Posted by
Zdenek Svindrych-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-Photon-Microscope-Low-Signal-Troubleshooting-tp7585738p7585744.html
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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
--
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, VA
http://www.kcci.virginia.edu/
tel: 434-982-4869
Annual FRET Workshop: http://kcci.virginia.edu/workshop-2017
---------- 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?