Short-exposure annular LED array-based digital refocusing microscopy via multiplexed illumination

Digital refocusing microscopy based on light-emitting diode (LED) arrays extends the depth of field of conventional microscopes without mechanical scanning. However, its sequential image acquisition process inherently limits temporal resolution. To overcome this limitation, we propose a short-exposure digital refocusing microscopy using multiplexed illumination. This approach not only shortens the imaging time of LED-array-based digital refocusing microscopy but also addresses the common issues of low contrast in dark regions and loss of fine image details under short-exposure conditions. Consequently, it enables achieving higher imaging speed while preserving high-quality digital refocusing images. To further reduce the number of required measurements, we propose a digital refocusing microscopy based on an annular LED array. Compared with conventional full circular arrays, the annular configuration reduces the measurements from 37 to 16 while enhancing image contrast and spatial resolution. Experimentally, the proposed method improves the structure similarity index measure (SSIM) of the refocused images in the dark regions by at least 3.16 times relative to sequential single-LED illumination. Collectively, these two strategies establish a practical and efficient framework that improves the temporal resolution of LED-array-based digital refocusing microscopy, thereby enabling its application to the dynamic observation of biological samples.