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The microfluidic-based, label-free image-guided cell sorter offers a low-cost, high information content, and disposable solution that overcomes many limitations in conventional cell sorters. However, flow confinement for most microfluidic devices is generally only one-dimensional using sheath flow. As a result, the equilibrium distribution of cells spreads beyond the focal plane of commonly used Gaussian laser excitation beams, resulting in a large number of blurred images that hinder subsequent cell sorting based on cell image features. To address this issue, we present a Bessel–Gaussian beam image-guided cell sorter with an ultra-long depth of focus, enabling focused images of >85% of passing cells. This system features label-free sorting capabilities based on features extracted from the output temporal waveform of a photomultiplier tube (PMT) detector. For the sorting of polystyrene beads, SKNO1 leukemia cells, and Scenedesmus green algae, our results indicate a sorting purity of 97%, 97%, and 98%, respectively, showing that the temporal waveforms from the PMT outputs have strong correlations with cell image features. These correlations are also confirmed by off-line reconstructed cell images from a temporal–spatial transformation algorithm tailored to the scanning Bessel–Gaussian beam.

Classification of cell types and isolation of targeted cells according to their imaging and spatial characteristics, beyond traditional fluorescently labeled biomarkers, enable the development of new biological insight and establishment of connections between phenotypical and morphological, and genomic cell information in normal and diseased states. Here, we demonstrate a microfluidic flow cytometer cell sorter that uses fast scanning laser excitation sources and photomultiplier tubes (PMTs), coupled with real-time image processing, to image and sort cells based on user-defined spatial features. Dozens of features, including label-free characteristics such as size, shape, scattering pattern, morphology, as well as the spatial distribution of fluorescently labeled biomarkers, greatly expand the available information space in cell analysis. The use of a disposable microfluidic cartridge that is compatible with the imaging system contributes to size reduction and lowered risk of cross contamination. The so-called cameraless design that uses PMTs to replace CCD makes the system more compatible with conventional fluorescence-activated cell sorters and user friendly, while enjoying the sensitivity and benefits of PMTs. The cell image features can be directly extracted from the temporal waveforms or from the reconstructed cell images via temporal–spatial transformation.