Collecting PSF files for Zeiss 100X oil objective

[Krishanu Ray]

Hello every one,

We are trying to use the deconvolutionJ plug-in to deconvolve an
epifluorescent image stack, collected using the Zeiss 100X NA 1.4
NeoFluar objective and a Hamamatsu ORCA camera.

Now we are wondering how to make the PSF file for the objective,
which is esssential for the deconvolution.

We will be grateful if someone could help us with a setp-by-step
instruction, or, it would be even better if someone could supply the file.

many thanks,

krishanu.

Krishanu Ray
Department of Biological Sciences
Tata Institute of Fundamental Research
Homi Bhabha Road, Colaba, Mumbai 400 005.
India.
Ph: [phone] ext 2730
Fax: [phone]

[Giancarlo Mascetti]

Hello,
         what do you need is to acquire a set of optical sections of
microspheres fluo sample of 0.1 micron in diameter and to collect data at
0.1 micron or less step along z-axis. I suggest to start 10 microns below
and to stop 10 microns above the focal plane using 512x512 and to follow
the method described in Agard&Sedat  paper:

Biophysical Journal, Vol 57, 325-333
Determination of three-dimensional imaging properties of a light microscope
system. Partial confocal behavior in epifluorescence microscopy
Y Hiraoka, JW Sedat and DA Agard

Giancarlo




At 19.39 15/12/2005 +0530, you wrote:

>Hello every one,
>
>We are trying to use the deconvolutionJ plug-in to deconvolve an
>epifluorescent image stack, collected using the Zeiss 100X NA 1.4
>NeoFluar objective and a Hamamatsu ORCA camera.
>
>Now we are wondering how to make the PSF file for the objective,
>which is esssential for the deconvolution.
>
>We will be grateful if someone could help us with a setp-by-step
>instruction, or, it would be even better if someone could supply the file.
>
>many thanks,
>
>krishanu.
>
>Krishanu Ray
>Department of Biological Sciences
>Tata Institute of Fundamental Research
>Homi Bhabha Road, Colaba, Mumbai 400 005.
>India.
>Ph: [phone] ext 2730
>Fax: [phone]

Giancarlo Mascetti, Ph.D.
X-Istituto di Calcolo Scientifico

c/o Paramed Health Services Ltd.
c.so F. M. Perrone, 73r - 16152 Genova, Italy
tel. [phone] - mob. [phone] - fax. [phone]
E-mail: [hidden email]




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