High Throughput Volume Flow Cytometry (parallel-iLIFE) Resolves Mitochondrial Network On the Go

Cell screening and viability studies are paramount to access cell morphology and intracellular molecular variations within large heterogeneous populations of cells. This forms the basis for diagnosis of infections, evaluating immunohistochemistry and routine histopathology. The proposed volume flow cytometry (also termed as, parallel Integrated Light-sheet imaging and flow-based enquiry (parallel-iLIFE)) is a powerful method that adds new capabilities (3D volume visualization, organelle-level resolution and multi-organelle screening) powered by light sheet based illumination. Unlike state-of-the-art point-illumination based imaging cytometry techniques, light sheet based parallel-iLIFE technique is capable of screening species with high throughput and near diffraction-limited resolution. The flow system was realized on a multichannel (Y-type) microfluidic chip that enables visualization of mitochondrial network of several cells in-parallel at a relatively high flow-rate of 2000 nl/min. The calibration of system requires study of point emitters (fluorescent beads) at physiologically relevant flow-rates (50−2000 nl/min) for determining flow-induced optical aberration in the system point spread function (PSF). Subsequently, recorded raw images and volumes were deconvolved with flow-variant PSF to reconstruct cellular mitochondrial network. High throughput investigation of HeLa cells were carried out at sub-cellular resolution in real-time and critical parameters (mitochondria count and size distribution, morphology and cell strain statistics) are determined on-the-go. These parameters determine the physiological state of cells and the changes in mitochondrial distribution over-time that may have consequences in disease diagnosis. The development of volume flow cytometry system (parallel-iLIFE) and its suitability to study sub-cellular components at high-throughput high-content capacity with organelle-level resolution may enable disease diagnosis on a single microfluidic chip.

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Light sheet based volume flow cytometry (VFC) for rapid volume reconstruction and parameter estimation on the go

Scientific Reports ◽

10.1038/s41598-021-03902-8 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Prashant Kumar ◽

Prakash Joshi ◽

Jigmi Basumatary ◽

Partha Pratim Mondal

Keyword(s):

Parameter Estimation ◽

Real Time ◽

High Throughput ◽

Physiological State ◽

Volume Visualization ◽

Disease Diagnosis ◽

Critical Parameters ◽

Single Shot ◽

Volume Reconstruction ◽

Over Time

AbstractOptical imaging is paramount for disease diagnosis and to access its progression over time. The proposed optical flow imaging (VFC/iLIFE) is a powerful technique that adds new capabilities (3D volume visualization, organelle-level resolution, and multi-organelle screening) to the existing system. Unlike state-of-the-art point-illumination-based biomedical imaging techniques, the sheet-based VFC technique is capable of single-shot sectional visualization, high throughput interrogation, real-time parameter estimation, and instant volume reconstruction with organelle-level resolution of live specimens. The specimen flow system was realized on a multichannel (Y-type) microfluidic chip that enables visualization of organelle distribution in several cells in-parallel at a relatively high flow-rate (2000 nl/min). The calibration of VFC system requires the study of point emitters (fluorescent beads) at physiologically relevant flow-rates (500–2000 nl/min) for determining flow-induced optical aberration in the system point spread function (PSF). Subsequently, the recorded raw images and volumes were computationally deconvolved with flow-variant PSF to reconstruct the cell volume. High throughput investigation of the mitochondrial network in HeLa cancer cell was carried out at sub-cellular resolution in real-time and critical parameters (mitochondria count and size distribution, morphology, entropy, and cell strain statistics) were determined on-the-go. These parameters determine the physiological state of cells, and the changes over-time, revealing the metastatic progression of diseases. Overall, the developed VFC system enables real-time monitoring of sub-cellular organelle organization at a high-throughput with high-content capacity.

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