Water/Glycerine immersion instead of oil immersion for darkfield

Here you can discuss different microscopic techniques and illumination methods, such as Brightfield, Darkfield, Phase Contrast, DIC, Oblique illumination, etc.
Message
Author
Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Water/Glycerine immersion instead of oil immersion for darkfield

#1 Post by Hobbyst46 » Tue Mar 23, 2021 10:13 pm

Following the notion of immersion darkfield in another post, here are some experiments with water, instead of oil.
The specimen is a Pleurosigma diatom, mounted in Pleurax.

Darkfield was obtained with the D position of the phase contrast condenser. Here is how Zeiss describes it:
For dark-field work, the N.A. 1.4 front lens of the condenser is immersed (object distance in glass 1.1-1.3mm) . lts aperture is then 1.1-1.4,
and it is suited for use in conjunction with N.A.0 .65 to 1.0.
The objective was Planapo 40X1.0 oil immersion, with iris, so its range of NA is 0.6-1.0
So I used distilled water, rather than oil, for both objective immersion and condenser top lens contact.
Images are untouched, except for cropping.
Pleurosigma is a fascinating optical device. Whenever I inspect it with top illumination, for example, under the stereoscope, it behaves like a prism or grating.
I forgot to turn off room lights, those caused some unwanted brightness of the dark background, in the bottom right corner. Apparently, good DF photos should be taken in a semi dark room.
Attachments
1) NA=0.6 full image.jpg
1) NA=0.6 full image.jpg (35.96 KiB) Viewed 7196 times
2) NA=0.6 Crop.JPG
2) NA=0.6 Crop.JPG (91.44 KiB) Viewed 7196 times
3) NA=1.0 full image.jpg
3) NA=1.0 full image.jpg (37.05 KiB) Viewed 7196 times
4) NA=1.0 Crop.JPG
4) NA=1.0 Crop.JPG (109.39 KiB) Viewed 7196 times
Last edited by Hobbyst46 on Thu Mar 25, 2021 10:37 am, edited 1 time in total.

hans
Posts: 986
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Water immersion instead of oil immersion for darkfield

#2 Post by hans » Wed Mar 24, 2021 2:42 am

Interesting test, thanks. I am working on plotting glass-water-glass transmission vs. NA (somewhat separate issue from aberrations) as mentioned in the other thread but not finished yet. Might be useful to try to separate effect of watering the condenser vs. objective in this sort of experiment? I haven't tried but watering the objective is expected to add quite a bit of spherical aberration at high NA, I think? Watering the condenser would presumably add some also, but not so obvious what effect SA on the illumination side should have on final image quality. I looked for a while but never found a clear technical explanation of the benefit of an aplanatic condenser.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#3 Post by Hobbyst46 » Wed Mar 24, 2021 11:59 am

hans wrote:
Wed Mar 24, 2021 2:42 am
... Might be useful to try to separate effect of watering the condenser vs. objective in this sort of experiment? I haven't tried but watering the objective is expected to add quite a bit of spherical aberration at high NA, I think? Watering the condenser would presumably add some also, but not so obvious what effect SA on the illumination side should have on final image quality. I looked for a while but never found a clear technical explanation of the benefit of an aplanatic condenser.
Just made some more tests to cover the (a) condenser vs objective and (b) spherical aberrations.

For such tests, diatoms are not really useful. So I pulled out the ancient Zeiss negative stage micrometer from the depth of the drawer.
negative stage micrometer.jpg
negative stage micrometer.jpg (37.7 KiB) Viewed 7143 times
Below, there are photos of the scale, using either the turret condenser (always watered to the slide bottom) or the Ultracondenser (always watered); and compared the Planapo 40X{0.7-0.8} (that is, objective iris set and fixed at 0.7-0.8), water immersed, to the Neofluar 40X0.75 Ph2 dry objective. Note that the Planapo 40X0.8 objective does not need a coverslip, whereas the Neofluar 40x0.75 requires a 0.17mm coverslip. So, in photo no. 7, a drop of water was placed on the stage micrometer, and a coverslip was added.
All photos were taken with the afocal setup, a 12.5X KPL eyepiece and a 50mm Prime camera lens. These limit the image width to 220um (through a 40X objective), vs ~450um through a 10X viewing eyepiece. So my conclusions are valid for the central area.
Some corner brightness in the otherwise black background originate from ambient lights and possibly from incomplete centration of the condenser, or maybe some fault in the condenser. These are very old condensers that I refurbished.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#4 Post by Hobbyst46 » Wed Mar 24, 2021 12:03 pm

