Dodging the infinity hump

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Beatsy
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Re: Dodging the infinity hump

Post by Beatsy »

Macro_Cosmos wrote:
Thu Nov 24, 2022 5:17 pm
Which adaptor part did you get? If it is the one on eBay for BH, it will not work on BX. ...
Oh dear. Fingers crossed I got a compatible one but it sounds like not. It's this one from Spain https://www.ebay.co.uk/itm/294543952457 - expensive for what it is but my usual "handy machinist" is busy - so I went for it. It was the last one in stock too which contributed to the "hurried grab".

On the hopeful side, the listing doesn't specifically mention "BH", only "Olympus scopes", and as far as I can tell the dimensions check out compared to the mounts on a BH 1.4 oil condenser and a BX 1.25 Abbe condenser. But I don't know how tight the tolerances are - or if a bit of bodgery with a file might "adjust" any inaccuracies.

I'll wait till it turns up to try out, but then I might (likely?) be back in touch. Do you have adapters available for sale?

Macro_Cosmos
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Re: Dodging the infinity hump

Post by Macro_Cosmos »

Beatsy wrote:
Fri Nov 25, 2022 1:10 am
Macro_Cosmos wrote:
Thu Nov 24, 2022 5:17 pm
Which adaptor part did you get? If it is the one on eBay for BH, it will not work on BX. ...
Oh dear. Fingers crossed I got a compatible one but it sounds like not. It's this one from Spain https://www.ebay.co.uk/itm/294543952457 - expensive for what it is but my usual "handy machinist" is busy - so I went for it. It was the last one in stock too which contributed to the "hurried grab".

On the hopeful side, the listing doesn't specifically mention "BH", only "Olympus scopes", and as far as I can tell the dimensions check out compared to the mounts on a BH 1.4 oil condenser and a BX 1.25 Abbe condenser. But I don't know how tight the tolerances are - or if a bit of bodgery with a file might "adjust" any inaccuracies.

I'll wait till it turns up to try out, but then I might (likely?) be back in touch. Do you have adapters available for sale?
Oops...
Unfortunately, that one will not work.
You can see my listing here, the last photo: https://www.ebay.com.au/itm/18565186743 ... media=COPY
Image
All the smudges and scratches were due to a Dremel tool, I bought the adaptor and spent a couple of hours manually digging out enough clearance so it can be mounted.

The one I made based on this also allows the original filter clip to be used. China is in lockdown, so I have no chance to get a batch made. I am debating between making adaptors for each brand or having one that allows the dovetail to be exchanged which will complicate things. Testing the thing is going to be an issue too, I no longer have access to Zeiss/Leica scopes.

Beatsy
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Re: Dodging the infinity hump

Post by Beatsy »

Not oops. Fortunately, it works perfectly. The adapter slips right in to the condenser holder on the BX61 and clamps firmly. It holds the Heine nice and straight with centering done using the Olympus controls. I was able to remove all the other centering gubbins that was attached to the Heine. It's not needed and fouled the underside of the stage in some orientations anyway.

The Heine works exactly as expected with dry objectives up to N.A. 0.75. BF, DF and COI are all superb. The 40x/0.75 requires the top cap fitted on the Heine condenser to achieve DF. Better image contrast resulted when that top cap was oiled to the slide. All dry lenses worked better with the Heine oiled to the slide like this - but only 10x/0.3 and 20x/0.5 worked well without oil.

The first quick look using immersion objectives and the Heine oiled to the slide (60/1.42 and 100/1.4) was not so promising. Couldn't get a good DF at all. Could be that the Heine can't deal with NA 1.4, but I was under the impression it should do. Condenser position looks hyper-critical here though, so I need a longer session with more intense twiddling before I posit any limitation. Update soon...

Pau
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Re: Dodging the infinity hump

Post by Pau »

You can't do dark field with 1.4NA
The inner cone of the condenser light must have an angle larger than the objective entrance angle.
This limits DF to 1.0 or maybe 1.1 objectives with a oil DF condenser

I can have nice DF with my 50/1.0 oil but not with higher NA using the Zeiss ultracondenser 1.2-1.4
Pau

viktor j nilsson
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Re: Dodging the infinity hump

Post by viktor j nilsson »

1.4 should be too much. I think the practical limit for darkfield with the Heine is quite a bit lower than that. 1.15 maybe?

