Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

Abstract

We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening compatible application, we automatized a digital holographic microscope for image acquisition of cells using commercially available 96-well plates. Data generated through both label-free DHM imaging and fluorescence-based methods were in good agreement for cell viability identification and a Z 0 -factor close to 0.9 was determined, validating the robustness of DHM assay for phenotypic screening. Further, an excellent correlation was obtained between experimental cytotoxicity dose-response curves and known IC 50 values for different toxic compounds. For comparable results, DHM has the major advantages of being label free and close to an order of magnitude faster than automated standard fluorescence microscopy.

Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

“…Consequently, QPM in general and in particular QP-DHM, thanks to its numerical flexibility, represent an appealing imaging modality to perform high content phenotypic screening.. This view is supported by some studies which have permitted to stress the possibility to develop in vitro label-free cytotoxicity screening assays based on QP-DHM [18][19][20][21] . Specifically, Pavillon et al 21 have explored in vitro the early detection of excitatory neuronal death induced by perfusion of glutamate, the main excitatory neurotransmitter in the brain (cf.…”

“…Indeed, although QP-DHM does not have the advantage of signal specificity provided by labeled approaches, it provides a non-invasive highly sensitive QPS allowing to directly quantify cytotoxicity 18 and permits a drastic improvement in scanning speed for multi-well plates thanks to its numerical autofocusing capability. Practically, instead of having to mechanically focus on each well, a re-focusing step is performed off line after the acquisition during the numerical reconstruction of the holograms, in parallel to the image analysis and quantification.…”

“…In addition, the use of free wandering particles would also inform precisely on the nature of binding forces and properties of the macromolecules. This detection of multiple biophysical cell parameters could even be scaled into a high-throughput cellular screening assay 18 allowing the monitoring of several cellular processes concurrently as well as their modulations as a function of the transcriptome and metabolome of different cell-types derived from controls, patients as well as high-risk subjects. This paves the way to explore cell biomarkers of risk for different diseases.…”

Holography: The Usefulness of Digital Holographic Microscopy for Clinical Diagnostics

“…Cell volume changes, resulting from cytotoxic treatments or apoptosis events, have recently been investigated with DH microscopy [11,[21][22][23][24][25]. When cells go in to early apoptosis, the first discernible indication is an increase in the cell phase shift followed by a decrease as the cell eventually dies [21].…”

Accurate single-shot quantitative phase imaging of biological specimens with telecentric digital holographic microscopy

“…This unique feature has been determinant to find applications of DHM in living cell screening, [1][2][3][4] particle tracking, [5][6][7][8][9][10][11] and MEMS evaluation, [12][13][14] among many others. [15][16][17][18][19][20][21][22][23][24] The ability of performing QPI has been extended to other microscopy methods like spatial light interference microscopy or spatial light interference tomography; 25,26 hence similar applications have been reported by the use of these imaging approaches.…”