Posted by
Zdenek Svindrych on
URL: http://confocal-microscopy-list.275.s1.nabble.com/discussion-is-open-continues-about-localization-and-super-resolution-tp7581644p7581647.html
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Hi,
also a brute force simulation of many images of many molecules and
subsequent localisation with your preferred method is a straightforward
(albeit a bit time consuming) way to get the localisation accuracy (and
precision...) at nearly real-life conditions. You can simulate any photon
counts, background, detector pixelation, effect of molecule density, biases
of the localisation algos and so on.
Hopefully our group (Guy Hagen et al) will soon publish a software tool to
greatly simplify this. But I still didn't find a satisfactory astigmatic PSF
model to include into the simulation. The cylindrical gaussian is really not
close approximation of our experimental results...
Best,
zdenek svindrych
lf1.cuni.cz
---------- Původní zpráva ----------
Od: Sripad Ram <
[hidden email]>
Komu:
[hidden email]
Datum: 4. 3. 2014 23:17:16
Předmět: Re: discussion is open...continues ... about localization and super
resolution
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Hi Chris,
The z-localization precision for astigmatism based 3D localization was
worked out by holtzer et al., applied physics letters, 2007, 90, 053902.
On a related note, the precise calculations for the X/Y localization
precision that includes background and pixilation was published in 2004 by
Raimund Ober group (Ober et al; Biophysical Journal, 2004, 86:1185-1200).
You can download the software to calculate the localization precision from
here (
http://www.wardoberlab.com/software/fandplimittool/).
Hope this helps.
Regards,
Sripad
-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On
Behalf Of Christophe Leterrier
Sent: Tuesday, March 04, 2014 3:16 PM
To:
[hidden email]
Subject: Re: discussion is open...continues ... about localization and super
resolution
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This is a great introduction to a question a wanted to ask to the SMLM
specialists (I did not find the answer in the above reference). I also
posted it on the Nanoscopy LinkedIn group, so I'm sorry for people that will
get it two times!
There are several formulas to estimate the lateral (XY) precision of
individual localization events in PALM/STORM microscopy. This goes from the
crude sigma(PSF)/√Nphotons to more elaborate formulas that includes
pixelation and background (such as Thomson & Webb Biophys J 2002, corrected
in Mortensen Nat Methods 2010). What I'm looking for is an equation that
allows to estimate the precision for the Z localization when using
astigmatism-based 3D PALM/STORM. The reference I have found where this
precision is studied theoretically (such as Badierostami Applied Phys Lett
2010, Shaevitz Int J Optics 2009) do not use a directly calculable formula.
I assume the precision depends on the photon number and the point on the Z
calibration curve with the respective X and Y PSF width. Is anyone aware of
such a formula?
Best Regards,
Christophe
2014-03-04 21:52 GMT+01:00 Alberto Diaspro <
[hidden email]>:
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>
http://www.nature.com/nmeth/journal/v11/n3/full/nmeth.2843.html>"