Proceedings Article | 27 July 2019
KEYWORDS: Phase imaging, Digital holography, Refractive index, Phase measurement, Water, Visualization, Holography, Microscopy, Interferometry, Molecules
Quantitative Phase Imaging (QPI) has recently emerged as a powerful new imaging modality to non-invasively visualize transparent specimens, including living cells in culture. Among different QPI techniques, Quantitative Phase Digital Holographic Microscopy (QP-DHM) is particularly well suited to explore, with a nanometric axial sensitivity, cell structure and dynamics. Concretely, accurate interferometric measurements of the phase retardation of a light wave when transmitted through living cells are performed. This phase retardation, namely the Quantitative Phase Signal (QPS) depends on both the thickness of the observed cells as well as the difference between its refractive index (RI) nc and that of the surrounding medium nm. This RI difference is generated by the presence of organic molecules, including proteins, DNA, organelles, nuclei present in cells. QPS provides thus information about both cell morphology and cell contents.
According to this intracellular RI dependency, QPI has proven to be successful in performing cell counting, recognition and classification, the monitoring of cellular dry mass, cell membrane fluctuations analysis as well as the reconstruction, through tomographic approaches, of the intracellular 3D refractive index distribution. Furthermore, thanks to the development of different experimental procedures, additional relevant biophysical cell parameters were successfully calculated, including membrane mechanical properties, osmotic membrane water permeability, transmembrane water movements and the RI of transmembrane solute flux. However, all these cell parameters can be quantitatively and accurately measured provided that both the QPS exhibits a high stability and the RI value of the surrounding medium nm is accurately known. Any changes of nm will significantly affect the measurements of all these cellular parameters, comprising thus the major advantage of QPI, its quantitative aspects. This particularly the case, for the applications claiming a quantitative evaluation of the cellular dry mass as well as when compounds are directly added to the perfusion solutions for performing either screening or specific pharmacological experiments dedicated to decipher specific cellular processes.
In this talk, we will present different substrates — coverslips and do-it-yourself 3D-printed flow chambers — that we have developed, which meet the challenge, when combined with QP-DHM of obtaining highly stable QPS as well of measuring in real time and with the accuracy of ±0.00004 the absolute values of refractive index of the surrounding medium in the vicinity of living cells. Specifically, such accuracy can be obtained thanks to the high QPS stability resulting from the QP-DHM capability to propagate the whole object wave (amplitude and phase) diffracted by the observed specimen during the numerical reconstruction of the digitally recorded holograms. Indeed, this 3D wavefront numerical reconstruction can efficiently compensate for experimental artifacts including lens defects, and noise originating from vibrations or thermal drift.
These results pave the way for developing, based on QP-DHM technology, label-free high-content screening approaches to study test compound effects in cellular disease‐modeling systems.
