Imaging red blood cell dynamics by quantitative phase microscopy

Abstract

Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.

Introduction

Optical microscopy has been a major tool for biological and biomedical research for centuries. Although other techniques such as electron microscopy offer significantly better spatial resolution, light microscopy occupies a central role in biomedical science because of its ease of use and the potential for non-invasive, live cell imaging. In particular, since the invention of the phase contrast microscope by Frederik (Frits) Zernike, for which he was awarded the Noble Prize in physics in 1953, this instrument and its related techniques have been a cornerstone of every cell biology laboratory. In spite of their enormous value as non-invasive investigational tools, however, traditional phase methods such as phase contrast and differential interference contrast (DIC) are inherently qualitative and lack subcellular specificity. At the same time, extrinsic contrast techniques such as fluorescence microscopy offer molecular specificity and high spatial resolution. Nevertheless, these methods generally require alteration or modification of cellular and molecular structures including cell permeabilization, chemical or immunostaining, or genetic modification, and are therefore less than ideal for characterization of live cells in their native physiological state.

Over the past several years, significant progress has been made in quantitative phase microscopy methods that promise to overcome limitation of traditional phase microscopy. In particular, full-field quantitative phase techniques that provide simultaneous information from a wide field of view offer an ideal experimental approach to characterize spatial and temporal behaviors of the sample [1], [2], [3], [4], [5], [6], [7], [8]. Our own laboratory has developed multiple experimental methods and instrumentation that enable full-field quantitative imaging of live cells using intrinsic cellular contrast such as refractive index and light scattering. These novel methods that typically have nanometer sensitivity over millisecond time scales have broken new ground in important areas of cellular biology, including characterization of structural and mechanical properties of cells [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], high-throughput particle tracking in live cells [18], nanoscale imaging of cell electromotility [19], and quantitative phase tomographic microscopy [20], [21]. In this report, we present an update on instrumentation and methods for imaging live red blood cell (RBC) mechanical and dynamical properties using field-based phase microscopy methods. These methods complement the existing techniques for the study of RBC dynamics such as micropipette aspiration [22], [23] and optical tweezers [24], [25], and for the first time provide the opportunity for high-throughput, real-time analysis of RBC mechanical properties in health and disease.