Abstract
Optical 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.
Highlights
Optical imaging is paramount for disease diagnosis and to access its progression over time
Data obtained by VFC imaging cytometry contain cellular, multicellular, and intracellular information of a large population, which can be used to classify cell types and access their physiological state
The data can be used for multiparameter analysis and gather large population statistics
Summary
Optical imaging is paramount for disease diagnosis and to access its progression over time. Unlike state-of-the-art point-illumination-based biomedical imaging techniques, the sheetbased 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. Conventional FCs are constrained by low-throughput (due to sequential cell screening), poor resolution (owing to flow-induced optical aberration and limited photon budget), and complex instrumentation (due to the necessity of sheath fluid-based hydrodynamic focussing)[7,11]. To overcome these limitations, a light sheetbased system is developed. VFC is a promising next-generation technology that may find use in biophysics and optical imaging
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