Re: Confocal Shape changes in Round beads

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:

> Two reasons come to mind from normal microscopy: the bead cooking 
> effect
> mainly at high UV but most likely the cover slip squash effect where 
> due
> to evaporation the volume under the cover slip becomes less and less,
> leading to considerable pressure on the beads. Should not happen when
> you use a slightly higher sample volume and seal the edges with a thin
> layer of Vaseline. I use a 1ml syringe and just go round the edges of
> the cover slip.
>
> Please tell me the final solution of the riddle when found.
>
> Gerhard
>
> -----Original Message-----
> From: Zucker.Robert@epamail.epa.gov
> [mailto:Zucker.Robert@epamail.epa.gov]
> Sent: 07 May 2004 20:06
> To: cyto-inbox
> Subject: Confocal Shape changes in Round beads
>
>
>
>
>
>
> Beads are normally used in flow cytometers to QA the instrument. The
> measurement of beads using light scatter or Coulter electronic volume
> can yield histogram distributions that are not always representative of
> the actual size of the particle due to various physical measurement
> factors. Using a confocal microscope, we observed that a round bead is
> not always a round bead when it comes to measurements.
>
> Beads are supposed to be round in XY scanning using a confocal
> microscope. What causes beads not to be round in an image.??
>
> While zooming 2-3 x, I noticed a single slice of the bead at the
> midsection was sometimes round, but other times looked oblong or had
> blebs protruding from the slice.
>
> Solid objects were changing their shape before my very eyes on my 400K
> confocal microscope !!!! How can this be?? They were definitely not
> round with some scans but were round in other scans. What is the cause
> of this confocal instability to yield irregular shaped beads ?
>
> Any one else observe this phenomenon?.
> Is the galvanometer in the system defective? Any other ideas?? It may
> also be interesting to discuss why flow beads are not always round in
> their distributions.
>
> Bob
>
>
> Robert M. Zucker, PhD
> U.S. Environmental Protection Agency
> Office of Research and Development
> National Health and Environmental Effects Research Laboratory
> Reproductive Toxicology Division, MD 72 Research Triangle Park, North
> Carolina, 27711
> Tel: 919-541-1585; fax 919-541-4017
> e-mail: zucker.robert@epa.gov
>
>
>
>
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