But I'd be interested to hear people's opinions about optimizing the Heine for high-NA df. I have 3D printed an adapter for the Heine to use it on my Vanox AH. It works very well without the cap, but I haven't gotten at all good results with the cap.

Recently, I picked up a proper oil darkfield condenser for the Vanox. And this made me realize that the settings for the light path before the condenser was much more important than I had previously realized. I thought that df condensers basically just needed to be fed enough light into their lower opening. But when I started playing around with the oil darkfield condenser I realized that I could get amazing high-NA darkfield only when adjusting the light as I would if I was using the ultrawide condenser. For the Vanox, this means flipping the auxiliary lens into the HI/UL position and flipping an extra ultra-low lens into the light path. But the thing is I can't use this setting with my Heine, as the condenser is too tall and interferes with the auxiliary lens when it is in the HI/UL position.

I guess most other systems have a bit less adjustments than my microscope, but I'm still interested to hear what configurations you use that lets you get the most from the Heine (and other high-NA darkfield condensers). I don't think I've seen this discussed much at all, and it surprised me that the settings had such a large effect.

Beatsy
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Re: Dodging the infinity hump

Post by Beatsy »

I mis-spook about DF. I know it usually starts to "drop out" above NA1.2 or so, but the resultant COI you get instead can look like a poor DF too. That's what I was (clumsily) referring to. Here's a test shot from last night - all dry, no oil. I decided to switch the blue filter cube in for this one, which only allows blue light to the sensor (and eyepieces). Definitely see the extra resolution from doing that (compared to white light).

I have my A7riv connected directly to the C-Mount trinocular using a C-mount lens adapter. This gives exactly 1x onto the sensor, so a 100x objective gives 100x reproduction ratio, which is handy. A single shot of these Stauroneis sp. comes out like this (using the Heine condenser). I used full frame to get the whole FoV in here (61 megapixels) but I'll generally use APS-C crop mode with this camera - that's still 26 megapixels. That gives you the full width of the FoV, but crops top and bottom a bit.
al1.jpg
Same image converted to B&W using only the blue channel, cropped square and inverted to negative.
al2.jpg
And finally, cropped in tighter to show details. Those rows of dots are approximately 320nm pitch. All images straight exports with a tiny bit of sharpening for screen on export. Otherwise this is just a straight, single shot (no stacking).
al3.jpg
All this with a simple Plan 40x/0.75 objective too. Way better than any results I ever got on my Zeiss with equivalent lenses. Me and the BX, and the Heine, will definitely be enjoying a happy and productive future relationship... :D

Edit: I forgot, this isn't QUITE a straight shot. It was a 4-shot pixel-shift capture. You only trigger the shutter once, but the camera takes 4 shots, shifting the sensor by one pixel for each shot to sample R, G, G and B at every pixel position. It doesn't change the image resolution, per-se, but does improve colour resolution by ensuring each pixel is fully sampled for R, G and B. The colours are not "guesstimated" from the Bayer array (important as we're only using the blue channel here. The Bayer approach would only get 1 pixel in 4 "correct" while the other three would be estimates of the blue contribution, but derived from red and green pixels).

Macro_Cosmos
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Re: Dodging the infinity hump

Post by Macro_Cosmos »

Great to hear that he refined the adaptor to allow BX mounting as well. In this case, I will not trouble myself with making a batch.

Dry darkfield tops out at 0.7 NA and oiled ones at 1.15 NA.
To test this out, I have tried 0.7 NA and 0.75 NA objectives, I was able to get good DF with the former. I have also tried 1.15 vs 1.2 for oil immersion darkfield condensers, same story. It seems like even an extra 0.05 is too much.

For Olympus, I highly recommend the 100x oil immersion apochromat with an iris. The 40x one is great too. They come in phase variants too, though very rare.

Sym P. le
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Re: Dodging the infinity hump

Post by Sym P. le »

Wonderful work, as usual. Thanks.

