Re: Optical slice thickness and number for PSF and deconvolution

Posted by Mark Cannell on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Optical-slice-thickness-and-number-for-PSF-and-deconvolution-tp5415740p5418195.html

Hi All

I'm sorry but this advice is wrong. The pinhole is a control that  
_should_ be used when decreased (mainly z) resolution is acceptable. The
lasers can then be turned down and, if desired, decon. can be used to
help clean up the image. The problem is that many users want a  "pretty
picture" but pretty pictures may not be needed for quantification of
(say) number of mitochondria.  As we say on the Vancouver course, "Every
photon is precious" and you may also increase signal by accepting a
wider spectral band or using an LP filter.  The key to good experimental
work is to understand what measurement you want and then to pick
conditions that allow you to get sufficient data with sufficient (not
too many) time points to answer your question. Do you need a full 3D
image or will a couple of slices suffice? Use a high NA lens. As others
have said, consider using widefield with a high QE CCD if you really
don't need the maximum possible resolution in 3D...

My 2c

Mark Cannell


Vincent wrote:

> *commercial interest*
>
>
> Dear Jan,
>
> The amount of the signal in images is mostly judged just after image
> acquisition. Based on this it is often decided to use a wider pinhole.
> As you probably know, when deconvolution is properly performed you will gain not
> only an increase in resolution but also in signal. Therefore, we advise to close
> the pinhole and use deconvolution for increasing the signal (to noise) before
> determining the quality of the image.
>
> As with imaging the object of interest it is important to follow the Nyquist
> criteria for imaging the bead images.
> We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator) to
> determine these rates. You can also create a picture here of your theoretical
> PSF to get an idea of its dimensions.
>
> In general it is best to really match the Nyquist criterion in xyz. Else you can
> go for 2x more. This however may introduce other problems like e.g., bleaching.
> If the bead images are differently sampled it requires interpolation for
> matching that, making the process of deconvolution more computationally
> demanding. Thus Nyquist is okay. Another important thing to keep in mind is that
> you need to image enough planes to cover your PSF.
>
> I hope this answers your questions.
> Best regards,
> Vincent
>
> ***********************************************************
> Vincent Schoonderwoert, PhD
> Scientific Volume Imaging bv
> Hilversum, The Netherlands
> [hidden email]
> [hidden email]
> Tel: + 31 35 646 8216
> ***********************************************************
>
>
>
>
>
> Jan Trnka wrote:
> > Dear list,
> >
> > this is probably a trivial question but so far I haven't found a good answer.
> > When taking 3D images of subresolution beads in a confocal microscope (for PSF
> > construction) does the number and thickness of slices in the z-stack need to
> > be exactly the same as that of a sample to be deconvolved? I understand the
> > x-y dimensions need to be the same but how does it work for z? Would a higher
> > number of thinner slices (finer z resolution) of the bead improve the
> > construction of the PSF? My actual samples are imaged with a rather wide
> > pinhole setting to limit the exposure of the sample (live cells) and thus
> > provide quite thick optical sections.
> >
> > Thanks,
> >
> > Jan
> >
> > Jan Trnka, MD, PhD
> > Department of Biochemistry
> > 3rd Medical Faculty
> > Ruska 87
> > 100 00 Praha 10
> > Czech Republic
> > [hidden email] <mailto:[hidden email]>
> > Tel.: [phone]
> >
> >
> >
>
>
>
>