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

Abstract

The measurement of the volume of intact, viable cells presents challenging problems in many areas of experimental and diagnostic science involved in the evaluation of cellular morphology, growth and function. This investigation details the implementation of a recently developed quantitative phase microscopy (QPM) method to measure the volume of erythrocytes under a range of osmotic conditions. QPM is a computational approach which utilizes simple bright field optics to generate cell phase maps which, together with knowledge of the cellular refractive index, may be used to measure cellular volume. Rat erythrocytes incubated in imidazole-buffered solutions (22°C) of graded tonicity were analysed using QPM (n=10 cells/group, x63, 0.8 NA objective). Erythrocyte refractive index (1.367) was measured using a combination of phase and morphological data obtained from cells adopting spherical geometry under hypotonic conditions. Phase-computed volume increased with decreasing solution osmolality: 42.8 ± 2.4, 48.7 ± 2.3, 62.6 ± 2.3, 90.8 ± 7.7 µm3 in solutions of 540, 400, 240, and 170 mosmol/kg respectively. These volume changes were associated with crenated, bi-concave and spherical morphological states associated with increasing tonicity. This investigation demonstrates that QPM is a valid, simple and non-destructive approach for measuring cellular phase properties and volume. QPM cell volume analysis represents a significant advance in viable cell experimental capability and provides for acquisition of ‘real-time’ data - an option not previously available using other approaches.

Frequency to Voltage Converter as a Phase Controller in Phase Shifting Interference Microscopy

“…Finally, the piezoelectric and its control system can be widely used in other setups for optical phase acquisition techniques, involving interferometry or controlled blurring of the optical system (Curl et al 2006). Additionally, the control system can be used for the control of a microscope automatic focus system.…”

One Platelet Dynamic Characteristics Analysis Method Based on the Phase Imaging

“…Therefore, the phase of light wave can be obtained by TIE, no matter in coherent or partially coherent light field [18][19][20][21][22][23][24].…”

Application of Phase Images for Estimation of Peritoneal Macrophages State

“…Interference microscopy and some noninterferometric methods (e.g. Curl et al, 2006;Roma et al, 2014) can provide quantitative estimates of the optical path difference (OPD) in each point of cell (phase images). The OPD varies with cell thickness and concentration of cellular substances.…”