| Summary: | 3D light‐sheet microscopy is a powerful tool to obtain content‐rich information of cell populations. However, the limited frame rate of high‐quantum‐efficiency cameras hinders its application in high‐throughput flow cytometry. Herein, a high‐throughput 3D‐imaging flow cytometry technique based on light‐sheet microscopy that can screen thousands of cells per second is introduced. This method enables fast, parallel optofluidic scanning of cells by performing 1D acoustofluidic focusing on multiple cells under wide‐field light‐sheet microscopy with a single objective lens. Multicolor 3D‐imaging flow cytometry with cell‐line samples at a record detection throughput of over 2000 cells s−1 is demonstrated. Furthermore, an unprecedentedly large‐scale 3D‐morphology‐based flow cytometric analysis at an order of 105 cells is demonstrated. Results show that the system is able to capture subcellular structures even at high throughputs and obtain cellular information that is overlooked under 2D‐imaging cytometry.
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