Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

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Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#1 Post by Hobbyst46 » Thu Jun 27, 2019 2:31 pm

Note: This is a continuation of the post
viewtopic.php?f=5&t=7653

My Zeiss Ultracondenser (cardiodid), combined with a 40X1.0 Planapo Zeiss objective, all oiled to the slide, gave great darkfield of diatoms. For other high NA objectives, the FOV was bright, without contrast.

Following zzffnn's idea, I inspected the back focal plane of the objectives and found that as expected, when a very very thin ring of light was visible on the perimeter, darkfield was obtained. Hence the setup is correct. See first drawing, I could not take a picture of it.

I double-checked the higher NA objectives, they are oil immersion and phase contrast. The 63X1.25 Neofluar Ph3 and the 100X1.3 Planapo Ph3 gave neither DF nor reasonable oblique. There was no point in trying the 63X1.4 Planapo Ph3.
Then I found my "rejected orphan" - the 100X1.25 achromat. Not plan, no special corrections, I have never used it for anything.
And it yields some darkfield ! maybe "darkened background" as called by Apochronaut, but not very bad. The 40X1.0 Planapo is shown for reference.

Notes:
1) None of the above four oil immersion objectives requires a coverslip.
2) The sample was strew diatoms, mounted in Pleurax or in NOA61 (no significant difference between them for DF), under a coverslip. They are 30-50 um long.
3) The slide itself is 1.09mm thick.
4) Illumination was the same for all tests: the 10W white LED plus a green interference filter to produce monochromatic, yet pleasant view. The violet filter would give better resolution but my eyes reject it.
5) None of the photos has been darkened or brightened in post processing.

So, is it that the phase rings are so deteriorating darkfield ? This effect is not visible on lower NAs. I mean, the 40X0.75 Neofluar Ph2 (dry), yields good DF. So does the 25X0.45 Ph2 (dry).
The "orphan" 100X1.25 has earned an extended stay with the others...
Two more stack images below.
Attachments
back focal plane objectives Ultracondenser.jpg
back focal plane objectives Ultracondenser.jpg (55.87 KiB) Viewed 2929 times
63X1.25 Neofluar Ph3.jpg
63X1.25 Neofluar Ph3.jpg (145.55 KiB) Viewed 2929 times
100X1.3 Planapo Ph3.JPG
100X1.3 Planapo Ph3.JPG (137.01 KiB) Viewed 2929 times
100X1.25 single image.JPG
100X1.25 single image.JPG (113.37 KiB) Viewed 2929 times
40X1.0 single image.JPG
40X1.0 single image.JPG (129.32 KiB) Viewed 2929 times
Last edited by Hobbyst46 on Thu Jun 27, 2019 3:30 pm, edited 2 times in total.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#2 Post by Hobbyst46 » Thu Jun 27, 2019 2:35 pm

two more stack examples
100X1.25 stack of 5.jpg
100X1.25 stack of 5.jpg (246.48 KiB) Viewed 2927 times
40X1.0 Planapo stack of 5.jpg
40X1.0 Planapo stack of 5.jpg (273.86 KiB) Viewed 2927 times
Bottom line: when one has an assortment of objectives, experimentation might pay.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#3 Post by apochronaut » Thu Jun 27, 2019 4:01 pm

All more or less as expected. I haven't found phase objectives to be very useful for DF. Only in the case of using a high magnification phase annuli as a DF stop for a low magnification phase objective.

There is a fairly easy method of lowering the N.A. of your objectives and making them usefull for DF. I say fairly easy because the alignment can be tricky. I have had success doing this but I never spent too much time with it because appropriate objectives with iris diaphragms came along easily. Some mfg. actually made these. They may go under various names but reduction diaphragm is one. Sometimes it is funnel stop but there is no funnel here; just a modification of the rear diaphragm.

