64 KiB) Viewed 521 times

This is using a phone with a 3d-printed holder on the 10x ocular, so not the best representation of what I see. The mycelium cells are 2-2.5µm wide, and the clamps I'm looking for about the same. You can see one potential clamp in the center of the image.

Well slides could work nicely for this. I was looking into these types of dishes before: https://www.thermofisher.com/order/cata ... uct/150680 I can easily go through 20-30 plates in a month though, and paying $200 a month for dishes is not an option.

I appreciate the help here, I'm learning a lot. I guess the discussion on petri dish imaging could be a separate topic. It's hard to find good information on Leica gear, all I have is a pdf manual with grainy pictures; no part numbers or detailed info on condensers etc. The manual mentions a flip-top condenser, but I haven't found much info on it. The dovetail on my condenser is 45mm at the widest, height is 6.8mm. Single slope as seen below.

IMG_3486.jpg (121.36 KiB) Viewed 521 times

[Hobbyst46]

...I was looking into these types of dishes before: https://www.thermofisher.com/order/cata ... uct/150680 I can easily go through 20-30 plates in a month though, and paying $200 a month for dishes is not an option...

If cost is the only reason for not using the glass-bottom petri dishes: they are sold as disposable, but CAN be cleaned and reused at least 5-10 rounds.

[snik]

Cleaned perhaps, but can they take 121C sterilization?

[apochronaut]

I recognize that you have a system that you have evolved that works for culturing and live mounting but I see a few major red flags that could limit your attempts to increase resolution with higher N.A. objectives.
10X .25 is very forgiving. Anything over .30 or .35 increasingly not so The other thing is your intention to use that modulation contrast objective in it's intended application. I doubt if you will be able to do that with a condenser immersion that is essentially consisting of air/glass/thick air, when it wants thin air or oil. Your dovetail by the way is different than the one on this condenser I have here. I had high hopes for that as an option for you. High N.A. dry or oil condensers achieve lower N.A's. through throttling the condenser with the iris diaphragm while maintaining a consistent working distance. Lower N.A. condensers achieve similar N.A's. through a lower magnification and longer working distance. With your current above the stage sample prep., a low N.A. , longer working distance condenser seems the best option. In the job lot of condensers I bought , there are some older .66 condensers that were used on microscopes that had a 4mm .66 objective as the big gun. They sat well below the stage, so had a long working distance. I can find out how much. One could be diy'd into your situation.
The plastic in the bottom of those dishes would need to be an optical grade plastic. That does exist but if yours is not, trying to resolve further details through that plastic bottom I can't see as very likely.

The well slides that I refer to are slides, not dishes. Also called cavity slides. To my knowledge they come as single cavity standard ( 1mm thick) slides, double cavity standard slides and thick slides of about 5mm thick with a deeper cavity. Are you using an agar block smear or just a thin substrate? In lieu of your desire to increase resolution and perhaps utilize the modulation plate in that objective, could you not scale down your gear? Would it not be possible to do an agar culture in a well slide and use a high grade, measured, optically perfect 1mm slide as a lid? That way your 40X .55 CORR objective would be pretty happy and your condenser too.

Well slides are about 1.00 ea.

[snik]

I haven't been at this for long, I got the scope in December and the petri dish project took place during the past 2 months.

I'd like to use the scope for all things mycology, such as identifying macroscopically similar species, single spore/single cell transfers, and cross breeding of strains. So looking at mycelium cells like this is typically done by mounting a tiny sample on a slide with coverglass, squished flat and usually with some stain at 1000x. This is time consuming and there's a risk of contaminating the culture when I open the lids to get a sample. What I've found is that I can spot most dikaryotic cultures with the 10x, so I can eliminate those and mount the one's I'm unsure about. I got the 40x on a poorly researched whim, not thinking about the WD of the condenser..

The petri dishes are the cheapest kind, most likely not very suited for microscopy. I can get pretty decent phase contrast with the 10x though, looking through the lid so the condenser is within it's WD. With the 40x I can't do that of course without taking the lid off, but I can use it for normal slides. I actually have a few thick slides with a meniscus shaped cavity. That was inspired by Oliver

[snik]

I made a quick test, making a slit out of aluminium foil pieces. I like this contrast technique a lot, and I'm sure I can get it better than this if manage to adjust the slit size and position with some precision.

