Re: Sampling rate for confocal microscopy

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Dear all,

 

Many thanks for the replies, it looks like opinion is divided with George suggesting 100 nm and Giulia from SVI suggesting the 46 nm of the Nyquist calculator. It would be best to do the test to see if there is any improvement with the 40 nm sampling, does anyone know of papers looking into this? I would have thought that there is a sweet-spot and further reducing the pixel size (at the same total acquisition time) would just lower the signal to noise ratio and any useful signal would be buried in noise. But I guess the deconvolution algorithm nicely sums up the pixels to get the signal back. Is there any advantage of acquiring e.g. one big pixel instead of four smaller ones? I am thinking of something similar like minimising read-out-noise of CCD cameras by binning, just for the confocal case, or does the PMT and read out electronics behave like CMOS where there is no advantage of binning?

 

best wishes

 

Andreas

 

-----Original Message-----
From: Giulia De Luca <[hidden email]>
To: CONFOCALMICROSCOPY <[hidden email]>
Sent: Thu, 22 Sep 2016 16:55
Subject: Re: Sampling rate for confocal microscopy

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Hello Andreas,

As imaging specialist at SVI I am happy to answer your question. We always suggest to use our Nyquist Calculator, that you nicely link, to calculate the ideal x, y and z sampling specific for your imaging settings. This will assure that you optimally preserve the spatial frequencies in your images for further image restoration with deconvolution. Such small sampling distances may arise the worry for bleaching. However, because the restoration procedure will improve the Signal to Noise Ratio of the images dramatically, this should not worry you! You can proceed in lowering the laser power or the exposure time/acquisition speed and then proceed with deconvolution afterwards. You will see that the noise will be taken care of in the deconvolution process.

Please do not hesitate to contact us if you have any further questions, we are happy to help you!

Best regards,

Giulia

2016-09-22 17:37 GMT+02:00 Tobias Rose <[hidden email]>:

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Just quickly:

* optimize laser power (low), detector settings (modest gain, maybe higher than you think), scan speed (fast is good, resonant scan is faster and therefore better) with averaging (median would be even better, if in your software or you are willing to make the extra effort).

Why median?

Tobias

 

On 9/22/2016 7:22 AM, [hidden email] wrote:

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Dear all,

What would be a sensible lateral sampling rate for a confocal z-stack when the sample has relatively weak fluorescence and one wishes to apply deconvolution to get the most out of the data? The Nyquist calculator https://svi.nl/NyquistCalculator gives 46 nm for an NA 1.3 oil immersion lens. I would think this causes too much bleaching and  think that about 80 - 100 nm should be enough, based on 230 nm resolution (limited by S/N ratio of the weak sample) and Nyquist sampling?

 

best wishes

 

Andreas