Measure the rear diaphragm diameter and then find or make( out of black paper or a thin card) a disc to overlay it beginning with a 5% reduction, 10% etc. The degree of reduction will vary according to the objective somewhat. I have one I use that is just around a 9% reduction: 5.5mm compared to 6mm that was originally in the objective. A few small rubber O-rings and various small thin washers will give you an array of pre cut circles to play with. Sometimes, they are dead on. When trying them out, stick them on with something thick, non hardening and water soluble, so they don't move when you install the objectives. Honey works. If you find you cannot acquire a spare rear diaphragm more cheaply, one that you can permanently modify, using honey is o.k. It comes off. A tiny bit of blu tack might do. I don't use that.

Sometimes, if the rear diaphragm is not integrated with the back lens and you have a series of objectives, they may have used the same o.d. and thread for the rear diaphragm on all the objectives in the series. Only the i.d. is different. A diaphragm for a 100X objective is only marginally smaller than a 63X for instance and can be temporarily swapped over and might work to use the 63 for DF.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#4 Post by zzffnn » Thu Jun 27, 2019 5:04 pm

Hobbyst46,

I will try to get you some photos from my COL in a few days.

Was visual view of COL more contrasty than those photos? If visual view is significantly better: play with camera's metering and exposure compensation. Use single dot metering and put that dot on your exact diatom. Then dial down exposure compensation to -0.7 or so.

I don't know what optical or mechanical changes can be made, if your visual view is not significantly better than photos.

I usually do not stack COL or DF images, as halos will confuse software and kill contrast.

If the black outside edge in your drawing and relative location of the illumination ring is similar to your actual back focal plane image, then your setup is close to optimum. You should not see any light ring at back focal plane for DF, but at NA 1.0, light spill is inevitable.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#5 Post by MicroBob » Thu Jun 27, 2019 5:12 pm

Hi Doron,
nice to see that you make progress with the Ultra condenser! I like the method of observing the objective back plane to adjust the dark field setup. So far I have just fiddled the adjustment together by feel.
Did you use all colour channels of the image?

@Phil: Thank you for your explanation of the diaphragm making process!


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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#6 Post by Hobbyst46 » Thu Jun 27, 2019 6:20 pm

Thanks Apochronaut, zzffnn and MicroBob for your comments. Frankly, I am quite happy with the new (antique) Ultra toy.
Apochronaut wrote:All more or less as expected...
I am just curious about the difference between the 63X1.25 objective and the 100X1.25 objective, that showed a partially contrasty image and an almost plain bright FOV, respectively, under the same conditions. Does th phase contrast ring do it, and how ? after all, they have the same NA (apart from small possible deviations, within the production tolerance).
Could it be that the results also depend on:
(a) the physical diameter of the front lens ? The diameter of the 100X1.25 is roughly 1/4 (or less) of that of the 63x1.25).
(b) the different complexities of the two objectives, namely, the number of elements in each ?
zzffnn wrote:Hobbyst46,
Was visual view of COL more contrasty than those photos? If visual view is significantly better: play with camera's metering and exposure compensation. Use single dot metering and put that dot on your exact diatom. Then dial down exposure compensation to -0.7 or so.
I don't know what optical or mechanical changes can be made, if your visual view is not significantly better than photos.
I usually do not stack COL or DF images, as halos will confuse software and kill contrast.
If the black outside edge in your drawing and relative location of the illumination ring is similar to your actual back focal plane image, then your setup is close to optimum. You should not see any light ring at back focal plane for DF, but at NA 1.0, light spill is inevitable.
The visual view of COL was as poor as it is in the photos.
Thanks for the light metering tip - I will try it.
The drawing is a genuine picture of the back focal plane .
MicroBob wrote:...nice to see that you make progress with the Ultra condenser! I like the method of observing the objective back plane to adjust the dark field setup. So far I have just fiddled the adjustment together by feel.
Did you use all colour channels of the image?
Thanks, Bob. Yes, I used all color channels of the image. Do you suggest to separate into R, G and B channels for better resolution ? Actually, since I used a filter, probably most of the image resides in the G channel...
Last edited by Hobbyst46 on Thu Jun 27, 2019 7:32 pm, edited 1 time in total.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#7 Post by zzffnn » Thu Jun 27, 2019 7:04 pm

You have to completely open up field diaphragm.

