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, 17 (1), 80-8

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

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

Benjamin Rappaz et al. Comb Chem High Throughput Screen.

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.

Figures

Fig. (1)
Fig. (1)
The Digital Holographic Microscopy (DHM) technology and experimental workflow. (A) Plate preparation procedure. (B) Image acquisition: First, a hologram (magnifying glass highlights the interference fringes) is recorded out of focus by a digital camera on a DHM T1001 system equipped with a motorized stage for automated multi-well plate experiments (7). Legend: M, mirror, BS, beam splitter, BE, beam expander, MO, microscope objective, C, condenser. Then, it is reconstructed by a computer to form an in-focus quantitative phase image (8). Contrast in DHM is provided by optical path length (OPL) variations in the specimen. For cell biology experiments, the measured optical path difference (OPD) is related to the thickness d and mean intracellular refractive index nc of the cultured cells, as well as to nm, the refractive index of the surrounding medium through Equation (1). (C) Image analysis is performed on the quantitative phase images using either on the global image (9) or on individual cell (10).
Fig. (2)
Fig. (2)
Compound induced phenotypes. Representative endpoint (24 h) images of HeLa (A, B, C) and H9c2 (D, E, F) cells in control condition (A, D) treated with 30 µM doxorubicin (B, E) or 30 µM chloroquine (C, F). Scale bar: 50 µm. All images are drawn with the same intensity levels (except inset image). Ring pattern in the doxorubicin images are dead cells detached from the well-plate.
Fig. (3)
Fig. (3)
Time-lapse measurements. HeLa (A, B) and H9c2 (C, D) treated with serial dilution of doxorubicin (A-C) or chloroquine (B-D) were imaged each 10 min for 24 h. For each condition, the average OPD and the percentage of round phenotype was measured. Doseresponse graphs (in blue) are area under the curve calculated from the graph on their left.
Fig. (4)
Fig. (4)
Imaging mode comparison. (A) Differential Interference Contrast (DIC), Phase Contrast (PC), Digital Holographic Microscope (DHM), Bright-field (BF) and Transport of Intensity Equation (TIE, generated from a z-stack of 3 BF images) from the same field of view. (B) normalized profile along the red path in DHM, DIC, PC, BF and TIE images illustrating the quantitative aspect of DHM and TIE and qualitative aspect of the other techniques. Horizontal scale bar = 50 µm, vertical scale bar = 200 nm OPD (DHM); arbitrary units (DIC, PC, BF and TIE).

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