2.5d Microscopy: Fast, High-Throughput Subcellular Imaging Via Volumetric Projection

Single-cell transcriptomics allows hundreds to thousands of genes to be measured simultaneously in individual cells, showing its tremendous power in understanding fundamental biological phenomena and studying cell functions. Imaging-based approaches, such as single-molecule RNA fluorescence in situ hybridization (smFISH), are powerful in quantitatively measuring the abundance of transcripts on the subcellular level and in revealing their spatial location with high resolution. However, its throughput is relatively low compared to other techniques such as imaging flow cytometry. To speed up three-dimensional (3D) imaging acquisition of fluorescence microscope, we have sought an approach to extend the depth of field toward no serial z-scanning while maintaining high spatial resolution and high photon collection efficiency. By encoding a binary axisymmetric phase plate at the conjugated back focal plane of the detection path, volumetric information is projected onto a 2D image plane with a single shot (2.5D) and the original image can be readily interpreted without complicated image post-processing. We demonstrated the potential capability of our 2.5D microscopy in fast high-throughput smFISH imaging which reduced imaging acquisition time by one order of magnitude compared to conventional approach. In addition, we applied it to visualize proteins and other subcellular markers via immunofluorescence. Our approach will open new possibilities in developing fast fluorescence imaging systems capable of performing high-throughput and high-content imaging analysis.