Our collaborator Peijun Zhang's laboratory recently published a new assay to automate correlative light and electron microscopy (CLEM) of HIV particles inside cells (see workflow below). CLEM is a method to image small fluorescent particles (in this case, fluorescently labeled HIV made by Zhou Zhong in the Ambrose lab) inside cells on electron microscopy grids using live-cell confocal microscopy. Then the cells are frozen without fixation and imaged by cryo-electron microscopy and tomography (cryoEM/ET). CLEM is a great tool that allows you to identify small fluorescent particles within a cell and zoom in on it by cryoEM/ET to get more structural information on it.
Because viral complexes are so small inside the relatively large cell, one needs to map their location precisely in order to know where to look for them by cryoEM/ET. This is like using a GPS system to locate a car within a neighborhood or a city! Unfortunately, the light microscope mapping coordinates are not the same as the electron microscopy mapping coordinates, so Peijun's lab had to "translate" the confocal coordinates to the "language" used by the electron microscope. To help with this, they used fluorescent beads that are a different color and size than the fluorescent HIV. These beads are added to the grid during the initial confocal imaging and used as visual landmarks during mapping. In the above GPS metaphor, it would be the same as using buildings as reference points to know the position of your car within the city.
Really cool work and we were happy to be a part of it! Check out the paper here. We hope to use AutoCLEM to visualize HIV complexes associated with cellular structures inside cells with high resolution.
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