Re: Confocal Shape changes in Round beads

Hi Jan,

That "oval effect" actually depends on the size of the beads, and the 
resolution of your optics.  If the size of the bead is smaller than the 
point-spread function of your optics, the image that you see will be the 
actual bead convolved with the point-spread-function (PSF) of your optics.

In the x-y direction, the pinhole of a properly tuned confocal microscope 
effectively eliminates the outer lobes of the point-spread-function so that 
you are only convolving your image with the main lobe. This has the effect 
of improving your resolution in the x-y direction as compared to 
conventional optics.

However, this does not affect the resolution in the z-direction, which 
stays basically the same. A well tuned machine, using 488 nm light, can 
typically achieve a resolution of about 0.3 um in the x-y direction and 0.7 
um in the z direction. What this means is that a bead smaller than 0.3 um 
will appear 0.3 um wide and 0.7 um high, hence your oval.

In order to obtain the proper spherical image, you have to "deconvolve" the 
output image. If compensation is considered a can of worms, deconvolution 
is a regular compost heap. The problem is how to accurately determine the 
point-spread-function of your object, in order to use this function for the 
convolution. The real PSF is almost always quite different from the 
theoretical one.

This is one of the main reasons that two-photon microscopy gives you higher 
resolution. The size of your spot is not determined only by the PSF of your 
objective, but can be tuned by adjusting the laser power and the cross 
section of the area where the two-photon affect occurs.

This smearing becomes insignificant when the beads become larger, because 
the size of the psf becomes negligibly small compared to the size of the 
bead, and the convolution does not affect the final image that much.

However, this effect has little to do with whatever problem Bob wrote about 
in the first place. Blebbing from one angle and not another could not be 
accounted for by this phenomenon. In that case, I would guess that there is 
some inconsistency between where the spot actually is, and where the 
computer thinks it is when it is reconstructing the image.

-Dave


At 12:17 AM 5/13/2004, Jan Bayer wrote:
>Excuse me for interfering in this discussion, I have no pretention 
>whatsoever of being knowledgeable in microscopy-related issues. 
>Nevertheless, I do remember having listened in on a discussion of this 
>same issue in my lab, years ago.
>
>If I remember correctly, the phenomenon is perfectly normal. Everey 
>perfectly round object will be imaged as an oval in the Z-axis on confocal 
>systems. It has to do with breaking index changes in the light trajectory 
>going from the microscope-objective to the object (and back). Movements 
>along the Z-axis would probably have to be corrected for this phenomenon, 
>in order to image perfect spheres as such.
>
>Please, could one of the image specialists explain this in sufficient 
>detail ??!!
>
>Jan Bayer
>On 11 May 2004, at 00:41, Nebe-Von-Caron, G wrote:
>Jan A. Bayer, Ph.D.
>Fondation Jean Dausset - CEPH
>27, Rue Juliette Dodu
>75010 Paris
>TEL: +33 1 53 72 51 14
>FAX: +33 1 53 72 51 28

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