(continued)
(note: the condenser in photo no. 9 is watered to the slide, as in all other images).
In all photos, the scale is not exactly horizontal, because the camera was slightly rotated.
IMO, SA is not that bad, maybe a very slight pincushion.
CA is awful, on the other hand, especially with the dry 40X0.75 objective.

An image of the same scale, taken with the Ultracondenser and OIL immersion, will be shown shortly.
Attachments
5) Watered turret D condenser, 40X0.8 water immersion.JPG
5) Watered turret D condenser, 40X0.8 water immersion.JPG (99.49 KiB) Viewed 7142 times
6) Watered turret D condenser, 40X0.75 dry, no coverslip.JPG
6) Watered turret D condenser, 40X0.75 dry, no coverslip.JPG (114.36 KiB) Viewed 7142 times
7) Watered turret D condenser, 40X0.75 dry, coverslip over water.JPG
7) Watered turret D condenser, 40X0.75 dry, coverslip over water.JPG (85.28 KiB) Viewed 7142 times
8) Watered Ultracondenser, 40X0.8 water immersion.JPG
8) Watered Ultracondenser, 40X0.8 water immersion.JPG (100.75 KiB) Viewed 7142 times
9) Ultracondenser, 40X0.75 dry, no coverslip.JPG
9) Ultracondenser, 40X0.75 dry, no coverslip.JPG (110.27 KiB) Viewed 7142 times
Last edited by Hobbyst46 on Wed Mar 24, 2021 12:26 pm, edited 1 time in total.

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#5 Post by viktor j nilsson » Wed Mar 24, 2021 12:23 pm

Well, that number 8) is a cracking combination!

A little puzzled by some of them.

- If your 40x 0.75 is designed to be used with a coverslip, then why is the image with coverslip (7) so much worse than the one without (6)?

- Very surprising that the water immersed 40x 0.8 perform worse with the turret D condenser (image 5) than the 40x 0.75 (image 6). Could image 5 suffer from vibrations?

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#6 Post by Hobbyst46 » Wed Mar 24, 2021 12:32 pm

Now for the "standard". Oil immersion of the Ultracondenser (as per its design), with the dry objective and the oil immersion objective. The latter is, likewise, properly oiled to the slide, not watered. No coverslips in both.
Visually, these arrangements yield the best images, clearly better than the water-immersion images. The stage marks appear sharp and clean, not fuzzy.
Attachments
10)  Oiled Ultracondenser, 40X0.75 dry.JPG
10) Oiled Ultracondenser, 40X0.75 dry.JPG (109.05 KiB) Viewed 7129 times
11) Oiled Ultracondenser, 40X0.8 oil immersion.JPG
11) Oiled Ultracondenser, 40X0.8 oil immersion.JPG (105.54 KiB) Viewed 7129 times
Last edited by Hobbyst46 on Wed Mar 24, 2021 1:17 pm, edited 1 time in total.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#7 Post by Hobbyst46 » Wed Mar 24, 2021 12:42 pm

viktor j nilsson wrote:
Wed Mar 24, 2021 12:23 pm
Well, that number 8) is a cracking combination!

A little puzzled by some of them.

- If your 40x 0.75 is designed to be used with a coverslip, then why is the image with coverslip (7) so much worse than the one without (6)?

- Very surprising that the water immersed 40x 0.8 perform worse with the turret D condenser (image 5) than the 40x 0.75 (image 6). Could image 5 suffer from vibrations?
Thanks for the comment.
Yes, the 40X0.75 is 160/0.17, and the result is weird indeed - yet, I am not sure about this negative stage micrometer. The glass disk within the metal plate - are they the same thickness ? what is the distance between the glass scale and the bottom of the coverslip (i.e. how thick is the liquid layer) ? I never found any specific info on this stage micrometer which I inherited ages ago.

