Review on methods of solving the refractive index–thickness coupling problem in digital holographic microscopy of biological cells

Abstract

KeywordsDigital holographic microscopy, quantitative phase microscopy, interferometric phase microscopy, decoupling, tomographic phase microscopy, refractive index. AbstractDigital holographic microscopy is a thriving imaging modality that attracted considerable research interest in quantitative biological cell imaging due to its ability to not only create excellent label-free contrast, but also supply valuable physical information regarding the density and dimensions of the sample with nanometer-scale axial sensitivity. This technique records the interference pattern between a sample beam and a reference beam, and by digitally processing it, one can reconstruct the optical path delay between these beams. Per each spatial point, the optical path delay map is proportional to the product of the sample physical thickness and the integral refractive index of the sample. Since the refractive index of the cell indicates its contents without the need for labeling, it is highly beneficial to decouple cell physical thickness from its refractive index profile. This manuscript reviews various approaches of extracting the refractive index from digital holographic microscopy measurements of cells. As soon as the refractive index of the cell is available, it can be used for either biological assays or medical diagnosis, as reviewed in this manuscript.

Dehydration of plant cells shoves nuclei rotation allowing for 3D phase-contrast tomography

“…In DH, the measured quantitative information is the optical thickness contrast, which is the integral product between the sample refractive index contrast and its physical thickness along the optical axis. In order to decouple this information, multiple channels have to be exploited, e.g., multiple wavelengths or illumination angles 15 . In particular, phase-contrast tomography (PCT) probes the sample from different directions to achieve resolving capability along the optical axis and to measure the sample refractive index 3D distribution 16 – 22 .…”

Towards the Development of Portable and In Situ Optical Devices for Detection of Micro-and Nanoplastics in Water: A Review on the Current Status

“…On the contrary, DH provides 3D imaging holograms as a function of time and monitors particles out of the focal plane as well. This allows more flexibility because it is possible to monitor, in addition to the shapes of the MPs, their locations and velocities in a water volume, yielding information on the particle thickness and the continuously varying refractive index of the ambient medium [ 131 ]. The practical implementation of a DH system can be based on the use of a high-power laser diode as a point light source to create a divergent beam.…”

Measuring Absolute Cell Volume Using Quantitative-Phase Digital Holographic Microscopy and a Low-Cost, Open-Source, and 3D-Printed Flow Chamber

“…Although temporal dependency is not explicitly indicated in Equation ( 4), it remains valid for each quantitative-phase image of a time-lapse. The dual dependence expressed by Equation (4) (known as the refractive index-thickness coupling problem [45]) results in a counterintuitive and challenging interpretation of the QPS.…”