Cell Volume Measurements by Optical Transmission Microscopy

Michael A. Model1

1 Department of Biological Sciences, Kent State University, Kent, Ohio
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.39
DOI:  10.1002/0471142956.cy1239s72
Online Posting Date:  April, 2015
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Abstract

Cell volume is an important parameter in cell adaptation to anisosmotic stress, in the development of apoptosis and necrosis, and in the pathogenesis of several diseases. This unit describes a method for measuring the volume of adherent cells using a standard light microscope. A coverslip with attached cells is placed in a shallow chamber in a medium containing a strongly absorbing and cell‐impermeant dye, Acid Blue 9. When such a sample is imaged in transmitted light at a wavelength of maximum dye absorption (630 nm), the resulting contrast quantitatively reflects cell thickness. Once the thickness is known at every point, the volume can be computed as well. Technical details, interpretation of data, and possible artifacts are discussed. Measurements in absolute units require knowledge of the absorption coefficient, and a similar procedure for the measurement of absorption coefficient is described. © 2015 by John Wiley & Sons, Inc.

Keywords: optical microscopy; transmission‐through‐dye microscopy; cell volume; cell topography; absorption; acid blue 9

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Image Acquisition
  • Support Protocol 1: Measurement of the Absorption Coefficient α
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Image Acquisition

  Materials
  • Cells
  • Appropriate cell medium
  • Acid Blue 9 (AB9) in sufficiently pure form can be obtained from TCI America (if using the chemical from other suppliers, verify that it is nontoxic for cells at the working concentration and that it does not change the pH of the medium)
  • Glass slides and coverslips can be obtained from numerous vendors, such as Fisher Scientific (http://www.fishersci.com), Tedd Pella (http://www.tedpella.com/), or Electron Microscopy Sciences (https://www.emsdiasum.com/)
  • Wet cotton swabs
  • Standard upright or inverted microscope
  • Bandpass filters: 630/10 (Andover, Salem, NH); 485/10 nm (Omega Optical) or similar (for strongly scattering samples, filters with antireflective coating are preferred, such as ET630‐10 bp (Chroma Technology) or Brightline 632/22 from Semrock

Support Protocol 1: Measurement of the Absorption Coefficient α

  Materials
  • Solution of dye to be measured
  • Glass slide
  • Pipets
  • Half‐ball lens with d = 10 mm (Edmund Optics, cat. no. 45‐937)
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Figures

Videos

Literature Cited

Literature Cited
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