Refractive index measurement in viable cells using quantitative phase-amplitude microscopy and confocal microscopy.
BACKGROUND
The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.


METHODS
The experimental strategy involved calculation of cell thickness by using confocal optical sectioning procedures, construction of a phase map of the same cell using quantitative phase microscopy, and selection of cellular regions of interest to solve for the cell RI.


RESULTS
Mean cell thickness and phase values for six cell regions (five cytoplasmic and one nuclear) were determined. The average refractive index calculated for cytoplasmic and nuclear regions was 1.360 +/- 0.004. The uncertainty in the final RI value represents the technique measurement error.


CONCLUSIONS
The methodology we describe for viable cell RI measurement with this prototype cell has broad generic application in the study of cell growth and functional responses. The RI value we report may be used in optical analyses of cultured cell structure and morphology.
Curl
Claire L
CL
Department of Physiology, University of Melbourne, Parkville, Victoria, Australia.
Bellair
Catherine J
CJ
Harris
Trudi
T
Allman
Brendan E
BE
Harris
Peter J
PJ
Stewart
Alastair G
AG
Roberts
Ann
A
Nugent
Keith A
KA
Delbridge
Lea M D
LM
D002470:Cell Survival / Q000502:physiology; D006801:Humans; D007399:Interphase / Q000502:physiology; D008433:Mathematics; D018613:Microscopy, Confocal / Q000379:methods; D008858:Microscopy, Phase-Contrast / Q000379:methods; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D012031:Refractometry / Q000379:methods*