Here are some tests on a mushroom sample, stained with methylene blue and mounted in KOH.

20240423-IMG_3590.jpg (111.56 KiB) Viewed 335 times

20240423-IMG_3589.jpg (171.85 KiB) Viewed 335 times

Here's a comparison with phase contrast and BF:

20240423-IMG_3597.jpg (190.13 KiB) Viewed 335 times

20240423-IMG_3594.jpg (194.76 KiB) Viewed 335 times

20240423-IMG_3593.jpg (130.22 KiB) Viewed 335 times

There's oil on the cover slip, shows up easily with Hoffman contrast. And again just a phone camera - I had no idea Leica trinocular heads cost more than the complete microscope.

[Hobbyst46]

Cleaned perhaps, but can they take 121C sterilization?

Don't think so. Can be sterilized with UV.

[apochronaut]

Are you able to view the slit in a bertrand lens? I was going to suggest using the jaws of a vernier caliper as a slit mock up or a small straight jawed clamp, such as a small Jögensen clamp, then transfer the dimensions to a filter disc.

[MichaelG.]

Your ‘quick test’ looks very encouraging, snik

MichaelG.

[snik]

Are you able to view the slit in a bertrand lens? I was going to suggest using the jaws of a vernier caliper as a slit mock up or a small straight jawed clamp, such as a small Jögensen clamp, then transfer the dimensions to a filter disc.

Thats a good idea, thanks! Taping up a filter disk was very fiddly. I'm using a phase telescope to view the slit and modulator, and I have to take out the filter holder each time I adjust.

I'm not using a polarizer here, so this is not really true Hoffmann. Being able to adjust the relief effect would be nice, so I need to get some thin polarizing foil that I can cut and mount in a filter holder.

[apochronaut]

PZO had a version of a slit condenser interference contrast system between about 1970 and the early 90's. The slit condenser has sadly become somewhat popular, so it is unlikely you will find one, nor the dovetailed polarizer that slid in under it for the $30 or so that they sold for only a couple of years ago. However, the rotating polarizer that slid under was being printed by someone in Europe as of about 2 years ago. He was asking only about 25.00, if I remember correctly but it was designed to slide in under that slit condenser on a fitted akehurst slide.
If I can still find a link to that, if he still makes them, is that of interest to you?

[snik]

I appreciate the offer, but I think I'm close to getting where I want with this setup. I'm pretty self sufficient when it comes to DIY, machining and whatnot. I've been thinking I could adapt other condensers to the DMLS, like achromat aplanatic or darkfield condensers, without paying Leica prices. What can a slit condenser do besides light control in modulation contrast?

[apochronaut]

Perhaps I didn't explain properly. The 360° polarizer and slit condenser are two separate units, mateable by virtue of an akehurst slide. Here is an authentic PZO polarizing attachment being sold on ebay for instance.
https://www.ebay.ca/itm/325886635516?it ... R4qL56PiYw

The knockoff one I saw functioned the same but was printed and only about 25.00. The seller wasn't selling condensers, just polarizers. I thought it might ease the job of fitting a polarizer if still available at a decent price.

[snik]

Ah, I see. My plan is to cannibalize an lcd to get a thin plastic polarizer for the slit, and just place a 55mm linear (camera) polarizer on top of the lamp housing. I also have a blue filter on top of the lamp, and at some point I'll probably print a holder for these filters. Having the rotation angle printed on the side like that is kinda neat.

[snik]

Maybe not a lot of help, but … this appears to feature one in the appropriate setting
https://americanlaboratorytrading.com/l ... cope-14736

MichaelG.
.
.
Edit: __ __ Which quickly led me to find this
https://mmcri.org/wp-content/uploads/2 ... Manual.pdf

Diving-in at page 97 would probably be your best bet !

I've been following this lead a bit, although I'm not keen on buying a complete inverted scope. I've found a few condensers that are used in these:
521500 0.53 S23
501037 0.50
505234 0.55 S28
505175 0.55 S28
As far as I can tell, this is the only optical component between the stage and field lens, so I should be a ble to make a holder that goes in my DMLS dovetail. I'm keeping an eye out for reasonably priced parts I can experiment with.