Single Cell Volume Measurement by Quantitative Phase Microscopy (QPM): A Case Study of Erythrocyte Morphology

Abstract

The measurement of the volume of intact, viable cellspresents challenging problems in many areas ofexperimental and diagnostic science involved in theevaluation of cellular morphology, growth andfunction. This investigation details the implementationof a recently developed quantitative phasemicroscopy (QPM) method to measure the volume oferythrocytes under a range of osmotic conditions.QPM is a computational approach which utilizes simplebright field optics to generate cell phase maps which,together with knowledge of the cellular refractive index,may be used to measure cellular volume. Raterythrocytes incubated in imidazole-bufferedsolutions (22°C) of graded tonicity were analysedusing QPM (n=10 cells/group, x63, 0.8 NA objective).Erythrocyte refractive index (1.367) was measuredusing a combination of phase and morphological dataobtained from cells adopting spherical geometry underhypotonic conditions. Phase-computed volumeincreased with decreasing solution osmolality: 42.8 ±2.4, 48.7 ± 2.3, 62.6 ± 2.3, 90.8 ± 7.7 µm3 in solutionsof 540, 400, 240, and 170 mosmol/kg respectively.These volume changes were associated withcrenated, bi-concave and spherical morphologicalstates associated with increasing tonicity. Thisinvestigation demonstrates that QPM is a valid, simpleand non-destructive approach for measuring cellularphase properties and volume. QPM cell volumeanalysis represents a significant advance in viablecell experimental capability and provides foracquisition of ‘real-time’ data - an option not previouslyavailable using other approaches.