Advanced Light Sheet Volume Flow Cytometry (VFC/iLIFE) and Spectroscopy System for Cell Biophysics and Clinical Biology

Includes bibliographical references

PhD;2023;Instrumentation and Applied Physics

Optical imaging is paramount for disease diagnosis and to access its progression over time. The developed light sheet-based optical flow imaging techniques, known as Volume Flow Cytometry(VFC/iLIFE), and related techniques (Multicolor Volume Flow Cytometry, and High-throughput Multichannel Multi-sheet Multicolor Imaging Cytometry) offer a range of advanced capabilities for disease diagnosis and monitoring disease progression over time. Unlike existing point-illumination-based biomedical imaging methods, these developed techniques use light sheet illumination, which enables single-shot sectional visualization, high throughput analysis, real-time parameter estimation, and instant volume reconstruction with an organelle-level resolution for live specimens. The detection is carried out in an orthogonal configuration. On the other hand, Spectral Integrated Light-sheet imaging and flow-based inquiry (Spectral iLIFE) is an innovative imaging/spectroscopy system that combines the benefits of spectroscopy and optical microscopy. The technique provides valuable insights into molecular interactions and other critical information alongside imaging. By utilizing the spectral characteristics of fluorophores, Spectral iLIFE enables the identification and analysis of specific molecular components within a biological sample. This integration can allow researchers to not only visualize cellular structures and organelles but also study molecular interactions, such as protein-protein interactions or the binding of specific molecules. The ability to simultaneously obtain imaging data and spectral information enhances our understanding of complex biological processes and provides a more comprehensive view of molecular dynamics within living systems. The stages of system development include the fabrication of a multichannel microfluidic chip, light sheet illumination, and multicolor widefield 4f detection. The experimentation involves the preparation of fluorescently-labeled cells, determination of flow-variant PSF, calibration of the light sheet, image deconvolution, and separation of spectrally-distinct fluorescent signals. The system offers the determination of physiologically relevant cell parameters (organelle count, morphology, and organelle distribution) on the go. Moreover, it facilitates changes over time, thereby revealing the metastatic progression of diseases. Overall, the developed imaging cytometry technique and its variants allow for real-time monitoring of sub-cellular organelle organization with high throughput and high-content capacity, enhancing our ability to understand disease progression and facilitate drug therapy.