Intensity Calibration and Flat‐Field Correction for Fluorescence Microscopes

Michael Model1

1 Department of Biological Sciences, Kent State University, Kent, Ohio
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 10.14
DOI:  10.1002/0471142956.cy1014s68
Online Posting Date:  April, 2014
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Standardization in fluorescence microscopy involves calibration of intensity in reproducible units and correction for spatial nonuniformity of illumination (flat‐field or shading correction). Both goals can be achieved using concentrated solutions of fluorescent dyes. When a drop of a highly concentrated fluorescent dye is placed between a slide and a coverslip it produces a spatially uniform field, resistant to photobleaching and with reproducible quantum yield; it can be used as a brightness standard for wide‐field and confocal microscopes. For wide‐field microscopes, calibration can be further extended to absolute molecular units. This can be done by imaging a solution of known concentration and known depth; the latter can be prepared by placing a small spherical lens in a diluted solution of the same fluorophore that is used in the biological specimen. Curr. Protoc. Cytom. 68:10.14.1‐10.14.10. © 2014 by John Wiley & Sons, Inc.

Keywords: fluorescence microscopy; confocal microscopy; standardization; calibration; shading correction; sodium fluorescein; Acid Fuschin; Rose Bengal; Acid Blue 9

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Concentrated standard solutions including:
    • Sodium fluorescein (Sigma‐Aldrich) for blue excitation, green emission
    • Rose Bengal (Sigma‐Aldrich) for green excitation, red emission
    • Acid Blue 9 (TCI America) for red excitation, far‐red emission
    • Prepare 0.25 g of the dye in 1 ml water; vortex initially, and then continue dissolving for 30 min on a shaker or in a sonication bath
  • Diluted solution of the fluorophore that was used to stain the specimen (approximately 20 μl of a 10 μM solution)
  • Widefield or confocal fluorescence microscope (upright widefield microscope for calibration in molecular units)
  • Slides and coverslips
  • Unmounted lens with at least one spherical surface (for example, half‐ball with 5 mm radius, Edmund Optics, cat. no. 45‐937)
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Literature Cited

Literature Cited
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