Fluorescence photobleaching-based image standardization for fluorescence microscopy

Authors

  • Ghauharali,

    1. Institute for Molecular Cell Biology, Section of Molecular Cytology, BioCentrum, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
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  • Brakenhoff

    1. Institute for Molecular Cell Biology, Section of Molecular Cytology, BioCentrum, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
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Professor Brakenhoff Tel: + 31 20 525 5189; fax: + 31 20 525 6271; e-mail: brakenhoff@bio.uva.nl

Abstract

A method is presented for the standardization of images acquired with fluorescence microscopy, based on the knowledge of spatial distributions proportional to the microscope's absolute excitation intensity and fluorescence detection efficiency distributions over the image field. These distributions are determined using a thin fluorescent test layer, employed under practically mono-exponential photobleaching conditions. It is demonstrated that these distributions can be used for (i) the quantitative evaluation of differences between both the excitation intensity and the fluorescence detection efficiency of different fluorescence microscopes and (ii) the standardization of images acquired with different microscopes, permitting the deduction of quantitative relationships between images obtained under different imaging conditions.

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