High‐Resolution Lensless Microscopy Imaging Based on Fluorescence Intermittency

Lensless imaging microscopy has gained extensive application with the merits of system compactness and cost efficiency; however, its spatial resolution is usually compromised compared to conventional lens-based microscopes. To further enhance the spatial resolution, we built a lensless imaging system integrating a phase mask and a CMOS image sensor, and employed fluorescence fluctuation super-resolution microscopy (FF-SRM) algorithms to fully exploit the fluorescence intermittency (FI) characteristics of fluorescent molecules for high-resolution lensless image reconstruction. The study demonstrates that lensless image sequences processed by the Wiener deconvolution method can effectively retain the original fluorescence intermittency information, allowing for high-resolution reconstruction using FF-SRM algorithms. Furthermore, by combining expansion microscopy (ExM) and leveraging multi-algorithm synergy, we obtained additional improvements in spatial resolution and image quality for lensless imaging, facilitating clear visualization of biological subcellular organelles. This scheme offers a new pathway to achieve high spatial resolution imaging with practical advantages in simplicity and affordability.