Diatom imaged with 313nm light

[jmc]

Great, I very much look forward to knowing the results! Thank you. I hope it at least has some useful properties, even without deep transmission.

The Nikon UV-F 100x NA 1.3 arrived (along with a 10x Fluor NA 0.5 as well) so I measured the transmission of them in the UV.

The Nikon UV-F 100x NA 1.3 is as follows (note that transmission is normalized 0-1 as the diameter of the rear element of this is smaller than the light beam in my measurement setup).

It transmits down to about 310-320nm which is in keeping with what the paper you shared mentioned.

The Fluor 10x NA 0.5 also had good UV reach.

Unlike the 100x UV-F, this is now absolute transmission as the rear element on it is huge. As the with UV-F this would be good down to about 320nm.

Both of these would offer great transmission for 365nm work (as for image quality at that wavelength that remains to be seen).

For those who haven't seen one, here's a photo of the Nikon 10x Fluor. Some big chunks of glass in there.

[Lou Jost]

That's better than I had expected! Thanks for doing these tests. I wonder what the image quality is like.

Did you do these tests with glycerine immersion? I wouldn't expect it to matter much, but the internal reflection off the front surface of the front lens would be reduced if it were immersed in glycerine.

[jmc]

That's better than I had expected! Thanks for doing these tests. I wonder what the image quality is like.

Did you do these tests with glycerine immersion? I wouldn't expect it to matter much, but the internal reflection off the front surface of the front lens would be reduced if it were immersed in glycerine.

No problem, if I do some imaging with them in the UV I'll share the results. Yes, they both have rather good UV reach.
The transmission was just the lens, no immersion fluid. It's on a setup I originally built for measurement of camera lenses, so can't test for things like immersion. It'd make a few percent difference to absolute transmission but wouldn't impact the depth of transmission.

[jmc]

That's better than I had expected! Thanks for doing these tests. I wonder what the image quality is like.

Did you do these tests with glycerine immersion? I wouldn't expect it to matter much, but the internal reflection off the front surface of the front lens would be reduced if it were immersed in glycerine.

Hi Lou, I had a play with the Nikon 100x UV-F for UV trasnmission imaging today. These images were of aluminium coated diatoms prepared by John Dale. Imaged at 365nm, in brightfield with glycerine immersion on the objective (but not on the condenser, which was a Zeiss quartz NA 0.85 one). Single images, no stacking, as I still haven't figured out a good way of stacking diatom images. Massively reduced in resolution for sharing here - originals are full frame on a Nikon d800 which is 7360x4912.

[Lou Jost]

Thanks for sharing these! They look pretty good and they seem like they would clean up rather well.

[jmc]

Thanks for sharing these! They look pretty good and they seem like they would clean up rather well.

No problem. I was quite happy with them. I am currently at about the limit of my ability with making my diatom images 'better' though - any pointers about where to look for advice is welcome.

[LouiseScot]

I have the same Nikon 100x - I must have another play with it too! I get distracted too easily but will try and have another go with it sometime this week. I did get some borosilicate coverslips from Laboquip https://londonlaboquip.com/product/labo ... 8mm-glass/ which have a refractive index close to glycerol. http://www.vidrasa.com/eng/products/duran/duran_pf.html The latter is usually quoted as 1.47 though I don't know what it is at shorter wavelengths - would be higher. I've not got around to trying Boro 3.3 cover slips yet, and I'm not sure if they'll make much difference anyway. I think the 0.85 NA of your condenser will be limiting. A higher NA one with glycerol coupling to the slide might be better. Without that, the high NA of the objective might be somewhat wasted even at short wavelengths? The max overall NA using air coupling will always be limited to 1 (probably a bit less in practice). See homogenous immersion system https://www.olympus-lifescience.com/en/ ... immersion/

Of course, a scanning electron microscope (SEM) would be good!

Louise

[jmc]

I have the same Nikon 100x - I must have another play with it too! I get distracted too easily but will try and have another go with it sometime this week. I did get some borosilicate coverslips from Laboquip https://londonlaboquip.com/product/labo ... 8mm-glass/ which have a refractive index close to glycerol. http://www.vidrasa.com/eng/products/duran/duran_pf.html The latter is usually quoted as 1.47 though I don't know what it is at shorter wavelengths - would be higher. I've not got around to trying Boro 3.3 cover slips yet, and I'm not sure if they'll make much difference anyway. I think the 0.85 NA of your condenser will be limiting. A higher NA one with glycerol coupling to the slide might be better. Without that, the high NA of the objective might be somewhat wasted even at short wavelengths? The max overall NA using air coupling will always be limited to 1 (probably a bit less in practice). See homogenous immersion system https://www.olympus-lifescience.com/en/ ... immersion/

Of course, a scanning electron microscope (SEM) would be good!

Louise

Interesting about the coverslips. I've not seen those before.

Yes, absolutely the NA of the objective will be an issue here and that it was used dry. I seem to recall I had the 100x UV-F turned down to 0.8 for these images anyway as I didn't have the condenser 'glycerined' to the underside of the slide. For some of my UV imaging I have used glycerine as a coupling fluid, but with these Al coated diatoms I did not.

Oh, and yes, an SEM at home would be lovely, but not something that is going to happen in the short term (or so I have been told by my wife).

