High-Throughput, Low Background, and Wide-Field Microscopy by Flat-Field Photobleaching Imprinting Microscopy

Wide-field photobleaching imprinting microscopy (PIM) can improve fluorescence image contrast by cleverly exploiting the fluorophores' photobleaching properties. However, as conventional wide-field PIM commonly adopts Gaussian illumination with a nonuniform lateral fluence distribution, the field-of-view (FOV) and sampling density are largely reduced. In addition, the slow axial fluence gradient of Gaussian illumination limits the signal-to-background ratio (SBR) improvement and optical sectioning capability of PIM. Here, we present flat-field photobleaching imprinting microscopy (ffPIM) with a uniform lateral excitation fluence and sharp axial intensity gradient at the focal plane. ffPIM demonstrates low background, large FOV, and thin optical section. More importantly, compared to either conventional wide-field PIM or light-sheet microscopy, ffPIM shows much better balance for FOV, sampling density, SBR, and optical sectioning capability. The performance of ffPIM is characterized by simulation and resolving multiple cellular structures. Finally, ffPIM can be easily implemented to a standard commercial wide-field microscope and, thereby, allow general laboratories to benefit from this technique.