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Knife-Edge Scanning Microscopy


Figure 4: KESM control screen. The operator controls the KESM through a browser-based interface. This interface allows operators to (1) set sample volume cutting parameters, (2) view the most recent images, and (3) vary cutting parameters, such as speed of slicing and slice thickness, to improve image quality. This view shows raw data as acquired including the knife edge, and non-tissue areas of the camera sensor, which are seen as black stripes. During image processing these are removed, and the resulting images are stitched together.


operator to set slicing parameters, such as slicing speed (mm/s), slice thickness (µm), clearance angle, water bath temperature, light level, and other parameters (panel 3). A major feature of the system is that the user interface displays the most recently acquired images in a tile viewer within tens of seconds aſt er slicing (panel 2). T e operator can also see the status of the microscope in real time on any device with an internet browser, including mobile devices and tablets. T is quick visual feedback allows the operators to adjust the slicing and imaging parameters to optimize the imaging and react promptly to any issues that might arise.


Figure 5 : Data fl ow in the KESM. (A) Data are received by the process, which retains the raw image data in RAM until all processing is completed. It shares this memory with the three: the Image Compressor, Image Tiler, and Image Analyzer. Image Compressor compresses the image to a PNG bytestream in memory and then sends the compressed image to the local storage cluster. Image Tiler generates a tile pyramid from the raw image. Image Analyzer generates basic image statistics from the raw data and inserts the statistics in a queryable database. These images are sent to the AWS S3 cloud. (B) Database reads commands from KESM operator to enable cluster synchronization and can control multiple KESMs on a web browser from any electronic device.


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Image acquisition and storage . As the KESM takes a slice and the camera records the data, the pixel data of each image go through multiple processing steps using a scalable framework across a cluster of image processing computers. T e KESM sends the image data to any machine in the cluster using 3Scan’s XACT communication protocol, a framework that facilitates transactional exchanges for high-throughput and high-fi delity communication among multiple computers ( Figure 5A ). Data are acquired by the KESM and passed to the imaging processing cluster via XACT, where the Receptionist process shares raw image data with three daughter processes: data are analyzed to generate basic image statistics, multi- resolution image tiles (pyramidal tiling) are produced, and the images are compressed in PNG format—all in parallel. Aſt er these processes are complete, image data are passed to local storage via the XACT protocol. A local copy is maintained for approximately two months to facilitate low-latency image


www.microscopy-today.com • 2017 July


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