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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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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Basic Protocol 1: Image Acquisition

  • 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

  • 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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Literature Cited

Literature Cited
  Boss, D., Kühn, J., Jourdain, P., Depeursinge, C., Magistretti, P.J., and Marquet, P. 2013. Measurement of absolute cell volume, osmotic membrane water permeability, and refractive index of transmembrane water and solute flux by digital holographic microscopy. J. Biomed. Opt. 18:036007.
  Bottier, C., Gabella, C., Vianay, B., Buscemi, L., Sbalzarini, I.F., Meister, J.J., and Verkhovsky, A.B. 2011. Dynamic measurement of the height and volume of migrating cells by a novel fluorescence microscopy technique. Lab. Chip 11:3855‐3863.
  Boudreault, F. and Grygorczyk, R. 2004. Evaluation of rapid volume changes of substrate‐adherent cells by conventional microscopy 3D imaging. J. Microsc. 215:302‐312.
  Chamberlin, M.E. and Strange, K. 1989. Anisosmotic cell volume regulation: A comparative view. Am. J. Physiol. 257:C159‐73.
  Gibson, S.F. and Lanni, F. 1992. Experimental test of an analytical model of aberration in an oil‐immersion objective lens used in three‐dimensional light microscopy. J. Opt. Soc. Am. A 9:154‐166.
  Gómez‐Angelats, M. and Cidlowski, J.A. 2002. Cell volume control and signal transduction in apoptosis. Toxicol. Pathol. 30:541‐551.
  Grebien, F., Dolznig, H., Beug, H., and Mullner, E.W. 2005. Cell size control: New evidence for a general mechanism. Cell Cycle 4:418‐421.
  Gregg, J.L., McGuire, K.M., Focht, D.C., and Mode, M.A. 2010. Measurement of the thickness and volume of adherent cells using transmission‐through‐dye microscopy. Pflugers Arch. 460:1097‐1104.
  Hessler, J.A., Budor, A., Putchakayala, K., Mecke, A., Rieger, D., Banaszak Holl, M.M., Orr, B.G., Bielinska, A., Beals, J., and Baker, J. Jr. 2005. Atomic force microscopy study of early morphological changes during apoptosis. Langmuir 21:9280‐9286.
  Hevia, D., Rodriguez‐Garcia, A., Alonso‐Gervós, M., Quirós‐González, I., Cimadevilla, H.M., Gómez‐Cordovés, C., Sainz, R.M., and Mayo, J.C. 2011. Cell volume and geometric parameters determination in living cells using confocal microscopy and 3D reconstruction. Protocol Exchange doi:10.1038/protex.2011.272.
  Hoffmann, E.K., Lambert, I.H., and Pedersen, S.F. 2009. Physiology of cell volume regulation in vertebrates. Physiol. Rev. 89:193‐277.
  Lang, F., Busch, G.L., Ritter, M., Völkl, H., Waldegger, S., Gulbins, E., and Häussinger, D. 1998. Functional significance of cell volume regulatory mechanisms. Physiol. Rev. 78:247‐306.
  McManus, M.L., Churchwell, K.B., and Strange, K. 1995. Regulation of cell volume in health and disease. N. Engl. J. Med. 333:1260‐1266.
  Model, M.A. 2012. Imaging the cell's third dimension. Microsc. Today 20:32‐37.
  Model, M.A. 2014. Possible causes of apoptotic volume decrease: An attempt at quantitative review. Am. J. Physiol. 306:C417‐C424.
  Model, M.A. and Schonbrun, E. 2013. Optical determination of intracellular water in apoptotic cells. J. Physiol. 591:5843‐6849.
  Model, M.A., Khitrin, A.K., and Blank, J.L. 2008. Measurement of the absorption of concentrated dyes and their use for quantitative imaging of surface topography. J. Microsc. 231:156‐167.
  Nash, G.B., Tatham, P.E., Powell, T., Twist, V.W., Speller, R.D., and Loverock, L.T. 1979. Size measurements on isolated rat heart cells using Coulter analysis and light scatter flow cytometry. Biochim. Biophys. Acta 587:99‐111.
  Okada, Y. 2004. Ion channels and transporters involved in cell volume regulation and sensor mechanisms. Cell. Biochem. Biophys. 41:233‐258.
  Pelts, M., Pandya, S.M., Oh, C.J., and Model, M.A. 2011. Thickness profiling of formaldehyde‐fixed cells by transmission‐through‐dye microscopy. BioTechniques 50:389‐396.
  Satoh, H., Delbridge, L.M., Blatter, L.A., and Bers, D.M. 1996. Surface: Volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: Species‐dependence and developmental effects. Biophys. J. 70:1494‐1504.
  Schliess, F., Reinehr, R., and Häussinger, D. 2007. Osmosensing and signaling in the regulation of mammalian cell function. FEBS J. 274:5799‐5803.
  Schonbrun, E., Gorthi, S.S., and Schaak, D. 2012. Microfabricated multiple field of view imaging flow cytometry. Lab Chip 12:268‐723.
  Schonbrun, E., Di Caprio, G., and Schaak, D. 2013. Dye exclusion microfluidic microscopy. Opt. Express. 21:8793‐8798.
  Schonbrun, E., Malka, R., Di Caprio, G., Schaak, D., and Higgins, J.M. 2014. Quantitative absorption cytometry for measuring red blood cell hemoglobin mass and volume. Cytometry A 85:332‐338.
  Sloot, P.M., Hoekstra, A.G., and Figdor, C.G. 1988. Osmotic response of lymphocytes measured by means of forward light scattering: Theoretical considerations. Cytometry 9:636‐641.
  Yu, R.C., Abrams, D.C, Alaibac, M., and Chu, A.C. 1994. Morphological and quantitative analyses of normal epidermal Langerhans cells using confocal scanning laser microscopy. Br. J. Dermatol. 131:843‐848.
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