Multicolor iLIFE (m-iLIFE) volume cytometry for high-throughput imaging of multiple organelles

To be able to resolve multiple organelles at high throughput is an incredible achievement. This will have immediate implications in a range of fields ranging from fundamental cell biology to translational medicine. To realize such a high-throughput multicolor interrogation modality, we have developed a light-sheet based flow imaging system that is capable of visualizing multiple sub-cellular components with organelle-level resolution. This is possible due to the unique optical design that combines an illumination system comprising two collinear light sheets illuminating the flowing cells and a dedicated dual-color 4f-detection, enabling simultaneous recording of multiple organelles. The system PSF sections up to 4 parallel microfluidic channels through which cells are flowing, and multicolor images of cell cross-sections are recorded. The data is then computationally processed (filtered using ML algorithm, shift-corrected, and merged) and combined to reconstruct the 3D multicolor volume. System testing is conducted using multicolor fluorescent nano-beads (size \(\sim 175\) $\sim 175$ nm) and flow-based imaging parameters (PSF size, motion-blur, flow rate, frame rate, and number of cell-sections) are determined for quality imaging. Drug treatment studies were carried out for healthy and cancerous HeLa cells to check the performance of the proposed system. The cells were treated with a drug (Vincristine, which is known to promote mitochondrial fission in cells), and the same is compared with untreated control cells. The proposed multicolor iLIFE system could screen \(\sim 800\) $\sim 800$ cells/min (at a flow speed of \(2490 ~\upmu\) $2490\mathrm{\mu }$ m/s), and the drug treatment studies were carried out up to 24 h. Studies showed the disintegration of mitochondrial network and dysfunctional lysosomes and their accumulation at the cell membrane, which is a clear indication of cell apoptosis. Compared to control cells (untreated), the mortality is highest at a concentration of 500 nM post 12 h of drug treatment. With the capability of multiorganelle interrogation and organelle-level resolution, the multicolor iLIFE cytometry system is suitably placed to assist optical imaging and biomedical research.