3D reconstruction from confocal microscopy data

Alex Sossick, Wellcome/CRC Institute, Cambridge, UK

Confocal laser scanning microscopy is a relatively non-destructive technique, with major advantages over conventional microscopy. A major feature of confocal microscopy is that only what is in focus is detected, and anything out of focus is not detected. Importantly this permits the three dimensional imaging of biological specimens. In the confocal microscope all structures being out of focus are suppressed at image formation. This is achieved because of the object not being illuminated and imaged as a whole at the same time, but at one point after the other. Rays from out-of-focus are suppressed by the detection pinhole. A sample therefore can be optically sectioned, and from this three dimensional reconstruction is possible. The depth of the focal plane is, besides the wavelength of light, determined in particular by the numerical aperture of the objective used and the diameter of the confocal diaphragm. At a wider detection pinhole the confocal effect can be reduced. Three dimensional imaging is therefore an important technique for examining confocal data.

References

Confocal laser scanning microscopy, Sheppard & Shotton	Handbook of Biological Confocal Microscopy, edited by J.B. Pawley
Confocal imaging - what is it? http://www.llt.de/conprin.html	Confocal Principles http://www.bocklabs.wisc.edu/imr/facility/confb.htm
Arabidopsis Web site MRC Laboratory of Molecular Biology http://brindabella.mrc-lmb.cam.ac.uk/ http://brindabella.mrc-lmb.cam.ac.uk/IndexConfcl.html	Chris Pudney's Bio-rad read/write module is available here