3D FLOW CYTOMETRY USING SNAPSHOT PROJECTION OPTICAL TOMOGRAPHY

Abstract

While flow cytometry is routinely used for high-throughput interrogation of suspended cells, it misses visual context provided by the rich spatial information inside the cell. On the other hand, fluorescence microscopy has been shown to capture the 3D images of cells with high spatial resolution. The existing methods to acquire a 3D image, however, are more suitable for adherently growing cells than suspending cells, and the low imaging throughput hinders their adaptation in a clinical workflow. We have successfully integrated the advantages of both systems to generate 3D images of living cells in suspension at a high throughput while maintaining relatively high spatial resolution. The enabling technology, known as snapshot optical tomography (SOT), can capture the 3D image of a microscopic specimen in a single snapshot. Our system acquires a set of 36 projection images simultaneously, each from a distinct viewing angle relative to the sample via a micro-lens array. A direct mapping of the projection images in the 3D Fourier space allows for fast-yet-accurate reconstruction of a 3D volumetric distribution of fluorescence intensity. The cell solution flows through a straight microfluidic channel driven by pneumatic pressure. So far, we have confirmed the system can run for 30 minutes at a rate of 13 cells/sec, resulting in over 20,000 3D images with spatial resolution of 1.2 μm. The imaging throughput is currently limited by the response time of the motion detection software, which triggers the camera to record the cell in the field of view. Our approach to 3D imaging flow cytometry may open a new door to high-throughput, high-content cell imaging in a clinical workflow.