Thin-sheet laser imaging microscopy for optical sectioning of thick tissues

Abstract

We report the development of a modular and optimized thin-sheet laser imaging microscope (TSLIM) for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear, and rat brain at a resolution that is comparable to wide-field fluorescence microscopy. TSLIM optically sections tissue using a thin sheet of light by inducing a plane of fluorescence in transparent or fixed and cleared tissues. Moving the specimen through the thinnest portion of the light sheet and stitching these image columns together results in optimal resolution and focus across the width of a large specimen. Dual light sheets and aberration-corrected objectives provide uniform section illumination and reduce absorption artifacts that are common in light-sheet microscopy. Construction details are provided for duplication of a TSLIM device by other investigators in order to encourage further use and development of this important technology.

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“…The inner ear was attached to a plastic rod and immersed in Spalteholz solution in the TSLIM specimen chamber. TSLIM uses a green laser to project a thin sheet of light through the transparent specimen and the fluorescent planes illuminated by the laser are recorded as optical sections through the tissue (Santi et al 2009). Movement of the specimen through the light-sheet nondestructively produces a complete stack of serial sections of the whole cochlea.…”

Implementation of a continuous scanning procedure and a line scan camera for thin-sheet laser imaging microscopy

“…A thin-sheet laser imaging microscope (TSLIM) is a device for nondestructive optical sectioning of organisms and thick tissues such as the mouse cochlea, zebrafish brain/inner ear and rat brain [1]. TSLIM is a modular device that can image transparent tissues such as zebrafish embryos or fixed and cleared transparent tissues that have been labeled with a fluorochrome.…”

“…However, since the light sheet is thinnest only in a narrow region (i.e., the beam waist) an x-axis scanning procedure was used to produce a well-focused composite image across the full width of the specimen. The original description of TSLIM [1] used a single green laser and a full-frame CCD camera in which the center 40 lines of the CCD array were collected using a start/stop procedure and an x-axis composite image was produced by stitching these image columns together. The present report describes hardware and software solutions that increase image quality and speed of optical sectioning, which also reduces photobleaching of the specimen.…”

“…It has been used to image organisms in a plane of water [5] but its most common application is to optically section thick tissues that have been labeled with a fluorochrome and made chemically transparent. Light-sheet microscopes have been developed by a number of different investigators [1][2][3][4][5][6][7][8] and this technology is rapidly evolving. TSLIM [1] was developed by optimizing components of previous light-sheet microscopes and this report describes software and hardware enhancements to TSLIM that significantly improve its capabilities.…”

“…Light-sheet microscopes have been developed by a number of different investigators [1][2][3][4][5][6][7][8] and this technology is rapidly evolving. TSLIM [1] was developed by optimizing components of previous light-sheet microscopes and this report describes software and hardware enhancements to TSLIM that significantly improve its capabilities. Specifically, TSLIM now features a line scan camera in addition to the full-frame CCD camera, hardware triggering of the cameras, and software that allows for continuous scanning of the image in the x-axis.…”