Also some diatoms (such as those very long ones in your photos) never sit flat on cover slips and are naturally low in contrast. Do not use them as resolution targets.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#8 Post by MicroBob » Thu Jun 27, 2019 8:28 pm

I think it would be worth a try to do this comparison:
- with filter, all colour channels
- with filter, green channel
- without filter, green channel

I can't really predict the result, but this would be an easy optimisation for colour less subjects, and cheaper than any hardware.

According the phase ring: It might cause internal reflection and affect the contrast this way.

To further optimize image quality it might be an advantage to use raw format. This way the image could be adjusted to show the extreme contrast in the raw image. I think a normal digital camera with it's light metering is really challenged by this king ot image. I use Rawtherapee, a freeware that works very well and supports most cameras, not just one brand.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#9 Post by Hobbyst46 » Thu Jun 27, 2019 8:35 pm

Thanks, Bob. All are worthy ideas, will be considered and tried..
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#10 Post by zzffnn » Thu Jun 27, 2019 8:41 pm

Hobbyst46,

Most likely, the diatoms that you used as resolution targets were too difficult to resolve. I viewed your images at 100% and think they may be good enough for those mounts.

Even from inverted cure (diatoms sticking to cover slips), you still cannot guarantee perfectly flat mount. Many diatoms are not flat and do not lay flat. Many do not have enough small internal dots to use as resolution targets.

If you have to use strew mounts, use a very highly diluted concentration. Reduce your concentration that you used above to 1/3 to start.

Better to use micromanipulated test diatoms that are flat and have many small internal dots.

I will upload images for you in a few hours.

Also, at 100x NA 1.3 or so, micro focus adjustment (I used magnified focus preview at around 5x to 14x to focus) and shutter vibration comes into play, significantly.
Last edited by zzffnn on Thu Jun 27, 2019 10:43 pm, edited 1 time in total.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#11 Post by zzffnn » Thu Jun 27, 2019 10:04 pm

COL 90x NA 1.3, LOMO oil apo non-plan, back focal plane (due to light spill, the light ring appears about 1.5x-2x wider that it actually was):
bfp.jpg
bfp.jpg (149.18 KiB) Viewed 2862 times
All photos presented herein are single (non-stacked) captures. Only one image was digitally enhanced (all others came straight from camera, except for cropping and resizing). Remember each diatom is different; some smaller individuals of the same species may be a lot harder to resolve than larger individuals of the same species.

Frustulia strew mount resized to 1024 pixel wide (note this was taken with a 35mm lens, instead of the correct 30mm lens for afocal for my micro 4/3 sensor, so it looks larger than actual visual view be at 900x):
Frustulia strew mount.jpg
Frustulia strew mount.jpg (122.62 KiB) Viewed 2862 times
Close to 100 percent crop of the original Frustulia file:
Frustulia 100 percent crop.jpg
Frustulia 100 percent crop.jpg (92.36 KiB) Viewed 2862 times
Same Frustulia in green light, at close to 100 percent crop:
Frustulia 100 percent crop gr.jpg
Frustulia 100 percent crop gr.jpg (92.59 KiB) Viewed 2862 times
Same Frustulia in green light, at close to 100 percent crop, with digital contrast enhancement and sharpening:
Frustulia 100 percent crop gr digi.jpg
Frustulia 100 percent crop gr digi.jpg (404 KiB) Viewed 2862 times
For the same Frustulia diatom, I changed condenser height, from almost crushing into slide to almost not immersed in oil. Neither image quality / dot resolving power nor back focal plane image changed significantly.
Last edited by zzffnn on Thu Jun 27, 2019 10:39 pm, edited 7 times in total.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#12 Post by zzffnn » Thu Jun 27, 2019 10:22 pm

The following images were taken with a 28mm afocal lens, so diatoms herein look slightly smaller than the visual view at 900x.

