2.5D microscopy with polarization independent SLM for enhanced detection efficiency and aberration correction

Fast, volumetric imaging by fluorescence microscopy is essential in studying biological phenomena and cellular functions. Recently, single-shot 2.5D microscopy showed promising results for high-throughput quantitative subcellular analysis via extended depth of field imaging without sequential z-scanning; however, the detection efficiency was limited and it lacked depth-induced aberration correction. Here we report that a spatial light modulator (SLM) in a polarization insensitive configuration can significantly improve the detection efficiency of 2.5D microscopy, while also compensating for aberrations at large imaging depths caused by the refractive index mismatch between the sample and the immersion medium. We highlight the improved efficiency via quantitative single-molecule RNA imaging of mammalian cells with a 2-fold improvement in the fluorescence intensity compared to a conventional SLM-based microscopy. We demonstrate the aberration correction capabilities and extended depth of field by imaging thick specimens with fewer z-scanning steps.