Document Detail

Measurement of the thickness and volume of adherent cells using transmission-through-dye microscopy.
MedLine Citation:
PMID:  20721672     Owner:  NLM     Status:  In-Process    
Cell volume is one of the basic characteristics of a cell and is being extensively studied in relationship to a variety of processes, such as proliferation, apoptosis, fertility, or locomotion. At the same time, its measurement under a microscope has not been well developed. The method we propose uses negative transmission contrast rendered to cells by a strongly absorbing dye present in the extracellular medium. Cells are placed in a shallow compartment, and a nontoxic and cell-impermeant dye, such as acid blue 9, is added to the medium. Transmission images are collected at the wavelength of maximum dye absorption (630 nm). Where the cell body displaces the dye, the thickness of the absorbing layer is reduced; thus, an increase in cell thickness produces brighter images and vice versa. The absolute values for cell thickness and volume can be easily extracted from the image by computing the logarithm of intensity and dividing it by the absorption coefficient. The method is fast, impervious to instability of the light source, and has a high signal-to-noise ratio; it can be realized either on a laser scanning or a conventional microscope equipped with a bandpass filter. For long-term experiments, we use a Bioptechs perfusion chamber fitted with a 0.03-mm spacer and an additional port to enable rapid switching of solutions. To show possible applications of this method, we investigated the kinetics of the cell volume response to a hypotonic buffer and to the apoptotic agents staurosporine and ionomycin.
Jennifer L Gregg; Karen M McGuire; Daniel C Focht; Michael A Model
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2010-08-19
Journal Detail:
Title:  Pflügers Archiv : European journal of physiology     Volume:  460     ISSN:  1432-2013     ISO Abbreviation:  Pflugers Arch.     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-10-18     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0154720     Medline TA:  Pflugers Arch     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  1097-104     Citation Subset:  IM    
School of Biomedical Sciences, Kent State University, Kent, OH 44242, USA.
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