[LouiseScot]

I have the same Nikon 100x - I must have another play with it too! I get distracted too easily but will try and have another go with it sometime this week. I did get some borosilicate coverslips from Laboquip https://londonlaboquip.com/product/labo ... 8mm-glass/ which have a refractive index close to glycerol. http://www.vidrasa.com/eng/products/duran/duran_pf.html The latter is usually quoted as 1.47 though I don't know what it is at shorter wavelengths - would be higher. I've not got around to trying Boro 3.3 cover slips yet, and I'm not sure if they'll make much difference anyway. I think the 0.85 NA of your condenser will be limiting. A higher NA one with glycerol coupling to the slide might be better. Without that, the high NA of the objective might be somewhat wasted even at short wavelengths? The max overall NA using air coupling will always be limited to 1 (probably a bit less in practice). See homogenous immersion system https://www.olympus-lifescience.com/en/ ... immersion/

Of course, a scanning electron microscope (SEM) would be good!

Louise

Interesting about the coverslips. I've not seen those before.

Yes, absolutely the NA of the objective will be an issue here and that it was used dry. I seem to recall I had the 100x UV-F turned down to 0.8 for these images anyway as I didn't have the condenser 'glycerined' to the underside of the slide. For some of my UV imaging I have used glycerine as a coupling fluid, but with these Al coated diatoms I did not.

Oh, and yes, an SEM at home would be lovely, but not something that is going to happen in the short term (or so I have been told by my wife).

I'll try and have a go with the glycerol immersion this week though I doubt I'll be able to produce such good images as you've made. I don't/won't have any slides I can try the borosilicate cover slips on, but I could try some glycerol on the condenser and maybe do a comparison without.

Louise

[Macro_Cosmos]

I did a comparison between 3 40x objectives with different specific immersion mediums (air, water, oil) to prove a silly point, it could be relevant here as well.
Many images to process. Overall, immersion gave better and brighter images despite similar NA (1 vs 0.95 for air).
This was some kind of vanity project because some guy I know kept saying a 40x oil immersion with NA 1 is pointless because a 40x dry exists.

[jmc]

I did a comparison between 3 40x objectives with different specific immersion mediums (air, water, oil) to prove a silly point, it could be relevant here as well.
Many images to process. Overall, immersion gave better and brighter images despite similar NA (1 vs 0.95 for air).
This was some kind of vanity project because some guy I know kept saying a 40x oil immersion with NA 1 is pointless because a 40x dry exists.

Your findings make sense to me. If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used. Out of interest, was whether or not the condenser was used with immersion fluid looked at as part of the test?

[rjlittlefield]

If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used.

Another possible factor: at same NA, the light cones at the subject will be narrower, in proportion to the RI of the immersion fluid.

So where NA 0.95 requires light rays that are angled at 71.8 degrees off-axis, NA 1.0 in RI 1.51 requires only 41.5 degrees off axis.

--Rik

[LouiseScot]

If nothing else there is less scattering/reflections because of the lack of air interfaces when the immersion fluid is used.

Another possible factor: at same NA, the light cones at the subject will be narrower, in proportion to the RI of the immersion fluid.

So where NA 0.95 requires light rays that are angled at 71.8 degrees off-axis, NA 1.0 in RI 1.51 requires only 41.5 degrees off axis.

--Rik

Interesting, Rik. Do you by any chance have a link to an explanatory source for the theory behind that?
Thanks
Louise

[rjlittlefield]

Another possible factor: at same NA, the light cones at the subject will be narrower, in proportion to the RI of the immersion fluid.

So where NA 0.95 requires light rays that are angled at 71.8 degrees off-axis, NA 1.0 in RI 1.51 requires only 41.5 degrees off axis.

--Rik

Interesting, Rik. Do you by any chance have a link to an explanatory source for the theory behind that?

I do not have a reference handy, and reviewing the thread, I see that the theory I used does not apply here.

The calculation that I used assumed that the diatoms themselves were dry versus immersed. In that case the standard formula that NA = n sin(θ) can be solved to give the angles that I listed.

But this thread is dealing with diatoms mounted under coverslips, in which case the diatoms themselves are always immersed and same NA will give same angles at the subject.

Brain glitch -- my bad. Been looking at too many images of dry diatoms recently...

--Rik

[LouiseScot]

Another possible factor: at same NA, the light cones at the subject will be narrower, in proportion to the RI of the immersion fluid.

So where NA 0.95 requires light rays that are angled at 71.8 degrees off-axis, NA 1.0 in RI 1.51 requires only 41.5 degrees off axis.

--Rik

Interesting, Rik. Do you by any chance have a link to an explanatory source for the theory behind that?

I do not have a reference handy, and reviewing the thread, I see that the theory I used does not apply here.

The calculation that I used assumed that the diatoms themselves were dry versus immersed. In that case the standard formula that NA = n sin(θ) can be solved to give the angles that I listed.

But this thread is dealing with diatoms mounted under coverslips, in which case the diatoms themselves are always immersed and same NA will give same angles at the subject.

Brain glitch -- my bad. Been looking at too many images of dry diatoms recently...

--Rik

Oh, ok - no worries. Thanks for the clarification also

Louise