65,000 shades of grey: Use of digital image files in light microscopy

Sidney L. Shaw, Edward H. Hinchcliffe

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations


Computers dominate image capture and analysis in modern light microscopy. The output of an imaging experiment is a binary coded file, called an image file, which contains the spatial, temporal and intensity information present in the sample. Understanding what comprises an image file, and how these files are generated is necessary in order to optimize the use of the digital light microscope. In this chapter, we discuss image file formats, and the various components of these files, such as bit-depth, sampling rate, color theory, and compression, from the perspective of the non-computer scientist. We also discuss the problem of proprietary file formats, and how these often are incompatible with certain types of imaging software. We present several solutions to this issue. Finally, we present the use of digital movie formats, compression routines, and provide some real world examples for optimizing the generation of digital movies.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
PublisherAcademic Press Inc.
Number of pages20
StatePublished - 2013

Publication series

NameMethods in Cell Biology
ISSN (Print)0091-679X

Bibliographical note

Funding Information:
We thank Katie Buchanan, Bill Burnip, Rich Cardullo, Paul Goodwin, Paul Jantzen, Karl Kilborn, Colin Monks, and Dee Sharma for providing helpful comments on the presentation of this material during the Analytical and Quantitative Light Microscopy (AQLM) course in Woods Hole. Both S. L. S. and E. H. H. are graduates of the 1995 AQLM course, and we would like to thank Dr. Ken Spring for his insight and humor during that course. Work in the authors’ labs is supported by research grants from the National Science Foundation and National Institutes of General Medicine.


  • Codec
  • Color
  • Compression
  • File format
  • Image file


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