Tomographic phase microscopy at single cell scale without a-priori knowledge of cell orientations: smart strategies for rotation angles recovery

Lisa Miccio; Daniele Pirone; Daniele Sirico; Francesco Merola; Pasquale Memmolo; Vittorio Bianco; Zhe Wang; Jaromir Behal; Danila Del Giudice; Martina Mugnano; Pietro Ferraro

doi:10.1117/12.2626452

20 May 2022 Tomographic phase microscopy at single cell scale without a-priori knowledge of cell orientations: smart strategies for rotation angles recovery

Lisa Miccio, Daniele Pirone, Daniele Sirico, Francesco Merola, Pasquale Memmolo, Vittorio Bianco, Zhe Wang, Jaromir Behal, Danila Del Giudice, Martina Mugnano, Pietro Ferraro

Author Affiliations +

Proceedings Volume 12136, Unconventional Optical Imaging III; 121360T (2022) https://doi.org/10.1117/12.2626452
Event: SPIE Photonics Europe, 2022, Strasbourg, France

Abstract

Tomographic phase microscopy in cytometry environment is feasible at single cell level and without the a-priori knowledge of the cell orientation. In the present paper we demonstrate different strategies for recovery the rotation angles of single cells and clusters when rotating into microfluidic channels, thus realistically opening to the implementation of marker-free cytofluorimeter for three-dimensional imaging of biological fluids. The pioneering developed strategies allows to measure quantitatively the inner distribution of the refractive indexes inside the cell volume avoiding the use of chemical and fluorescent tags. The imaging apparatus is based on label-free Digital Holography in microscopy setup designed in transmission geometry to image 700x700μm Field of View with lateral resolution of 0.5μm. Digital Holography is perfectly suited for imaging in microchannels as it allows the numerical refocusing of sample into a three-dimensional volume. In the present paper, such imaging arrangement is combined with a high-precision pumping system connected to a microfluidic channel that allow the complete rotation of the flowing cells into the Field of View. High-speed 25Megapixel camera acquires holographic set measurements of all rotating cells that are numerically processed to obtain quantitative two-dimensional phase-contrast maps at different view angles. Accurate numerical algorithms allow to tag each phasecontrast maps with the rotation angle in the microchannel. The couples made of phase-contrast map and measured angle are given as input at tomography algorithms to obtain the refractive index distribution into the cell volume. The approach in principles works properly for any kind of biological matter subjected to rotation as already demonstrated in case of nuclei of plant cells during dehydration. Furthermore, the same approach allows to show the three-dimensional distribution of internalized nano-particles as in case of nano-graphene oxide. The most important achievement and innovation of such strategy is the high-throughput phase-contrast tomography at single cell level that opens to new diagnostic tool thanks to the possibility to have statistically relevant measurement on cell population and also for the possibility to use artificial intelligence architecture for cell identification and classification.

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Lisa Miccio, Daniele Pirone, Daniele Sirico, Francesco Merola, Pasquale Memmolo, Vittorio Bianco, Zhe Wang, Jaromir Behal, Danila Del Giudice, Martina Mugnano, and Pietro Ferraro "Tomographic phase microscopy at single cell scale without a-priori knowledge of cell orientations: smart strategies for rotation angles recovery", Proc. SPIE 12136, Unconventional Optical Imaging III, 121360T (20 May 2022); https://doi.org/10.1117/12.2626452

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KEYWORDS

Tomography

Microfluidics

Microscopy

Digital holography

Holography

3D image processing

Artificial intelligence

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