25D microscopy with polarization independent SLM for enhanced detection efficiency and aberration correction

Abstract:
“Fast, volumetric imaging by fluorescence microscopy is essential in studying bi-ological phenomena and cellular functions. Recently, single-shot 2.5D microscopy showedpromising results for high-throughput quantitative subcellular analysis via extended depth offield imaging without sequentialz-scanning; however, the detection efficiency was limited and itlacked depth-induced aberration correction. Here we report that a spatial light modulator (SLM)in a polarization insensitive configuration can significantly improve the detection efficiency of2.5D microscopy, while also compensating for aberrations at large imaging depths caused bythe refractive index mismatch between the sample and the immersion medium. We highlightthe improved efficiency via quantitative single-molecule RNA imaging of mammalian cellswith a 2-fold improvement in the fluorescence intensity compared to a conventional SLM-basedmicroscopy. We demonstrate the aberration correction capabilities and extended depth of field byimaging thick specimens with fewerz-scanning steps.”