2/2/2024 0 Comments The lost watch 3d 2.1 serialNext, live imaging allowed us to track an infected cell over 5 days, by which time it was clear that the bacteria were alive, growing and dividing (the EGFP signal was increasing in area) despite being located in an LC3+ compartment, which is conventionally associated with Mtb killing. First, we identified lymphatic endothelial cells that had been transduced with LC3–RFP (the LC3B form, also known as MAP1LC3B) and also contained EGFP-expressing bacteria. We hypothesised that the bacteria were growing in autophagosomes, and this was investigated using this 3D CLEM workflow. In our study, we determined that there were fewer intracellular bacteria when the process of autophagy was inhibited. The workflow is illustrated using our recent analysis of primary human lymphatic endothelial cells (hLECs) infected with Mycobacterium tuberculosis (Mtb), a newly identified niche for the bacterium in the lymph nodes of patients with tuberculosis ( Lerner et al., 2016). In both cases, the revealed surface of the block is imaged using a backscattered electron (BSE) detector, and the process repeated sequentially to build up a stack of images through the volume of the sample. In FIB-SEM, a gallium ion beam sputters slices of material from the blockface, whereas in SBF-SEM a diamond knife in a miniaturised ultramicrotome removes thin slices from the blockface. In array tomography, sections are cut manually or automatically ( Hayworth et al., 2006) and placed in an array on a silicon wafer for large area imaging in the SEM. These innovative ‘volume electron microscopy’ techniques ( Kremer et al., 2015 Peddie and Collinson, 2014) include array tomography ( Micheva and Smith, 2007 Wacker and Schroeder, 2013), focused ion beam SEM (FIB-SEM) ( Heymann et al., 2006) and serial blockface SEM (SBF-SEM) ( Denk and Horstmann, 2004). However, automated systems based on the scanning electron microscopy (SEM) are gaining in popularity. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research.Įlectron microscopy for CLEM was traditionally performed by manually serial sectioning the sample and imaging each section using transmission electron microscopy (TEM), usually requiring more than 100 sections to image a single cell. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. An upgraded video transmission system is now capable of dual signal frequency and dual channel, streaming video from an onboard FPV camera and the main camera simultaneously, for better pilot and camera operator collaboration.The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Increased intelligence adds multiple intelligent flight modes, including Spotlight Pro, giving even single pilots the ability to create complex, dramatic shots. FlightAutonomy has been revised and developed specifically for the Inspire 2, providing two directions of obstacle avoidance and sensor redundancy. A dual battery system prolongs the flight time to a maximum of 27 minutes (with an X4S), while self-heating technology allows it to fly even in low temperatures. It goes from 0 to 50 mph (80 kph) in just 5 seconds and hits a maximum speed of 58 mph (94 kph) and has a max descent speed of 9m/s for unheard of speed and agility in an aircraft this size. The image processing system CineCore2.0 has been upgraded to CineCore2.1, recording video at up to 6K in CinemaDNG/RAW and 5.2K in Apple ProRes when used with Zenmuse X7 camera. The Inspire 2 takes everything that was good about the Inspire 1 and improves it. The launches of the Zenmuse X5 and X5R cameras further cemented the Inspire as a critical tool for filmmakers around the globe. The first filmmaking drone in the world to integrate an HD video transmission system, 360° rotating gimbal and a 4K camera, as well as the simplicity of app control.
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