Tube lens for STED microscopy?

[JanTonnesen]

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Hello everyone

I am building a STED microscope around a Leica DMi8 body and will primarily use a glycerol objective with a correction collar to image in live mouse brain slices, within the first 0 to 50 microns of tissue. The excitation wavelength is 485 nm with stimulated emission at 595 nm. Down the line, I may also want to do 2-photon FRAP or uncaging, at wavelengths of 700-900 nm, though this is of secondary importance.  While I am working to obtain a designated scan lens and tube lens set from Leica, I am in parallel exploring alternative options.

In this graphic https://imgur.com/a/d6t6xEJ I am comparing specs for two Thorlabs tube lenses with a considerable price difference (1300 vs 4900 Euros). The cheaper one appears to be slightly superior in terms of modulation transfer function and RMS wavefront error. However, the more expensive one is superior in terms of transmission and axial color shift.

Given the shown data, can anyone enlighten me which parameter, RMS wavefront error or axial focus, will have the biggest impact on PSF quality and excitation PSF / STED PSF co-alignment in the sample, taking into account that I may image dynamically over a 50 micron z-range while correcting aberrations on the objective collar. I am particularly looking to avoid introducing a tissue depth dependent z-shift between the STED PSF and excitation PSF based on their spectral differences.

Any input is very welcome!
Jan

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Hi Jan,

Good luck in building the STED! It can get frustrating at times, but it is really fun once everything works out. Anyway, here is my opinion:

First of all, I would try to get the Leica tube lens, if possible/reasonable. Typically, they finely tune their tube lenses and objectives to complement each other. That being said, my previous Leica tube lens hat some very noticeable astigmatism, that made my STED PSF a little ugly - even though it did not seem to affect the efficiency noticeably. If, however, you need to go with one of the Thorlabs tube lenses, I would opt for the more expensive CLS2. My reasoning is as follows. Both lenses show a good RMS wavefront error, which is a hard value to assess, anyway. Specific aberrations have more pronounced effects on the STED PSF than others, so just lumping them all into one value can be misleading. What will affect your STED setup very noticeably, however, is the chromatic shift in your setup. You are, after all, aiming to perfectly align the center of two different color beams in space, with any discrepancy possibly devastating. If you want to go and image multicolored samples, you have to align at least 3 beams - more, if you want to add red/far-red to the mix. And even more, if you consider your detection path as an additional color path. Especially if you want to go deep into tissue, this effect can really be nasty. You can precompensate it, but that requires you to add some slight defocus to your beam to begin with - which can add more aberrations down the road. The second consideration is transmission, which immediately affects both your available STED power, as well as your detection signal. It looks like the cheaper tube lens has almost 10% less transmission at the intended wavelengths, which is really significant if you want to optimize your beam. More STED power means more resolution, and more signal is obviously always better, as well.

If you do need to test tube lenses, maybe you can ask if you can test them out in your system first - that is, assuming you already have it up and running (which might be difficult w/o the tube lens to begin with).

Good luck with your setup! Feel free to contact me with any questions - maybe I can give you some useful tips.

Greetings from Florida,
Nicolai

---------------------------------------------------------------------
Dr. Nicolai T. Urban
Imaging Center @ Max Planck Florida Institute for Neuroscience
1 Max Planck Way
FL 33418, USA




-----Original Message-----
From: Confocal Microscopy List <[hidden email]> On Behalf Of Jan Tonnesen
Sent: Friday, 25 October 2019 10:47
To: [hidden email]
Subject: Tube lens for STED microscopy?

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Hello everyone

I am building a STED microscope around a Leica DMi8 body and will primarily use a glycerol objective with a correction collar to image in live mouse brain slices, within the first 0 to 50 microns of tissue. The excitation wavelength is 485 nm with stimulated emission at 595 nm. Down the line, I may also want to do 2-photon FRAP or uncaging, at wavelengths of 700-900 nm, though this is of secondary importance.  While I am working to obtain a designated scan lens and tube lens set from Leica, I am in parallel exploring alternative options.

In this graphic https://nam03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fimgur.com%2Fa%2Fd6t6xEJ&amp;data=02%7C01%7CNicolai.Urban%40MPFI.ORG%7C7a28615571484e54467708d7595c5157%7C947b45517db44636a5fd1bdcad603ed0%7C0%7C0%7C637076125379357298&amp;sdata=bMws4mcToYEWiRmlu5egetdGBUf2gKTYfxQCRRdt9xY%3D&amp;reserved=0 I am comparing specs for two Thorlabs tube lenses with a considerable price difference (1300 vs 4900 Euros). The cheaper one appears to be slightly superior in terms of modulation transfer function and RMS wavefront error. However, the more expensive one is superior in terms of transmission and axial color shift.

Given the shown data, can anyone enlighten me which parameter, RMS wavefront error or axial focus, will have the biggest impact on PSF quality and excitation PSF / STED PSF co-alignment in the sample, taking into account that I may image dynamically over a 50 micron z-range while correcting aberrations on the objective collar. I am particularly looking to avoid introducing a tissue depth dependent z-shift between the STED PSF and excitation PSF based on their spectral differences.

Any input is very welcome!
Jan

[JanTonnesen]

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Hi Nicolai, I hope everything goes well!

Thank you for the input. Indeed I am hoping that I can get hold of a Leica scan lens/ tube lens pair. I also lean towards buying the more expensive one if it comes to that. I figure that even if the more expensive lens has notably higher RMS wavefront error at the excitation wavelength, then the more important thing is that it performs well at the stimulated emission wavelength.
I will definitely keep you in mind in case I encounter further issues where your experience could be useful.

All the best
Jan