About the image 5 vs image 6 - agreed, calls for verification. These exposures are long, since I set the camera ISO to 200.

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#8 Post by viktor j nilsson » Wed Mar 24, 2021 12:49 pm

Wait, are the white lines supposed to have a dark center? Then why are the lines all white in #8 and #9. Now I am even more confused!

Are the lines scribed into a dark-colored glass? Or are they cut straight through?

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#9 Post by Hobbyst46 » Wed Mar 24, 2021 1:12 pm

Hobbyst46 wrote:
Wed Mar 24, 2021 12:42 pm
viktor j nilsson wrote:
Wed Mar 24, 2021 12:23 pm
Well, that number 8) is a cracking combination!

A little puzzled by some of them.

- If your 40x 0.75 is designed to be used with a coverslip, then why is the image with coverslip (7) so much worse than the one without (6)?

- Very surprising that the water immersed 40x 0.8 perform worse with the turret D condenser (image 5) than the 40x 0.75 (image 6). Could image 5 suffer from vibrations?
Thanks for the comment.
Yes, the 40X0.75 is 160/0.17, and the result is weird indeed - yet, I am not sure about this negative stage micrometer. The glass disk within the metal plate - are they the same thickness ? what is the distance between the glass scale and the bottom of the coverslip (i.e. how thick is the liquid layer) ? I never found any specific info on this stage micrometer which I inherited ages ago.

About the image 5 vs image 6 - agreed, calls for verification. These exposures are long, since I set the camera ISO to 200.
Just to eliminate doubts about vibrations etc, repeated images 5 and 6 above.
Attachments
14) repeated Watered turret D condenser, 40X0.8, water immersion.JPG
14) repeated Watered turret D condenser, 40X0.8, water immersion.JPG (109.42 KiB) Viewed 7130 times
15) repeated Watered turret D condenser, 40X0.75 dry, no coverslip.JPG
15) repeated Watered turret D condenser, 40X0.75 dry, no coverslip.JPG (110.73 KiB) Viewed 7130 times

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#10 Post by Hobbyst46 » Wed Mar 24, 2021 1:18 pm

Please note that the titles of photos 10 and 11 are now corrected. They were switched by mistake. Sorry.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#11 Post by Hobbyst46 » Wed Mar 24, 2021 1:59 pm

viktor j nilsson wrote:
Wed Mar 24, 2021 12:49 pm
Wait, are the white lines supposed to have a dark center? Then why are the lines all white in #8 and #9. Now I am even more confused!

Are the lines scribed into a dark-colored glass? Or are they cut straight through?
The stage micrometer is made of a glass disk that forms an opaque background, and the grid lines are somehow inscribed on it - or they are slits - and should be bright. Perhaps the disk is coated with metal and the thin lines were formed by etching. Have no idea, would be interesting to know. They are very thin and elegant lines.
Their image under brightfield is just bright full uniform strips, without any "center". Plain narrow strips. However, under dark field, they appear to be "hollow", narrow elongated frames. Since the light hits the slits at an angle, possibly the slit walls are illuminated whereas the core remains relatively dark.
So, when resolution is high, under dark field, the frames are well defined. Under lower resolution, the frame limits seem to coalesce and merge.

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#12 Post by viktor j nilsson » Wed Mar 24, 2021 2:58 pm

Hobbyst46 wrote:
Wed Mar 24, 2021 1:59 pm
viktor j nilsson wrote:
Wed Mar 24, 2021 12:49 pm
Wait, are the white lines supposed to have a dark center? Then why are the lines all white in #8 and #9. Now I am even more confused!

