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Project ANCD 20.70086.16/COV Biomedical application of polarized digital holographic microscopy

Type : Project within the "Offer of research-innovation solutions regarding the fight and mitigation of the impact of the COVID-19 pandemic" with the number 20.70086.16, financed by the NARD of the RM within the H2020 Program - acronym BIOHOLO
Head : Elena Achimova, D.Sci.
Duration : 2020 - 2021
Division(s) : Laboratory of Materials for Photovoltaics and Photonics

The coronavirus epidemic (COVID-19) has affected all aspects of life around the world. This has changed the scientific field situation offering of the research-innovation solutions on combating and mitigating the impact of the COVID-19 pandemic. For example, european scientists in photonics are now developing a nano-interferometric biosensor that detects coronavirus at the earliest point of infection from a saliva or nasal swab.

 Microscopic examination of cell morphology is one of the main research methods in many areas of biomedicine, such as cancer research, discovery of new drugs, cell behavior, phenotypic screening, the study of pathological processes, etc. Non-invasive high-resolution imaging of living cells in their natural environment is the main prerequisite to visualize the structure and processes taking place in bio samples.

The light microscopy is a noninvasive means that used to study bio samples in vivo, in situ and in vitro. Measurement of quantitative phase and optical anisotropy information has long been of interest for biomedical researchers. Compared to classical phase contrast and differential interference contrast microscopes, widely used in biology for the visualization of unstained transparent specimens, interferometric techniques present the significant advantage of yielding quantitative measurements of the 3D phase image of samples. The measured phase shift depends on both the refractive index and the thickness of the samples, two quantities linked to the nature of the intracellular content, cell’s morphometry and structure of the samples, respectively. The refractive index of tissue’s structures relates with polarized light propagating in bio samples, but in many optical imaging methods polarization effects are ignored.

The polarized light microscopy (PLM) reveals orientation order in native molecular structures inside living cells, tissues, and whole organisms. The PLM means exhibits birefringence (anisotropy of the refractive index) and dichroism (anisotropy of the absorption coefficient) that are important characteristic of molecular architecture of bio samples. While PLM is not sensitive to the chemical nature of the constituent molecules, it responds to the structural, anisotropic nature of macromolecular assemblies, such as the submicroscopic alignment of molecular bonds and filaments.

The digital holographic microscopy (DHM) techniques have become increasingly applied to fields of biophotonics, life sciences and medicine since they offer several compelling advantages over other imaging methods:

- Non-destructive imaging,

- Marker-free,

- “Full-field” imaging (no scanning required),

- Quantitative phase recovery (important for imaging transparent cells),

- Numerical refocusing (image focus can be changed without additional scanning),

- Simultaneous online monitoring.

DHM can be used to extract the 3D information of a biological organism using a single recorded digital hologram. The quantitative phase and amplitude information of the object wavefront can be digitally obtained along with the depth of the object from the recorded hologram.

So, synergy of both PLM and DHM microscopic techniques enables major advances in biological imaging, significantly increasing its functionalities and performance, and allowing it to emerge from a laboratory-driven to practical biomedical applications.

For example, viruses have a dual nature, the particle and the infected cell. Cells elicited by virus infection may have distinct features, such as loss of membrane integrity, cell shrinkage, density changes, cell detachment from the substratum, loss or enforcement of the cytoskeleton, and reorganization of intracellular membranes. Despite the predictive nature of viruses for clinical and biological infections, deep leaning 3D analyses of virus-induced cell's changes are missing.

In this context this project directs to advance the polarized-sensitive DHM beginning from optical image processing and acquiring digital phase holograms with polarized properties (hardware) and to digital image processing with considering their polarization performances (software).

The overall aim of the project was engineering of polarized-sensitive DHM for its application in biomedicine to study the structure and polarization properties of phase bio samples.

To reach the project’s aim the following objectives  were developed:

1. To modify DHM including real-time optical and digital image acquisition and processing for quantitative measurements of the structural characteristics, such as optical anisotropy and morphology of transparent and phase bio samples.

2. To elaborate the algorithms for reconstructing of phase digital holograms considering anisotropy to reveal molecular architecture of bio samples.

To carry out these objectives, it was necessary to develop original and adapted methodologies at different stages of the project implementation such as:

a) the design of optical setup of polarized-sensitive DHM for study of anisotropy of transparent phase objects;

b) the mathematical simulation and modeling of high-performance polarized-sensitive DHM;

c) the development of digital image processing algorithms for reconstructing digital polarization holograms;

d) the combine of the developed optical and digital image processing in one holographic microscopic technique suitable for studying the features including polarization of bio samples.

The expected outcomes of the proposed investigations were:

- The polarized-sensitive DHM for quantitative measuring of the optical phase and polarization distribution into transparent bio samples;

- The mathematical modeling and analysis based on the theory of light diffraction for reconstruction of digital holograms with maximal meaningful information, including 3D image of samples;

- The assessment of potentialities and limitations of elaborated polarized-sensitive DHM technology for biomedical applications.

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