Knife-edge scanning microscopy for connectomics research

Abstract

In this paper, we will review a novel microscopy modality called Knife-Edge Scanning Microscopy (KESM) that we have developed over the past twelve years (since 1999) and discuss its relevance to connectomics and neural networks research. The operational principle of KESM is to simultaneously section and image small animal brains embedded in hard polymer resin so that a near-isotropic, sub-micrometer voxel size of 0.6 µm × 0.7 µm × 1.0 µm can be achieved over ∼1 cm 3 volume of tissue which is enough to hold an entire mouse brain. At this resolution, morphological details such as dendrites, dendritic spines, and axons are visible (for sparse stains like Golgi). KESM has been successfully used to scan whole mouse brains stained in Golgi (neuronal morphology), Nissl (somata), and India ink (vasculature), providing unprecedented insights into the system-level architectural layout of microstructures within the mouse brain. In this paper, we will present whole-brain-scale data sets from KESM and discuss challenges and opportunities posed to connectomics and neural networks research by such detailed yet system-level data.

Automated neurovascular tracing and analysis of the knife-edge scanning microscope Rat Nissl data set using a computing cluster

“…whole-brain scale data [17]. KESM has acquired three different mouse brain data stained in Golgi, India ink, and Nissl [6]. Nissl stains all cell bodies of neurons leaving vasculatures unstained in the brain.…”

“…This whole process generates a thin section of tissue over the diamond knife. Line-scan imaging is done near the tip of the diamond knife as the tissue gets cut, resulting in high resolution images of the cut tissue [6]. (1) high-speed line-scan camera, (2) microscope objective, (3) diamond knife assembly and light collimator, (4) specimen tank (for water immersion imaging), (5) three-axis 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.…”

“…(1) high-speed line-scan camera, (2) microscope objective, (3) diamond knife assembly and light collimator, (4) specimen tank (for water immersion imaging), (5) three-axis 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. Reprinted from [6]. …”

“…In 2008, the earliest data were obtained from Golgi-stained (neuronal morphology) and India ink-stained (vasculature) mouse brain. In early 2010, a whole mouse brain stained with Nissl was scanned to observe its cell body distribution [6].…”

Scalable, incremental learning with MapReduce parallelization for cell detection in high-resolution 3D microscopy data

“…1, is one of the first instruments to produce sub-micrometer resolution (∼1µm 3 ) data from whole small animal brains. We successfully imaged, using the KESM, entire mouse brains stained with Golgi (neuronal morphology) [6], [7], [5], India ink (vascular network) [8], and Nissl (soma distribution) [7], [9]. The Nissl data set in particular enables detailed studies of whole-brain cortical and subcortical distribution of neuronal cell bodies.…”