Are the lines scribed into a dark-colored glass? Or are they cut straight through?
The stage micrometer is made of a glass disk that forms an opaque background, and the grid lines are somehow inscribed on it - or they are slits - and should be bright. Perhaps the disk is coated with metal and the thin lines were formed by etching. Have no idea, would be interesting to know. They are very thin and elegant lines.
Their image under brightfield is just bright full uniform strips, without any "center". Plain narrow strips. However, under dark field, they appear to be "hollow", narrow elongated frames. Since the light hits the slits at an angle, possibly the slit walls are illuminated whereas the core remains relatively dark.
So, when resolution is high, under dark field, the frames are well defined. Under lower resolution, the frame limits seem to coalesce and merge.
It does sound like they may have been made with the method Rowland invented in 1882 to produce diffraction gratings: to scratch thin parallel grooves in metal deposited on a flat piece of glass using a diamond point. Maybe the dark centers arise because the grooves are triangular in profile? Interesting to see this effect.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#13 Post by Hobbyst46 » Wed Mar 24, 2021 3:15 pm

viktor j nilsson wrote:
Wed Mar 24, 2021 2:58 pm
Hobbyst46 wrote:
Wed Mar 24, 2021 1:59 pm
viktor j nilsson wrote:
Wed Mar 24, 2021 12:49 pm
Wait, are the white lines supposed to have a dark center? Then why are the lines all white in #8 and #9. Now I am even more confused!

Are the lines scribed into a dark-colored glass? Or are they cut straight through?
The stage micrometer is made of a glass disk that forms an opaque background, and the grid lines are somehow inscribed on it - or they are slits - and should be bright. Perhaps the disk is coated with metal and the thin lines were formed by etching. Have no idea, would be interesting to know. They are very thin and elegant lines.
Their image under brightfield is just bright full uniform strips, without any "center". Plain narrow strips. However, under dark field, they appear to be "hollow", narrow elongated frames. Since the light hits the slits at an angle, possibly the slit walls are illuminated whereas the core remains relatively dark.
So, when resolution is high, under dark field, the frames are well defined. Under lower resolution, the frame limits seem to coalesce and merge.
It does sound like they may have been made with the method Rowland invented in 1882 to produce diffraction gratings: to scratch thin parallel grooves in metal deposited on a flat piece of glass using a diamond point. Maybe the dark centers arise because the grooves are triangular in profile? Interesting to see this effect.
Like the Zeiss positive stage micrometer, they are very fine, I admire it. The modern Chinese micrometer lines are many times thicker (though cheaper...).

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#14 Post by Hobbyst46 » Wed Mar 24, 2021 5:48 pm

So, what can one learn from the test ?
Within the present framework, especially the center portion of the FOV, using 40x0(0.7-0.8) objectives, we see that:

The chromatic corrections of the two different objectives are as expected from a fluorite vs planapo.
Water immersion, instead of oil immersion, yielded a decent image from the oil-immersion objective.
For both the turret condenser and the dedicated DF condenser, water immersion yielded decent DF. Without immersion, images are hardly visible.
Image 10 is better than image 9, so oil immersion is visually better than water immersion (for the same condenser and dry objective).
Image 11 is better than image 14 (or 5), so the combination of oil immersion on condenser and objective is visually better than the combination of water immersion of both components.
Spherical aberration is small, if at all, in all images.
Given that water immersion is less messy than oil, it appears inviting, at least for non-critical work. Especially for the condenser.

hans
Posts: 986
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Water immersion instead of oil immersion for darkfield

#15 Post by hans » Wed Mar 24, 2021 7:14 pm

Hobbyst46 wrote:
Wed Mar 24, 2021 12:42 pm
Yes, the 40X0.75 is 160/0.17, and the result is weird indeed - yet, I am not sure about this negative stage micrometer. The glass disk within the metal plate - are they the same thickness ? what is the distance between the glass scale and the bottom of the coverslip (i.e. how thick is the liquid layer) ? I never found any specific info on this stage micrometer which I inherited ages ago.
If there is any dirt on the micrometer can you tell focusing up and down whether the ruler pattern is exactly on the top surface vs. already under some thickness of glass?
viktor j nilsson wrote:
Wed Mar 24, 2021 12:49 pm
Wait, are the white lines supposed to have a dark center? Then why are the lines all white in #8 and #9. Now I am even more confused!

Are the lines scribed into a dark-colored glass? Or are they cut straight through?
viktor j nilsson wrote:
Wed Mar 24, 2021 2:58 pm
Maybe the dark centers arise because the grooves are triangular in profile?
If the lines are transparent openings in the metallization then the dark center would be expected with darkfield illumination since only light reflected/diffracted from the edges of the openings would make it into the objective?

