Reflected Beam Light-Sheet Microscopy for Whole-Cell 3D Super-Resolution Imaging

Abstract

Single molecule super-resolution (SM-SR) imaging is performed by localizing individual fluorophores within a densely labeled sample. SM-SR in 3D throughout a whole cell is hampered by wide-field activation because imaging light cannot be targeted to the in-focus image plane as done with TIRF. This results in high background fluorescence that severely degrades detection and localization precision. We have developed a technique, which we termed Reflected Beam Light-Sheet Microscopy (RB-LSM), that uses a common, single objective, inverted epi-fluorescence microscope along with a reflective surface to generate illumination only in the image focal plane. We use a reflective, planar surface which is placed at an angle of 45° on a cover slip to reflect the beam. This surface forms the side wall of a microfluidic channel incorporated into a microfluidic device. A light-sheet is generated through the objective and reflected by the surface such that it illuminates only the in-focal plane of the cell. The light-sheet is scanned through the cell for whole-cell imaging and an astigmatic lens in the emission light path enables the 3D localization of individual emitters, creating a whole-cell 3D super-resolution image. RB-LSM provides unprecedented localization accuracy due to ∼20 fold background reduction. An additional benefit comes from reduced photobleaching of un-imaged, out of focus fluorophores. The microfluidics device provides a closed environment that allows for fast and automated buffer exchange, which is utilized to maintain a reduced oxygen environment needed for many SM-SR probes. The RB-LSM is a system that is easily implemented on a regular wide-field fluorescence microscope. The development of this microscope and its imaging and analysis routines will therefore be of great value to investigators seeking a relatively straightforward, inexpensive method for whole-cell 3D super-resolution microscopy and high-speed live cell imaging.