Reflectance confocal microscopy of red blood cells: simulation and experiment.

Adel Zeidan,Dvir Yelin

doi:10.1364/boe.6.004335

Abstract

Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology.

Highlights

The properties of red blood cells (RBCs) are remarkable indicators of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology [1] is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease [1, 2]
In vivo imaging of RBCs, which is ideal for measuring the physiological properties of the cells within their natural environment, was first demonstrated using reflectance confocal microscopy (RCM) [9, 10] at high-resolution and without the need for fluorescence labeling [9]
We develop a numerical model that simulates the confocal reflectance image of a single RBC for various relative axial positions and tilt angles, and compare our results to in-vitro spectrally encoded flow cytometry (SEFC) images of flowing RBCs

Summary

Introduction

The properties of red blood cells (RBCs) are remarkable indicators of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology [1] is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease [1, 2]. In vivo imaging of RBCs, which is ideal for measuring the physiological properties of the cells within their natural environment, was first demonstrated using reflectance confocal microscopy (RCM) [9, 10] at high-resolution and without the need for fluorescence labeling [9]. A main challenge of all reflectance confocal imaging techniques, including laser-scanning RCM, SECM and SEFC, are interference effects that stem from the high spatial coherence of the focused beam. When imaging RBCs, the rounded, smooth plasma membrane and the relatively homogenous cytoplasm result in a characteristic appearance of smooth rings and curved features [20] These features stem directly from the three-dimensional shapes of these cells, and could provide invaluable information on their morphology and physiological state. This method allows us to evaluate the shape of the cells in three dimensions, with potential diagnostic capabilities that are based on accurate measurement of RBC morphology

Theoretical model and numerical simulation

Experiment – cell imaging using SEFC

Findings

Discussion and conclusion