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. 2006 Jun 6;5:36. doi: 10.1186/1475-925X-5-36

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Copyright © 2006 Diaspro et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MPE fluorescence emission distribution. Confinement of the emission process, in green, under red 2PE. Under 2PE the only molecules excited are those confined in a small subfemtoliter volume at the illumination beam focus position. This is particularly relevant for the photobleaching process that is globally reduced with respect to the 1PE case. The capacity of the volume can be approximatively calculated by using the resolution parameters of the optical system, since they are indicators of the volume containing the maximum photon density. This is valid only for non saturated processes. Under saturation of fluorescence beam intensity plays an important role in the emission shape and optical resolution.