the standard way to model a confocal PSF would be to take a
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi Milton,
the standard way to model a confocal PSF would be to take a widefield PSF (at
the excitation wavelength) for the excitation, and use a widefield PSF (at the
mean emmission wavelength) convolved with the pinhole shape as the detection and
to multiply the two together. Beyond this basic principle it really depends on
how realistic you want to get, as this will determine how sophisticated your PSF
model needs to be. The questions you need to ask are:
- do you want to model spherical abberation/ refractive index mismatch
- do you need a full vectorial model (high numeric aperture objectives or
polarised excitation sources)
- do you want to consider possible misalignment between excitation and emission
PSFs
The answer to all these questions is probably yes, but will depend on what your
exact application is. Using the mean emission wavelength is usually seen as a
good approximation, but if you've got a particularly broad emission spectrum you
might want to consider this as well.
If you don't need a full vectorial model I've got some matlab code floating
around which generates PSFs based on the paraxial assumption (low NA,
unpolarised) and includes refractive index mismatch effects. Once I'm back home
(middle of the coming week) I might be able to dig this up.
Another hint is that any measured profile will be based on a finite voxel size /
ROI - so the standard equations (ie sinc^2 etc ...) don't really work. A better
approach is to generate a theoretical 3D PSF and extract the profile from this
in the same way you would from your experimental data.
cheers,
David
----- Original Message ----
From: "
[hidden email]" <
[hidden email]>
To:
[hidden email]
Sent: Wed, 4 May, 2011 11:19:04 PM
Subject: Curves of intensity versus axial position
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
Hi everybody,
I apologize for this basic question but I really need help in order to be
able to represent in Matlab the curves of intensity versus z for non-ideal
cases.
In fact I made the representation of these curves for confocal (ideal
pinhole) and slit detection but I have to make it for real values (not
approximate to zero) of pinhole radius and slit widths to compare with
experimental results.
All inputs will be greatly appreciated. Of course Matlab routines would be
great!
Many thanks,
Milton
Locked