This diatom is not flat against cover slip (even though it came from a inverted cure) and is therefore difficult to resolve:
not flat difficult.JPG
not flat difficult.JPG (148.92 KiB) Viewed 2856 times
Here you can see an oval diatom, which does not look resolved (to dots) without zooming in:
oval #3.jpg
oval #3.jpg (132.01 KiB) Viewed 2856 times
But you can see its dots were in fact resolved, from 100 percent crop:
oval#3 100percent crop.jpg
oval#3 100percent crop.jpg (205.8 KiB) Viewed 2856 times
To illustrate my point that smaller individual of a similar-looking species may be a lot harder to resolve (this is a 100 percent crop):
oval small 100crop.jpg
oval small 100crop.jpg (104.77 KiB) Viewed 2856 times
Here is another oval diatom, probably of a different species (or viewed from another side), that looks a lot more contrasty and a lot easier to resolve (this is a 100 percent crop):
oval contrasty 100percent crop.jpg
oval contrasty 100percent crop.jpg (116.17 KiB) Viewed 2856 times
I hopes this helps. The take home message is that Hobbyst46's COL light might already be good enough (based on his back focal plane drawing, which looks good to my eyes). He probably picked very difficult diatoms to resolve and were expecting too much.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#13 Post by zzffnn » Fri Jun 28, 2019 3:42 am

For darkfield, I also want to echo what apochronaut has said before: try to remove all air bubbles in your immersion oil, between condenser and slide especially. One single bubble near your subject can cause significant flare and contrast lost.

For the COL imaging I did today, I had some bubbles over condenser, but they did not affect COL much and I was too lazy to remove them. But when I switched to lower power objective in darkfield, holy crab!

I usually use a super fine insulin needle (28 gauge is finer than 26 gauge) to puncture those air bubbles. Also pre-oil slide bottom will reduce bubble formation.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#14 Post by Hobbyst46 » Fri Jun 28, 2019 7:57 pm

zzffnn wrote:...
Many thanks, zzffnn, for your analysis and for taking the time and trouble to acquire and post the COL images. They pour some more light (pun) on my Darkfield experiments.

I take so much fun in the process of collection, processing and mounting my samples...that "standard" prepared slides are less attractive. Yet, I agree that the diatoms shown in my supposedly "COL" images were too difficult to resolve. I am glad to be reassured that the optical setup was essentially OK. The high concentration was chosen with the purpose of easily finding targets, since centration of the Ultracondenser is not easy. After the initial experience, I can switch to lower diatom concentrations. I will use 40X1.0 oil objective as auxiliary survey objective, to find potential target diatoms on the slide.

I do use 10X magnified focus preview on the camera. I think that shutter vibration is insignificant, but the sensitivity of the camera sensor is significant.
I shot at ISO 400 or 800. Exposure times were of the order of 1/10 S. The camera 3" LCD screen shows A LOT of noise at darkfield. That might have something to do with the metering. Or, the "noise" is a true representation of flare in the image, which my eyes somehow ignore or average out. So, I will try different modes of light metering. This feature of the camera I have mostly ignored so far.

Imperfect observed "COL" or "DF" might be a mixture of the two different effects. The fact, that in your own COL images of the Frustulia diatom, image quality and dot resolving power did not change when the condenser height changed, indicates, IMO, that it was indeed pure COL, without DF. I got the same result with my 100X1.3 and 63X1.25 phase contrast objectives. Yet the contrast in your COL images, on the whole, is better than in mine. Perhaps I can do better with other slides, and select the appropriate flat and thin diatom for resolution.

So far, I had no bubble problems. Bubbles in immersion oil tend to float and disappear as the viscosity drops. My immersion oil is Cargille Type A, of relatively low viscosity. Local temperatures are 24-32C (day and night), so, bubbles are rarely retained. I do not use oil Type B which may be better for inverted scopes.

