Knife‐edge scanning microscopy for imaging and reconstruction of three‐dimensional anatomical structures of the mouse brain

Abstract

Summary Anatomical information at the cellular level is important in many fields, including organ systems development, computational biology and informatics. Creating data sets at resolutions that provide enough detail to reconstruct cellular structures across tissue volumes from 1 to 100 mm3 has proven to be difficult and time‐consuming. In this paper, we describe a new method for staining and imaging large volumes of tissue at sub‐micron resolutions. Serial sections are cut using an automated ultra‐microtome, whereas concurrently each section is imaged through a light microscope with a high‐speed line‐scan camera. This technique, knife‐edge scanning microscopy, allows us to view and record large volumes of tissue in a relatively small amount of time (approximately 7 mm2 s−1). The resolution and scanning speed of knife‐edge scanning microscopy provides a new method for imaging tissue at sufficient resolution to reconstruct maps of cellular distribution and morphology. We show that these techniques preserve the alignment of serial sections accurately enough to allow for reconstruction of neuronal processes and microvasculature. Expanding these techniques to other tissues opens up the possibility of creating fully reconstructed cellular maps of entire organs.

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

“…Other optical methods for whole brain imaging afford higher resolution than CLSM, but at the price of longer preparation and/or imaging time [2][3][4] , or of a sparse z sampling 5 .…”

“…Actually, many technological efforts are devoted to reconstruct the mouse brain with microscopic resolution. Optical methods based on serial sectioning of resin-embedded samples, as Knife-Edge Scanning Microscopy (KESM) 2 , Micro-Optical Sectioning Tomography (MOST) 3 and fluorescence-MOST (fMOST) 4 , can provide sub-micron three-dimensional resolution imaging of entire mouse brains. However, the total time needed for the preparation and imaging of a single sample is of the order of several weeks, limiting the practical application of such methods.…”

Visualization of Cellular and Microvascular Relationships

“…The advent of high-throughput microscopy allows researchers to quickly produce large volumetric data sets representing high-resolution biological tissue. Knife-Edge Scanning Microscopy (KESM) [14] is capable of imaging large specimens at microscopic resolution producing data at a rate in excess of 100MB/second. Imaging entire organs such as the mouse brain produces several terabytes of data.…”

Knife-edge scanning microscopy for connectomics research

“…1 shows a photo of the KESM with its major components: (1) high-speed line-scan camera, (2) microscope objective, (3) diamond knife assembly and light collimator, (4) specimen tank (for water immersion imaging), (5) threeaxis precision air-bearing stage, (6) white-light microscope illuminator, (7) water pump (in the back) for the removal of sectioned tissue, (8) PC server for stage control and image acquisition, (9) granite base, and (10) granite bridge. See [10], [20] for technical details. The imaging principle of KESM is shown in Fig.…”

“…An emerging alternative to optical sectioning is physical sectioning. These approaches include Knife-Edge Scanning Microscopy (KESM) [10], [11], [3], [12] (cf. [13] that adopted the same principles as KESM), Array Tomography [14], and All-Optical Histology [15] that use light microscopy (LM) or fluorescence imaging (see [16] for a general overview of LM), while Serial Block-Face Scanning Electron Microscopy (SBF-SEM) [17], Automatic Tape-Collecting Lathe Ultramicrotome (ATLUM) [18], and Focused Ion Beam Scanning Electron Microscopy (FIB/SEM) [19] utilize electron microscopy (EM) for the actual imaging.…”