biostudies-literatureRen JNational Institutes of HealthNIGMS NIH HHSNational Science Foundation933-942https://www.ebi.ac.uk/biostudies/studies/S-EPMC8412410biostudies-literatureUnknown8(3)Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2×2 mm² region within an imaging depth of ~5 μm for mammalian cells in <10 min and immunofluorescence imaging at a >30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multi-color imaging, depth control and super-resolution imaging.ACS photonics2.5D microscopy: Fast, high-throughput imaging via volumetric projection for quantitative subcellular analysis.PMC8412410R35 GM1380391805200R35GM138039Ren JHan KYfalse2.5D microscopy: Fast, high-throughput imaging via volumetric projection for quantitative subcellular analysis.Imaging-based single-cell analysis is essential to study the expression level and functions of biomolecules at subcellular resolution. However, its low throughput has prevented the measurement of numerous cellular features from multiples cells in a rapid and efficient manner. Here we report 2.5D microscopy that significantly improves the throughput of fluorescence imaging systems while maintaining high-resolution and single-molecule sensitivity. Instead of sequential z-scanning, volumetric information is projected onto a 2D image plane in a single shot by engineering the emitted fluorescence light. Our approach provides an improved imaging speed and uniform focal response within a specific imaging depth, which enabled us to perform quantitative single-molecule RNA measurements over a 2×2 mm² region within an imaging depth of ~5 μm for mammalian cells in <10 min and immunofluorescence imaging at a >30 Hz volumetric frame rate with reduced photobleaching. Our microscope also offers the ability of multi-color imaging, depth control and super-resolution imaging.2021-01-01T00:00:00Z2021 Mar2025-04-05T14:05:04.416Z2025-04-05T14:05:04.416ZS-EPMC84124103448561410.1021/acsphotonics.1c00012