The result which I especially like is the "nearly DF" images from the plain, non plan, non-phase 100X1.25 objective. IMHO, the brightly lit diatoms are mainly visible due to true DF, diffraction, not direct oblique and sidebands. If true, this is better than expected, although not ideal. Perhaps this partial DF was achieved by virtue of the very small diameter of the front lens of this very old objective. Alternatively, the phase contrast objectives perhaps reflect direct light into the objective, as suggested above by MicroBob, although I cannot envisage how.

Anyway,I will try to reproduce these "nearly darkfield" images with other old objectives from the Past. And try crude fluorescence with the Ultracondenser, just for fun.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#15 Post by zzffnn » Fri Jun 28, 2019 9:29 pm

Hobbyst46,

Your M10 camera's sensor is APSC and larger than my micro 43 sensor (supposedly your camera's iso performance will be better). I suggest using iso 200, aperture priority mode (let camera meter exposure based on dot or center-weighted metering mode and determine which shutter speed to use, even if shutter speed can be as long as a few seconds; mounted diatoms don't move). Then set your lens (if using any) to its sharpest aperture, as long as it is wider than f/8 and shoot with both jpg and raw. Lower iso and raw processing using external software will let you squeeze out more contrast and dot details from diatoms.

I double checked my iso and shutter values for you. For the Frustulia photos, I was at iso 200, shutter speed around 1/60-1/80, without maxing out my LED. For those small oval diatoms, I accidentally activated my bird photography presets and mistakenly had iso at 640. That might have degraded those images further.

Here is the same Frustulia diatom imaged with COL or darkfield using Leitz Heine condenser:
https://www.photomacrography.net/forum/ ... ulia+heine

You can see that dot resolving ability of DF is not as good as COL, at the same NA of 1.1.

With Leitz Heine, it is easy to achieve the transition state between DF and COL. However, such transition state reveals so much halo that its diatom dotting ability is worse than either DF or COL, based on my experiments. It works well to provide a 3D look for subjects without much internal details, such as an amoeba, but does not work well with diatoms.

A NA 1.25 objective would work closer to that transition state, than a NA 1.3 objective, from a 1.2-1.4 regular DF condenser. Many times, when the COL cone is close to objective edge and very narrow, it tends to produce more halos and poor contrast. A wider COL cone at around 80-85% as wide as objective outer edge tends to resolve more diatom dots for me.

Nearly perfect centration is required for DF and a good start for COL. With offset COL, many times I got worse dot resolution than that obtained from perfect centration, even though occasionally offset may resolve better. To make a good offset, I often have to examine both the diatom dots (through eyepiece or magnified camera focus preview) and the back focal plane (without eyepiece peeking down eye tube). That may be very inconvenient time consuming.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#16 Post by MicroBob » Sat Jun 29, 2019 6:30 am

Hi Doron,
in my experience the live view image is considerably moisier than the captured image in dim conditions.
The microscope image at these magnifications doesn't hold that much resolution. Especially for forum resolution 1024 pixel it should be ok to use ISO 800. For general use the lowest ISO setting is significantly better. Interestingly my old 6 megapixel Pentax K110D had about the same image quality between 200 and 800 ISO. Where is the progress here???
The exposure automatic does have a hard time with the extreme contrast in dark field images. I ended up with a high exposure compensation in aperture priority mode or manual exposure.
Your Canon probably has EFSC - make sure to have it switched on.

When I tried to resolve the dots of Amphipleura pellucida I got the best results with a decentered dark field condenser. Once the lighting was right the objective didn't matter that much any more.

Bob

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#17 Post by Hobbyst46 » Sat Jun 29, 2019 6:21 pm

Some more photos, after several try-and-errors with the camera settings, of diatoms through the Ultracondenser and high NA objectives..
I had read a general recommendation for afocal setups of photomicrography, to leave the camera lens aperture fully open, although in ordinary photography, closing the aperture about 2-3 stops improves the photo. I prefer fully Manual exposure over Av mode. For darkfield, since the light level is low, I think that F1.8 on the prime 50mm, 1.8 makes sense. Anyway, I find it most convenient for live view. For DF, I switched to ISO 200, and the shutter speed was about 0.5".