User avatar
Wes
Posts: 1027
Joined: Sat Mar 09, 2019 12:58 pm

Re: Water immersion instead of oil immersion for darkfield

#16 Post by Wes » Wed Mar 24, 2021 7:52 pm

Now that is a well controlled comparison. Thanks for posting Hobbyst. Number 11) looks like a top of the line homogenous immersion system, really nice and crispy.

I only have the universal condenser D position darkfield option at my disposal and I've used it with water which produced ok results at low to medium NA (up to about 0.75)

This video was taken with with planapo 10/0,32 and watered D stop: https://www.youtube.com/watch?v=AnIPV7qQmOw
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
Youtube channel

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#17 Post by viktor j nilsson » Wed Mar 24, 2021 8:24 pm

Agree, 11) looks perfect.

Now that we know what it is capable of - would you be interested in doing that one with glycerol instead? I would love to see that.

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#18 Post by viktor j nilsson » Wed Mar 24, 2021 8:27 pm

hans wrote: If the lines are transparent openings in the metallization then the dark center would be expected with darkfield illumination since only light reflected/diffracted from the edges of the openings would make it into the objective?
Ah, yes that's a reasonable interpretation.

User avatar
Wes
Posts: 1027
Joined: Sat Mar 09, 2019 12:58 pm

Re: Water immersion instead of oil immersion for darkfield

#19 Post by Wes » Wed Mar 24, 2021 9:44 pm

hans wrote:
Wed Mar 24, 2021 2:42 am
Interesting test, thanks. I am working on plotting glass-water-glass transmission vs. NA (somewhat separate issue from aberrations) as mentioned in the other thread but not finished yet. Might be useful to try to separate effect of watering the condenser vs. objective in this sort of experiment? I haven't tried but watering the objective is expected to add quite a bit of spherical aberration at high NA, I think? Watering the condenser would presumably add some also, but not so obvious what effect SA on the illumination side should have on final image quality. I looked for a while but never found a clear technical explanation of the benefit of an aplanatic condenser.
Different wavelengths experience differences in refractive index within the same material which gives rise to dispersion so I'm curious if it will have an effect on the NA of the condenser - sample - objective system.
Zeiss Photomicroscope III BF/DF/Pol/Ph/DIC/FL/Jamin-Lebedeff
Youtube channel

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#20 Post by Hobbyst46 » Wed Mar 24, 2021 9:47 pm

Wes wrote:
Wed Mar 24, 2021 7:52 pm
Now that is a well controlled comparison. Thanks for posting Hobbyst. Number 11) looks like a top of the line homogenous immersion system, really nice and crispy.

I only have the universal condenser D position darkfield option at my disposal and I've used it with water which produced ok results at low to medium NA (up to about 0.75)

This video was taken with with planapo 10/0,32 and watered D stop: https://www.youtube.com/watch?v=AnIPV7qQmOw
Great video ! I concentrated on the 40X, although I have some 25X including an immersion. 10X is too weak for "my" diatoms...
Last edited by Hobbyst46 on Wed Mar 24, 2021 9:52 pm, edited 1 time in total.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#21 Post by Hobbyst46 » Wed Mar 24, 2021 9:51 pm

viktor j nilsson wrote:
Wed Mar 24, 2021 8:24 pm
Agree, 11) looks perfect.

Now that we know what it is capable of - would you be interested in doing that one with glycerol instead? I would love to see that.
My glycerol is old so it contains some water, I will give it a try though and in addition, try and find drier glycerol. Pure glycerol is MUCH more viscous and tacky than good old Water...

apochronaut
Posts: 6272
Joined: Fri May 15, 2015 12:15 am

Re: Water immersion instead of oil immersion for darkfield

#22 Post by apochronaut » Wed Mar 24, 2021 10:22 pm

hans wrote:
Wed Mar 24, 2021 2:42 am
I looked for a while but never found a clear technical explanation of the benefit of an aplanatic condenser.
Just an older more expensive way of doing what an aspheric lens does.

