Sorry.
Wrong line cited the last time. I wanted to ask George why
he suggests to use the median pixel intensity for frame
averaging.
Tobias
From:
Confocal Microscopy List
[[hidden email]]
On Behalf Of George McNamara
Sent: Thursday, September 22, 2016 4:10 PM
To: [hidden email]
Subject: Re: Sampling rate for confocal microscopy
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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
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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?
-----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.
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
--
George McNamara, PhD
Houston, TX 77054
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75/
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650