Re: Two Photon Microscope Low Signal Troubleshooting

Posted by Michael Giacomelli on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-Photon-Microscope-Low-Signal-Troubleshooting-tp7585738p7585752.html

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A word of caution about scan lenses:  they're typically designed for a narrow range of wavelengths at relatively high F/# where the effects of spherical and chromatic aberration are not pronounced.  When operated outside of their design range, performance is decreased.  You can get a sense of this effect just looking at the Thor catalog's scan angle vs spot size plots and comparing different wavelengths.  Thorlabs is essentially the only company out there that posts (accurate) Zemax model files of their objectives on their website.  Unless you are going to run them at exactly the specified wavelength and aperture, I would simulate before you build and check that everything is ok.

Also worth noting, Thor's Advanced Imaging group sells scan/tube pairs that are optimized for the Ti:S range (650-1200) and for very large field numbers at very low dispersion.  You used to buy individual lenses separately from the scan heads, but checking the catalog they're no longer listed.  I suspect if you call and ask you can still get one at a reasonable price.  These are significantly better than you will be able to construct on your own (and yes I've tried...) 

Mike

On Thu, Sep 29, 2016 at 10:32 AM, <[hidden email]> wrote:

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Hi Tim,

nice scan lens! I was always wondering whether that's necessary, it seems like an overkill for me. I though an achromatic doublet would do a good job, especially in intravital imaging, where the flatness of the field is not so critical (the mouse brain is not flat, after all).

Some people use beam cleanup before the scanner to make sure they're using just the fundamental mode for excitation. But I can't comment on the benefits in terms of reducing the incident laser power while maintaining the fluorescence intensity. In theory, if the laser is good (low M^2) and all mirrors flat, it shouldn't be necessary.

Another place where you can loose a lot of light is the detectors. Are you sure you're collecting all the light the objective can gather? The PMT's you're using have much smaller effective area then the old multialkali tubes (5 mm diameter vs maybe 20 mm), so you want to make sure your optical design is perfect! I've spent a number of sleepless nights trying to image the BFP aperture of my objective (just over 10 mm dia) onto my detectors (R10467U inside HPM-100 module, just 3 mm diameter sensitive area)...
Good luck!

zdenek

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Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
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---------- Původní zpráva ----------
Od: Tim <[hidden email]>
Komu: [hidden email]
Datum: 29. 9. 2016 2:25:39
Předmět: Re: Two Photon Microscope Low Signal Troubleshooting

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Thank you all for your responses.

@ Michael:

Our Ti-Sapphire has 100 fs pulses and is tuned to 900 nm. We use only protected silver mirrors and the following lenses from Thorlabs:

Initial Beam expansion with:

https://www.thorlabs.com/thorproduct.cfm?partnumber=AC254-030-B
https://www.thorlabs.com/thorproduct.cfm?partnumber=AC254-060-B

Scan lens: https://www.thorlabs.com/thorproduct.cfm?partnumber=LSM05-BB
Tube lens: https://www.thorlabs.com/thorproduct.cfm?partnumber=AC508-400-B

Do you think this would this constitute too many achromats?

@ Craig:

I am measuring power after objective using a power meter from Thorlabs (https://www.thorlabs.com/thorproduct.cfm?partnumber=S121C) held up against output end of the objective.

@ Andreas:

For the PS Speck beads on a slide, when you say that the image is not too far off the expected values do you mean the expected spatial resolution values of the system?