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#23 Post by viktor j nilsson » Wed Mar 24, 2021 10:38 pm

Hobbyst46 wrote:
Wed Mar 24, 2021 9:51 pm
viktor j nilsson wrote:
Wed Mar 24, 2021 8:24 pm
Agree, 11) looks perfect.

Now that we know what it is capable of - would you be interested in doing that one with glycerol instead? I would love to see that.
My glycerol is old so it contains some water, I will give it a try though and in addition, try and find drier glycerol. Pure glycerol is MUCH more viscous and tacky than good old Water...
Yes, that's an annoying habit of glycerol. Mine is also old. Not sure how much water to expect in a half-full almost 10 years old bottle.

Some folks seem to get it pretty dry by drying it in the oven at 175°C:
[Link removed]

It cites this paper:
http://orgsyn.org/demo.aspx?prep=CV1P0015
Screenshot_20210324-234343~2.png
Screenshot_20210324-234343~2.png (108 KiB) Viewed 7019 times
Might be worth a try. I don't need it to be completely anhydrous, but it would be nice to know roughly what concentration it is when I mix water:glycerine for my silicone oil objective tests.

Edit: I removed the link to the forum post, but kept the reliable article. Don't want to promote stupid behavior.
Last edited by viktor j nilsson on Wed Mar 24, 2021 11:57 pm, edited 1 time in total.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water immersion instead of oil immersion for darkfield

#24 Post by Hobbyst46 » Wed Mar 24, 2021 11:04 pm

viktor j nilsson wrote:
Wed Mar 24, 2021 10:38 pm
...glycerol...
I shall try to work with glycerol of known humidity. I hope you do not even think of doing that stupid heating of glycerol in a home oven. The cited reliable article says "in a sand bath, in the hood" and tells you to monitor the temperature of the liquid. Glycerol might decompose or even catch fire in a closed poorly-controlled oven. The RI of even 90% glycerine (that is, quite wet) should not be that different from that of pure glycerol. And the shelf life of very dry glycerol in an open container is short...

viktor j nilsson
Posts: 760
Joined: Thu Aug 23, 2018 10:12 pm
Location: Lund, Sweden

Re: Water immersion instead of oil immersion for darkfield

#25 Post by viktor j nilsson » Wed Mar 24, 2021 11:17 pm

Hobbyst46 wrote:
Wed Mar 24, 2021 11:04 pm
viktor j nilsson wrote:
Wed Mar 24, 2021 10:38 pm
...glycerol...
I hope you do not even think of doing that stupid heating of glycerol in a home oven. The cited reliable article says "in a sand bath, in the hood" and tells you to monitor the temperature of the liquid. Glycerol might decompose or even catch fire in a closed poorly-controlled oven.
Oh really? It's been a good long while since I took organic chemistry, but I really can't see how such terrible things would happen with a small quality of glycerol kept at 175°C for three hours?

Edit: the flash point of pure glycerol is 176°C, so it could in theory catch fire at those temperatures if there's a spark.
Last edited by viktor j nilsson on Thu Mar 25, 2021 9:33 am, edited 1 time in total.

hans
Posts: 986
Joined: Thu May 28, 2020 11:10 pm
Location: Southern California

Re: Water immersion instead of oil immersion for darkfield

#26 Post by hans » Wed Mar 24, 2021 11:38 pm

Wes wrote:
Wed Mar 24, 2021 9:44 pm
Different wavelengths experience differences in refractive index within the same material which gives rise to dispersion so I'm curious if it will have an effect on the NA of the condenser - sample - objective system.
Interesting point, and a bit confusing to think about in terms of NA since the definition of NA factors in refractive index. Abbe numbers for glass mostly in the 30-70 range, and water is 55, which is a couple percent variation in refractive index across the visible spectrum, so seems like the basic effect on overall intensity of the image and resolution would be pretty small. But of course closely related to axial CA and achromatic condensers presumably exist for a reason, although as with aplanatic I have not found a clear technical description of how axial CA in the condenser affects the final image.
apochronaut wrote:
Wed Mar 24, 2021 10:22 pm
hans wrote:
Wed Mar 24, 2021 2:42 am
I looked for a while but never found a clear technical explanation of the benefit of an aplanatic condenser.
Just an older more expensive way of doing what an aspheric lens does.
I guess I was thinking of aplanatic as specifying the degree of correction (free from lowest-order spherical and coma) independent of specific implementation (more spherical surfaces vs. one aspheric surface) and would like to find a technical description of how exactly aberrations in the condenser show up in the final image.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water/Glycerine immersion instead of oil immersion for darkfield

