Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

Benjamin Rappaz,Etienne Shaffer,Gerardo Turcatti,Billy Breton

doi:10.2174/13862073113166660062

Abstract

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

Highlights

Phenotypic screens [1,2,3] rely on image-based cellular assays for analyzing cell morphological variations or alterations
Those transparent specimens, on the other hand, generally alter—or shift—the phase of light more significantly. Examination of such phase objects has led to the development of optical contrast-enhancing imaging techniques like phase contrast (PC), initially proposed by Zernike [7], as well as Nomarski Differential Interference Contrast (DIC) [8]
Digital holographic microscopy is a label-free microscopy technique in which contrast is related to biophysical parameters [9]

Summary

Introduction

Phenotypic screens [1,2,3] rely on image-based cellular assays for analyzing cell morphological variations or alterations. Most cells are transparent or generate only modest changes in the amplitude of light, which makes them hard to image with the simplest label-free optical imaging techniques such as bright field microscopy Those transparent specimens, on the other hand, generally alter—or shift—the phase of light more significantly. Examination of such phase objects has led to the development of optical contrast-enhancing imaging techniques like phase contrast (PC), initially proposed by Zernike [7], as well as Nomarski Differential Interference Contrast (DIC) [8]. These methods do not readily provide quantitative information on the specimen-induced phase shifts. Their inherent contrast mechanism and related artifacts [9] make automated cell segmentation very difficult and hardly robust

Methods

Results

Conclusion