Confocal Microscopy

Abstract

By using apertures reduced to the physical limit imposed by the numerical aperture and magnification of the objective lens as well as the wavelength of the light, confocal fluorescence microscopy effectively achieves point illumination and point detection. This permits ‘optical sectioning’; a stack of sections can be used to derive a three‐dimensional intensity distribution and to calculate projections along arbitrary axes.

Keywords: microscopy; confocal; resolution; two‐photon; laser; fluorescence; fluorophore; photobleaching

Figure 1.

Axial discrimination in conventional and confocal fluorescence microscopy. The microtubules in a fibroblast were labelled with an Alexa488‐tagged antibody. Two images were recorded in the same plane close to the coverslip. Image (a) was recorded with a large pinhole diameter and indicates the situation encountered in conventional microscopy. Image (b) was recorded with the optimal pinhole diameter for confocal fluorescence microscopy. Thin areas of the cell (small arrow) are very well resolved in both images; here, the conventional microscope provides no obvious advantage over the confocal microscope. The thick parts surrounding the nucleus can be resolved only with the confocal microscope; in this plane, only the microtubules close to the coverslip are visible. Other parts of the object become visible by moving the lens into a different focal plane. Sample from Luisa Pieroni and Eric Karsenti; images by James Jonkman (Cell Biology and Biophysics Programme, EMBL, Heidelberg).

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References

Pawley JB (ed.) (1995) Handbook of Biological Confocal Microscopy. London: Plenum Press.

Stelzer EHK (1998) Contrast, resolution and the signal to noise ratio in fluorescence microscopy. Journal of Microscopy 189: 15–24.

White J, Johannes L, Mallard F et al. (1999) Rab6 coordinates a novel Golgi to ER retrograde transport pathway in live cells. Journal of Cell Biology 147(4): 743–759.

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How to Cite close
Stelzer, Ernst HK(Nov 2001) Confocal Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002639]