Images taken with 40X1.0 Planapo oil, and 100X1.25 oil, respectively, at F1.8. I think that the latter objective, despite not as dark bckg as the former, demonstrates slightly better resolution. The Cocconeis (elliptical) diatom is about 25um long.

The COL was taken with the 63X1.4 Planapo Ph3 oil, and F8. The condenser was slightly decentered. The contrast is better than my previous experience with the phase contrast objectives. I think that this effect of decentering is similar to what zzffnn had found. By cnotradistinction, the darkfield images taken with the 100X1.25 objective are very sensitive to condenser centration and condenser height.

Post processing: only crop and resize.
I will surf my slides to find more challenging diatoms for COL (smaller and denser dots and striae).
Attachments
100X1.25(1).jpg
100X1.25(1).jpg (135.59 KiB) Viewed 2721 times
40X1.0(1).jpg
40X1.0(1).jpg (158.35 KiB) Viewed 2721 times
63x1.4(1).jpg
63x1.4(1).jpg (152.95 KiB) Viewed 2721 times
Last edited by Hobbyst46 on Sat Jun 29, 2019 10:18 pm, edited 2 times in total.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#18 Post by Hobbyst46 » Sat Jun 29, 2019 7:00 pm

Yet another example, two images taken at ISO=200, F=1.8 and 5.6 respectively.
Post processing: crop and resize.
70um diatom, 100X1.3 oil, ISO-200, F-1.8.jpg
70um diatom, 100X1.3 oil, ISO-200, F-1.8.jpg (94.08 KiB) Viewed 2711 times
70um diatom, 100X1.3 oil, ISO-200, F-5.6.jpg
70um diatom, 100X1.3 oil, ISO-200, F-5.6.jpg (88.58 KiB) Viewed 2711 times
EDIT: SORRY ABOUT THE TYPO ERROR IN THE IMAGE TITLES - IT IS 100X 1.25 NA OBJECTIVE, NOT 1.3 NA.
Last edited by Hobbyst46 on Sun Jun 30, 2019 5:44 am, edited 2 times in total.
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#19 Post by Hobbyst46 » Sat Jun 29, 2019 9:41 pm

Here is a small note about diatom mounting.
For a while, I mounted diatoms in NOA61 glass adhesive. It has a quite high nD, is colorless, mechanically and chemically stable and easy to apply.
But...ocassionaly, if applied to the slide when it is cold, air bubbles are trapped among the diatoms. Moreover, sometimes air is trapped within diatoms. The air bubbles are evident in darkfield images (40X1.0 Planapo oil objective). Not all diatoms are infected. Heating the slides to remove the trapped bubbles is pointless, since NOA61 is not stable at temperatures above 60-70C.
Attachments
air filled diatoms.jpg
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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#20 Post by zzffnn » Sat Jun 29, 2019 11:12 pm

Hobbyst46,

They are look good to me.

f/5.6 seems slightly sharper than f/1.8 without 100% crop, but difference is not significant. I think since front lens (100x NA 1.3) of your system is already deep into diffraction, you won't lose anything when stopping down from f/1.8 to f/5.6, you may actually gain some contrast and flare reduction. I can't remember the math, but Pau at Photomacrography said you can safely stop down to f/8 in most cases.

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Re: Performance of high NA objectives with Zeiss Utracondenser (cardiodid)

#21 Post by Hobbyst46 » Sun Jun 30, 2019 7:00 am

zzffnn wrote:...
Thanks zzffnn.

I have just re-read the chapter about DF in the link below:
https://micro.magnet.fsu.edu/primer/tec ... field.html
It reminded me, that the NA of the DF illumination should be lower when the observed specimen is mounted in water (the swimming protists) than when it is mounted in a more refractive medium (such as my NOA61, n=>1.56, or Pleurax, n=>1.7). Just a thought.
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