Intensity Calibration and Flat‐Field Correction for Fluorescence Microscopes

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.

[1]  Robert M Zucker,et al.  Evaluation of confocal microscopy system performance. , 2001, Methods in molecular biology.

[2]  Brian Herman,et al.  Quantitative Fluorescence Microscopy , 2000, Fluorescence Microscopy.

[3]  K. Jalink,et al.  Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart‐based calibration procedures , 2008, Journal of microscopy.

[4]  John T Elliott,et al.  Automated live cell imaging of green fluorescent protein degradation in individual fibroblasts , 2007, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[5]  Karl Garsha,et al.  Quantitative Fluorescence Microscopy: Considerations and Controls , 2008 .

[6]  B. Herman,et al.  Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study. , 1994, Biophysical journal.

[7]  Armin Schneider,et al.  Expression of Hemoglobin in Rodent Neurons , 2009, Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism.

[8]  M. Model,et al.  A standard for calibration and shading correction of a fluorescence microscope. , 2001, Cytometry.

[9]  K. Healy,et al.  Quantification of the surface density of a fluorescent label with the optical microscope. , 2000, Journal of biomedical materials research.

[10]  R. Cardullo,et al.  Introduction to image processing. , 1998, Methods in cell biology.

[11]  M. Model.  Imaging the Cell's Third Dimension , 2012, Microscopy Today.

[12]  S. Shaw.  Imaging the live plant cell. , 2006, The Plant journal : for cell and molecular biology.

[13]  J. Vrba,et al.  Specific activity of cell-surface acid phosphatase in different bacterioplankton morphotypes in an acidified mountain lake. , 2006, Environmental microbiology.

[14]  M. Model,et al.  Measurement of wheat germ agglutinin binding with a fluorescence microscope , 2009, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[15]  J. Sisken.  Fluorescent standards. , 1989, Methods in cell biology.

[16]  U. Resch-Genger,et al.  How to Improve Quality Assurance in Fluorometry: Fluorescence-Inherent Sources of Error and Suited Fluorescence Standards , 2005, Journal of Fluorescence.

[17]  John T Elliott,et al.  Cell cycle dependent TN‐C promoter activity determined by live cell imaging , 2011, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[18]  J. Pawley,et al.  The 39 steps: a cautionary tale of quantitative 3-D fluorescence microscopy. , 2000, BioTechniques.

[19]  J. Groves,et al.  Quantitative fluorescence microscopy using supported lipid bilayer standards. , 2008, Biophysical journal.

[20]  I. Mandic-Mulec,et al.  Exopolymer Diversity and the Role of Levan in Bacillus subtilis Biofilms , 2013, PloS one.

[21]  Martin Oberholzer,et al.  Methods in quantitative image analysis , 1996, Histochemistry and Cell Biology.

[22]  Lili Wang,et al.  Impact of pre-existing immunity on gene transfer to nonhuman primate liver with adeno-associated virus 8 vectors. , 2011, Human gene therapy.

[23]  J. Sisken.  Chapter 4 Fluorescent Standards , 1989 .

[24]  Biliang Zhang,et al.  Characterizing Cell–Cell Interactions Induced Spatial Organization of Cell Phenotypes: Application to Density-Dependent Protein Nucleocytoplasmic Distribution , 2012, Cell Biochemistry and Biophysics.

[25]  Anne L Plant,et al.  Surface plasmon resonance imaging of cells and surface-associated fibronectin , 2009, BMC Cell Biology.

[26]  Talita Perciano,et al.  Introduction to Image Processing Using R , 2013, SpringerBriefs in Computer Science.