In 2-photon microscopy your resolution (FWHM) should be 0.5 * wavelength / (1.414 * NA) . At 900nm and NA 1.05 this works out at 303nm. Surely if you are
just imaging beads you should be able to do better than 410nm?
Guy
Guy Cox, Honorary Associate Professor
School of Medical Sciences
Australian Centre for Microscopy and Microanalysis,
Madsen, F09, University of Sydney, NSW 2006
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Andreas Bruckbauer
Sent: Thursday, 29 September 2016 5:18 PM
To: [hidden email]
Subject: Re: Two Photon Microscope Low Signal Troubleshooting
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@ 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?
Yes, I get about 410 nm fwhm in xy and about 1.2 um in z with a 25x 1.05 NA objective when the back aperture is nicely filled. If the z resolution is bad, you might not see small objects but will do just fine imaging larger ones. Also check if you are bleaching
the sample before you actually see it, but in this case I would expect a short spike in fluorescence.
Best wishes
Andreas
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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?
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