Re: Sampling rate for confocal microscopy

Posted by George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Shearing-Interferometer-with-Ti-Sapph-tp7585684p7585697.html

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Hi Andreas,

I suggest ALL of:

* X,Y pixel size = 100 nm

* Z pixel size = 300 nm

(you can evaluate smaller values later, if the below help).

* Re-evaluate the fluorophore(s) you are using.

* optimize mounting medium to minimize photobleaching of the fluorophore(s) you are using, also match R.I. with the immersion medium (R.I. ~1.515).

* 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).

* Evaluate all of the current deconvolution vendors, SVI, AutoQuant, Microvolution, to get demo's (once you've done the other things).

* Use a Booster ... treat your current current (or post-re-evaluation) fluorophore as a hapten. For example, if you are stuck with Alexa Fluor 488, detect with an anti-X and then detect that (FASER has all the reagents):

AntiAlexa Fluor® 488, Rabbit IgG Fraction (A-11094)

https://tools.thermofisher.com/content/sfs/manuals/mp11094.pdf

if you are still using fluorescein (and have optimized pH 8.0 and it is still dim), Molecular Probes has anti-fluorescein but I'll use as examples,

Anti-Fluorescein antibody (ab19491)
http://www.abcam.com/fluorescein-antibody-ab19491.html

Miltenyi Biotec's FASER kits (for APC, "FITC", PE ... Fluorescence Amplification by Sequential Employment of Reagents, check out all the tabs on the product page)

http://www.miltenyibiotec.com/en/products-and-services/macs-flow-cytometry/reagents/support-reagents/faser-kits.aspx

Consider detecting with a 'state of the art' fluorophore, like Brilliant Violet BV421 (BD Biosciences), though may require very low laser power. Be sure to use their buffer that comes with the kits, especially if expanding the experiments to multiple Brilliant's (they tend to aggregate in water, hence their inclusion of the buffer). You could also "change colors" to quantum dots, of which QD625 is the brightest of the Molecular Probes product line (shorter excitation wavelength has higher extinction coefficient and longer effective Stokes shift ... maybe we should call the difference of wavelength used and emission peak wavelength the "Bruchez shift").

May as many of your photons as possible be detected and PSFs symmetrical,

George

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

 

 

-----Original Message-----
From: Craig Brideau [hidden email]
To: CONFOCALMICROSCOPY [hidden email]
Sent: Wed, Sep 21, 2016 5:30 pm
Subject: Re: Shearing Interferometer with Ti-Sapph

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The way I check collimation in a Ti:Saph is using a cheap CCD or CMOS camera placed at a few different points in the beam. As long as the IR filter has been removed, the camera should be able to 'see' the beam projected onto the array. Do note you will want some good reflective ND filters on there to keep from burning the camera! You want to take pictures at several points along the path, or just use mirrors to extend the path while leaving the sensor stationary. You can get a rough measurement of spot size vs. distance which will give you some indication of where the Rayleigh range of the beam is located.

And yes, coherence length of a Ti:Saph is typically ~30um, so it won't work with a shearing interferometer. Also, running the Ti:Saph in CW may produce a slightly different vergence out of the laser. Finally, don't sweat collimation too much, you can always put a pair of good achromats into the beam as a telescope to adjust the beam waist position.

Craig

On Wed, Sep 21, 2016 at 8:50 AM, Michael Giacomelli <[hidden email]> wrote:

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Hi Tim,

A shearing interferometer is not going to work with an ultrafast
laser.  There is a note in the thorlabs catalog that can help you
figure out what it will work with:

https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=2970

The model you have has a 21.16 mm difference in optical pathlength, so
you will need a coherence length at least that long.  Try your laser
in CW mode, although even that may not be quite 2 cm depending on how
stable it is without a mode lock.  Usually though testing for
collimation after a beam expander doesn't require an interferometer
since the beam is large the divergence is low.  Have you tried just
bouncing it across the room and back with a mirror?

Mike

On Wed, Sep 21, 2016 at 12:07 AM, Tim <[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Hello, we recently purchased a shearing interferometer from Thorlabs (Part # SI254) to test collimation after beam expansion in our two-photon microscope setup. The strange thing is we cannot seem to get an interference pattern either before or after beam expansion (varying the distance between the lenses in the beam expander). We do see the pattern clearly if we try on a simple laser diode. Does anyone have experience looking at the shearing interferometer pattern with a Ti-Sapphire laser or have any ideas what may be causing our problem?
>
> Thanks for any help,
> Tim