Beatsy
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Re: Dodging the infinity hump

Post by Beatsy »

Macro_Cosmos wrote:
Sun Dec 04, 2022 5:29 pm
Great to hear that he refined the adaptor to allow BX mounting as well. In this case, I will not trouble myself with making a batch.
I bought the last Olympus adapter he had in stock. Just FYI.
Dry darkfield tops out at 0.7 NA and oiled ones at 1.15 NA.
To test this out, I have tried 0.7 NA and 0.75 NA objectives, I was able to get good DF with the former. I have also tried 1.15 vs 1.2 for oil immersion darkfield condensers, same story. It seems like even an extra 0.05 is too much.
I'd put the numbers slightly higher than that. Mainly because there isn't a completely hard cutoff where an illumination effect (BF, DF or COI) stops dead. There's a short transition where the effect fades, but still gives some contrast.
For Olympus, I highly recommend the 100x oil immersion apochromat with an iris. The 40x one is great too. They come in phase variants too, though very rare.
Actually, the setup was advertised as having a 100x/1.4 PlanAPO with collar, but the seller had made a mistake (no collar). I was advised before the sale, and a downward price adjustment was made. Not the end of the world. I put a bit extra into buying a Olympus 40x/0.75 UPlanFL N (dry). That added a useful "middle" to the range of objectives fitted, at the cost of introducing a risk of dipping the 40x in oil at some point. The risk is worth it as 10x, 20x then move to oil is far less convenient than 10x, 20x, 40x before oil. As the result above shows, oil will be required FAR less often thanks to that 40x being in the set.

I've pretty much met all my diatom imaging needs and wants with this scope. Very satisfying. Now onto the endless but enjoyable tinkering to make it sing and dance to the best of it's abilities...

chris_ma
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Re: Dodging the infinity hump

Post by chris_ma »

looks great, thanks for showing these!
Beatsy wrote:
Sun Dec 04, 2022 2:23 am
Edit: I forgot, this isn't QUITE a straight shot. It was a 4-shot pixel-shift capture. You only trigger the shutter once, but the camera takes 4 shots, shifting the sensor by one pixel for each shot to sample R, G, G and B at every pixel position. It doesn't change the image resolution, per-se, but does improve colour resolution by ensuring each pixel is fully sampled for R, G and B. The colours are not "guesstimated" from the Bayer array (important as we're only using the blue channel here. The Bayer approach would only get 1 pixel in 4 "correct" while the other three would be estimates of the blue contribution, but derived from red and green pixels).
one minor remark on this: pixel-shift *does* in fact increase image resolution, it's just that with a lot ob shootings situation it's not really showing up significantly because the subject doesn't have enough detail or the lens is not good enough to show it.
but in your case with shooting in blue light only, it should result in nearly twice the image resolution if the lens is up to it.
chris

houstontx
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Re: Dodging the infinity hump

Post by houstontx »

I bought the same adapter from ebay and using it now on my bx51. Fits perfectly. I found i must use the heine with caps to get a good image. Was not able to get good results with cap off. The dry cap must almost be touching the slide to get the best phase effect with a plan 40x phase objective. But oiled is the way to go...I have a 10, 20 and 40x apo and a plan 40x phase. The seamless switch to hunting in 10 and 20x in darkfield then going to phase with 40x is really nice. Also have a uplan FL 60x oil iris i'll make an attempt with this week...Overall its a bit frustrating getting a good image and even lighting so far. I'll have it for a bit and then lose it... I'm using a BD nosepiece with thorlabs 26mm to rms adapters, I wonder if that could be causing some issues...

Great diatom images to both of you, hope to get on your levels eventually!

Beatsy
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Re: Dodging the infinity hump

Post by Beatsy »

houstontx wrote:
Mon Dec 12, 2022 9:04 pm
I bought the same adapter from ebay and using it now on my bx51. Fits perfectly. I found i must use the heine with caps to get a good image. Was not able to get good results with cap off. The dry cap must almost be touching the slide to get the best phase effect with a plan 40x phase objective. But oiled is the way to go...I have a 10, 20 and 40x apo and a plan 40x phase. The seamless switch to hunting in 10 and 20x in darkfield then going to phase with 40x is really nice. Also have a uplan FL 60x oil iris i'll make an attempt with this week...Overall its a bit frustrating getting a good image and even lighting so far. I'll have it for a bit and then lose it... I'm using a BD nosepiece with thorlabs 26mm to rms adapters, I wonder if that could be causing some issues...
I agree, the cap needs to be fitted for best DF/COI results with most objectives, and oil needed for anything over about NA0.75. But without cap works better for lower powers and lower NA if you're after a bright field look. I'd say 20x/0.5 is about the limit for no cap work.

I've only tried it a couple of times, but using glycerin or even water between the Heine and the slide appears to improve the resolution and evenness across the FoV with otherwise-dry objectives too. Oil still works best though, and higher NA always needs oil between objective and slide too.

