2, Technische Universität, Ilmenau, Germany
3, Electrical and Computer Engineering, University of Minnesota, Minneapolis, MN, USA
For: Thin-sheet laser imaging microscopy for optical sectioning of thick tissues
This file can be viewed as an interactive 3-D PDF file in Adobe Acrobat 9 (Adobe Systems Incorporated, San Jose, CA, USA). Dual-laser illuminators are positioned along an x axis optical bench rail with the specimen chamber positioned between the illuminators. The specimen is moved in the x, y, and z directions by three micropositioners and by a rotating stage which is attached to the specimen rod that extends into the fluid filled chamber.
A movie showing a stack of 134 1-μm serial sections of the scala media of the adult mouse cochlea that has been virtually resectioned in an oblique plane along the outer hair cells.
Supplementary Movie 2. TSLIM optical sections of the spiral ganglion. A movie showing a stack of 82 1-μm serial sections through the spiral ganglion of the mouse cochlea showing neuron cell bodies, nuclei, and their axons.
A movie showing a stack of 100, 20 μm serial transverse sections through the head of a Casper zebrafish showing the brain and a 3D reconstruction of he inner ear.
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.
The ability to selectively visualize three-dimensional (3-D) biological structures is useful for understanding structure, function, and dysfunction relationships in tissues and organs, and is one of the initiatives of the National Institutes of Health (NIH) Roadmap for Medical Research. Three-dimensional reconstruction of anatomical structures requires that tissues be sectioned (either mechanically or nondestructively) and structures segmented for 3-D rendering. Nondestructive optical sectioning produces well-aligned sections and can be performed, at high resolution using confocal and multiphoton microscopy, but imaging depth is limited to a few hundred microns. For thick specimens, micro MRI or micro CT can be used, but at lower resolution and without the advantages offered by immunofluorescence. Light sheet–based microscopy is an important, innovative tool that offers nondestructive optical sectioning of selectively stained thick tissues at a spatial resolution between that of micro MRI and confocal microscopy. Development of this technology has been hindered by the lack of a commercial device, although several investigators have constructed light sheet–based microscopes.
Optical sectioning using a plane of light was described as early as 1903 by Siedentopf and Zsigmondy (1), but given the unfortunate name of ultramicroscopy, which now refers to electron microscopy. The basis for light-sheet optical sectioning and the materials for the construction of a device are quite simple. A thin light sheet is produced using a cylindrical lens and is projected through either a transparent or fixed and cleared specimen to illuminate a thin plane (i.e., optical section) within the tissue. The optical section is observed orthogonal to the light sheet, and by moving the specimen through the thin plane of light, a z stack of serial sections is produced. Pioneering work on a light sheet microscope was done by Voie et al. (2,3,4) and called orthogonal-plane fluorescence optical sectioning (OPFOS). Their device collected real-time, 2-D optical sections in cleared tissues using a light sheet that was produced using a single laser, beam expander, and cylindrical lens. Fuchs et al. (5) constructed a similar device, called thin–light sheet microscopy (TLSM) to optically section specimens in seawater. Another device was developed by Huisken et al. (6) called selective plane illumination microscopy (SPIM). This method rotates an agarose-embedded specimen through a single laser light sheet, but requires complex algorithms to produce a z stack of optical sections. Dodt et al. (7) called their method ultramicroscopy and added dual light sheet illumination for the optical sectioning of larger specimens such as the mouse brain.
The quality of the optical sections produced by all of these devices is dependent upon the optical geometry of the light sheet. A light sheet produced by a cylindrical lens has a Gaussian intensity profile and reaches a minimal thickness (called the beam waist) in close proximity to the focal plane of the lens (see Supplementary Materials and Supplementary Figure 1 for details). The beam waist has a relatively constant thickness over a region called the confocal parameter, which is also twice the Raleigh range. For small specimens whose dimensions do not exceed the confocal parameter, the optical section appears to have nearly uniform resolution across the width of the specimen. However, for large specimens, optimized resolution and focus is present only in the tissue region illuminated within the confocal parameter of the light sheet. For large specimens and full-frame imaging, one can choose lenses that result in a larger confocal parameter, but this configuration also produces thicker light sheets and lower image resolution. In order to achieve high-resolution across the full width of large specimens, Buytaert and Dirckx (8) moved a specimen through the beam waist and stitched image columns together to produce a high-resolution, well-focused composite image. However, a light sheet–based device has not yet been developed which incorporates the best features of the previous devices (2,3,4,5,6,7,8). This study reports on the development of such a device, which is modular and optimized, and called a thin-sheet laser imaging microscope (TSLIM). In addition, we provide details for the construction of a TSLIM device by other investigators to encourage its further use and development (see Supplementary Materials).