Single-molecule imaging is a valuable tool to study subcellular structure, gene expression and dynamics of biomolecules. Despite advancement of fluorescence imaging techniques, it has been challenging to image single-molecules in thick samples. Here we employ an inclined beam swept over the imaging area and a confocal slit to reject out-of-focus background, which enables high contrast and large field-of-view single-molecule imaging over a depth of 50 μm. Our method provides &gt;40-fold larger imaging area than highly inclined and laminated optical sheet (HILO) microscopy, and shows significantly improved optical sectioning and sensitivity compared to spinning disk microscopy. We present the performance of our method and demonstrate a few examples in transcription study, live-cell imaging and super-resolution microscopy. Our microscopy uses only a single high numerical aperture objective and requires neither an additional illuminator nor remote detection objectives, thereby fully compatible with inverted microscopes.

Extended field-of-view single-molecule imaging with a deep penetration depth

2019 Spring National Meeting

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