Development for Dynamic Live Cell Imaging by Cryo-Electron Tomography and Stem

Abstract

Cryo-EM tomography of intact cells is an emerging technology that compliments crystallography, NMR and single molecule imaging techniques. Its strength is in that it reveals the spatial arrangements of key proteins and complexes during intracellular signaling and mechanical events like motility and division. In this meeting, we describe dynamical Live Cell imaging by Cryo-EM tomography using the Titan Krios microscope with 300 kV electron column and HAADEF detector. Observed living cells were grown directly on Holey Carbon Support Film. In order to make sure that the cells are suitable for observation we analyzed the some parameters of cell movement and cell division on Quantifoil using Fiji and compared these properties with those of cells grown on a normal cell culture plastic plate. After phosphate-buffered saline washing, the cells were done rapid freeze fixation (vitrification) by dropping in liquid ethane using VitrobotTM Mark IV instead of the usual chemical fixation and were transferred onto the microscope immediately while keeping the environment under liquid N2. Cells were imaged over an angular range from −70 degrees to 70 degrees at 2 degrees x cos θ tilt increments automatically and analyzed with Inspect 3D and Amira software to provide 3D images and Volume rendering respectively.We observed some unique architectures of a part of Lamellipodium in GFP-Myosin X expressed COS 7 cell and them from near the nuclear membrane to plasma membrane in COS 7 and several eukaryotic cells (HeLa, NIH3T3⋯ etc.) by Cryo-EM tomography using STEM using intact cells and vitreous cell sections.At future, our system can provide any new information about many kinds of cells and organelles during important events.