2.5D Microscopy: Fast, High-Throughput Imaging via Volumetric Projection for Quantitative Subcellular Analysis

Abstract

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 mm 2 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 multicolor imaging, depth control, and super-resolution imaging.

On the validity domain of maximum likelihood estimators for depth-of-field extension in single-molecule localization microscopy

“…Because the lateral resolution is inversely proportional to the numerical aperture (NA), this technique employs high-NA objectives, which considerably limits its natural depth-of-field (DoF). Several groups have demonstrated that placing a phase mask in the aperture stop of a localization microscope can significantly increase its DoF [2][3][4][5]. Recently, we have proposed to use annular binary phase masks to extend 2Dlocalization performance within a prescribed DoF range [6,7] (see Fig.…”

E2E-BPF microscope: Extended depth-of-field microscopy using learning-based implementation of binary phase filter and image deconvolution

“…2, E2E-BPF outperforms all the reference pupil designs in terms of RMSE, SSIM, and DoF-extension ability. Notably, compared with the binary phase structure that has recently been applied to DoF-extension uorescence microscope 37 Experimental performance evaluation: uorescence microspheres…”

Point spread function engineering based on wavefront phase modulation

“…Lensless imaging systems enable large DOF extension 138 , broadband achromatic imaging 139 , 3D imaging 140 , and microscopy 141 . Planar optics can also be used as phase modulation devices in super resolution microscopy 142 or wavefront coding systems 81,[143][144][145] . Similarly, with the addition of deep optics, the speed and quality of lensless imaging can be enhanced 146,147 .…”