Confocal Microscopy List - Re: Two Photon Microscope Low Signal Troubleshooting

Re: Two Photon Microscope Low Signal Troubleshooting

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URL: http://confocal-microscopy-list.275.s1.nabble.com/Two-Photon-Microscope-Low-Signal-Troubleshooting-tp7585738p7585753.html

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Guy,
As I imaged at 800 and this should be treated as high NA, one would use 0.325 * lambda / (sqrt(2) * NA^0.91 and multiply with 2*sqrt(ln2) to convert from 1/e radius to fwhm (Zipfel et al 2003) which then gives 293 nm. So l calculated my own PSFs taking the vector properties and polarisation into account and got 320 nm. Convolution with the bead size of 175 nm gives roughly 365 nm (assuming Gaussian profiles). The difference between theory (365 nm) and real life (410 nm)was only 12%. It did not matter as the cells were 5 um diameter and this was all we wanted to image.

Best wishes

Andreas

From: [hidden email]
Sent: ‎29/‎09/‎2016 18:04
To: [hidden email]
Subject: Re: Two Photon Microscope Low Signal Troubleshooting

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

@ 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

From: [hidden email]
Sent: ‎29/‎09/‎2016 07:25
To: [hidden email]
Subject: 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?