#27 Post by Hobbyst46 » Thu Mar 25, 2021 10:49 am

Modified the post title to include glycerine.
So I discovered a few ml's of pure dry glycerine in my Lock-Lock storage box and gave it a shot with the D position of the turret condenser.
Following are photos under the same conditions as above, only that glycerine replaces oil.
16) Glycerinated turret D condenser, Planapo 40X0.8 glycerine immersion.JPG
16) Glycerinated turret D condenser, Planapo 40X0.8 glycerine immersion.JPG (106.14 KiB) Viewed 6959 times
17) Glycerinated turret D condenser, Neofluar 40X0.75 dry.JPG
17) Glycerinated turret D condenser, Neofluar 40X0.75 dry.JPG (97.31 KiB) Viewed 6959 times
Again, the immersion 40X objective performs better than the dry objective. Yet the main point is, that very good DF is obtained from the glycerinized condenser. Slightly inferior than that of oil immersion, as can be judged from the longitudinal "cores" of the stripes. And better than that of water immersion. No surprise, but nice to know.

Whether glycerine is less messy than oil is, I think, a matter of personal taste. Glycerine can be completely removed with water; oil can be completely removed with solvents.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water/Glycerine immersion instead of oil immersion for darkfield

#28 Post by Hobbyst46 » Thu Mar 25, 2021 10:54 am

BTW, when I can get access to true epi-illumination, will try and find the shape of the grooves (?) in that negative stage micrometer.
Looking again at the images of the diatom above, obtained with the Planapo 40X0.8 immersion objective, vs the image of the stage micrometer through the same objective, it is evident (I think) that the wonderful spectrum of colors from the diatom mainly originates from the object, not the optics.

apochronaut
Posts: 6272
Joined: Fri May 15, 2015 12:15 am

Re: Water/Glycerine immersion instead of oil immersion for darkfield

#29 Post by apochronaut » Thu Mar 25, 2021 11:26 am

Oil doesn't need to be completely removed if the immersion objective or condenser is being used in immersion frequently. It is nice and makes one feel better but a light wipe with a soft tissue is all that is needed. Any residue will easily become amalgamated during the next use. Oil is also useful, where the front lens of an objective or the top lens of a condenser have suffered some abuse over the years. I have a Bausch & Lomb 100X phase objective that looks like it was bombarded by micrometeorites, yet works perfectly under oil immersion. It is hard to overlook the value of high N.A. immersion.
For a universal condenser, maybe yes , one would need to clean it well after immersion in oil because it will be used dry sometimes.
I am looking into the cost of quartz slides and coverslips from China. They are prohibitively expensive from standard suppliers. Ideal with glycerin immersion, since the index of refraction is so similar. One would then have 1.52 -1.46-1.45-1.33-1.45-1.46-1.52, when viewing an aqueous sample. Presumably, the close homogeneity in the lens to lens interspace would result in better imaging.
Last edited by apochronaut on Thu Mar 25, 2021 8:02 pm, edited 1 time in total.

Hobbyst46
Posts: 4277
Joined: Mon Aug 21, 2017 9:02 pm

Re: Water/Glycerine immersion instead of oil immersion for darkfield

#30 Post by Hobbyst46 » Thu Mar 25, 2021 11:45 am

apochronaut wrote:
Thu Mar 25, 2021 11:26 am
Oil doesn't need to be completely removed if the immersion objective or condenser...
Glycerine, I think would be better remove completely since it is hygroscopic.
Borosilicate glass also has an RI of 1.47. Are there slides made of such glass ? they might be less expensive than quartz. The major advantage of quartz is transparency to UV.

Post Reply