Now I finally got to use it, 5 years after buying it, I really like the Heine. It's a bit like using a concave sub-stage mirror and the edge of a light beam to fish out detail from low-contrast specimens. Fun and rewarding for sure - I do that all the time with my travel scope (Tami). You certainly get to lift out a surprising amount of detail, but the overall illumination can be very patchy and uneven so is mainly suitable for visual examination - not so much for images.

I guess that's my main complaint about the Heine - it's very difficult to get a consistent and even "look" across images of different mounts. I can control that to some extent by putting several different groups of specimens in the same mount - so one Heine setting can be used for more shots in succession. That's close enough for me, for now. Practice might make that unnecessary over time. We'll see...

Edit: I intend to mount some diatoms on a coverslip but then stick that onto a large coverslip instead of a slide. The thinner substrate (0.17mm instead of 1.0mm) would allow more of the cone of light from the Heine to "reach" the specimen. That's for sometime later though - and I'll report on a new thread once I get a result.

Macro_Cosmos
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Re: Dodging the infinity hump

Post by Macro_Cosmos »

The oil cap must be used in conjunction with immersion oil for the best results and proper centring is important for phase contrast.
I have yet to find a low magnification (10x) phase contrast objective that works with the Heine. The official one has an immersion cap addon to the objective that uses oil which is convoluted and apparently produces suboptimal results. I have a 10x phase SApo which I can get good phase contrast with the heine, but the illumination is a complete mess, which I guess is due to NA mismatch.

Hopefully, I can find a 20x Ph2 soon.

patta
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Re: Dodging the infinity hump

Post by patta »

late to the party, here some stray, know-it-all comments:

- The old Solid Immersion, I buy small spheres from Mowen /BohrOptics on Aliespress. For Sapphire they list only full spheres but likely they can grind some to half for moderate $$, or the OP showed that it can be done at home... Another similar road is "microsphere superresolution": smear the slide with mayonnaise, the little droplet of oil in mayonnaise are micro-lenses that magnify what's underneath; somehow works but no great pictures.

- It is possible indeed to get Darkfield with NA 1.4 objective :shock: - condenser made on order or DIY :lol: https://www.thingiverse.com/thing:5241935
At NA 1.4 needs the specimen to be mounted between two coverslip, so it can sit near to the condenser, like observed for the Heine. For standard NA 1.25 objective, it is possible to get the light cone trough a normal glass slide.

- I've tried last year to DIY a confocal setup, that in principle could boost resolution by 1.4x; but gave up. Still think it may be feasible.

- Problems with the Heine as last post, my understanding is that it is the "focal position" of the light ring that gives trouble. Meaning, the borders of the light ring/hollow cone from the condenser are not reimaged/focused exactly over the phase ring inside the objective, but a bit above or below; at the phase ring, the illumination annulus is out of focus. Think of normal phase condenser, the image of the condenser annulus is projected - in focus - over the phase ring of the objective. That the focus is right there, doesn't happen by chance. If not in focus, some light gets out, the phase contrast work in the center of the field but not at the borders. It is just speculation I don't have the Heine; this issue pops up when DIY a phase annulus, it needs to be placed at a specific distance below the condenser so it is refocused exactly over the objective phase ring. Checking with "phase telescope" if the two rings are centered AND both in focus is difficult because usually phase telescopes are "stopped down" so only the center of the field is examined and the out of focus is not apparent. If one examine the border of the field, the ring would appear off-center.

Pau
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Re: Dodging the infinity hump

Post by Pau »

patta wrote:
Sat Dec 17, 2022 7:04 am
- It is possible indeed to get Darkfield with NA 1.4 objective :shock: - condenser made on order or DIY :lol: https://www.thingiverse.com/thing:5241935
At NA 1.4 needs the specimen to be mounted between two coverslip, so it can sit near to the condenser, like observed for the Heine. For standard NA 1.25 objective, it is possible to get the light cone trough a normal glass slide.
Likely you can have another source but what I can read is:
The print in the photos works at 1.35-1.45, so it gives nice DF with a 100x 1.25
The principle stating that the inner angle of the light hollow cone must be wider that the entrance cone of the objective remains. Even here I would like to see scientific papers and also other people experiences with it, I'm pretty skeptic.

Another approach is without condenser, profiting the total internal reflection of the light inside the glass slide laterally illuminated, although it also has NA limitation, see https://www.ncbi.nlm.nih.gov/pmc/articl ... 058344.pdf I think that this can't work with oil immersion objectives although in the linked paper it's unclear
Pau

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