This video will focus on preparation of yeast salmonella for cryo-electron tomography We will go through workflow from cell cultivation to cryo-electron tomography Saccharomyces cerevisiae are cultivated for ion specimen preparation in suspension It is recommended to work in sterile conditions inside of the laminar flow box ten milliliters of sterile yeast extracted to medium for cell cultivation is added to sterilize cultivation flask one milliliter of sterile twenty percent glucose is added to the flask with cultivation medium one yeast colony is carefully picked from our cultivation plate and transferred to the prepared growth medium in cultivation flask cultivation flask is then covered with foil and mixed well to disperse cells cell culture is placed in the incubator and cultivated at thirty degrees with constant agitation until the exponential phase is reached yeast cell culture is grown up to exponential phase and cell physical density is measured by holding meter against a clean medium as a reference sample cell culture is concentrated to OD1 or OD30 in addition of five percent of glycerol before blanche freezing Electro-microscopic grids surface is cleaned and activated for application of cell suspension by plasma grids are placed in glow discharging chamber facing carbon side up and procedure of plasma cleaning is set to thirty to forty-five seconds EM grids have now and they are ready for cell sample application Vitrification of yeast cell suspension on EM grids is done by blanche freezing into the liquid ethane using Vitrobot machine The preparation of liquid ethane takes place by liquefying ethane gas in a metal cup cooled with liquid nitrogen EM grid with cell suspension is blotted by filter paper from the back side and by non adhesive plastic bent from the front carbon side Temperature inside of the Vitrobot chamber is set to eighteen degrees and humidity to one hundred percent waiting time, blot time and blot force are set on Vitrobot screen as well The glow discharge grid is picked by Vitrobot tweezers and mounted to the instrument three point five microliters of Saccharomyces cerevisiae suspension is applied to the carbon side of the grid inside of the Vitrobot climate chamber Suspension needs to be properly mixed before every application of the grid grid is blanche frozen into the liquid ethane electro-microscopy grids with vitrified cells are stored under liquid nitrogen conditions or mounted into the grid cartridge for loading into the cryo SEM microscope Grid with vitrified cells is then secured into the grid cartridge suitable for loading to both SEM and TEM microscopes C-clip ring is mounted to the clipping tool and cooled down in liquid nitrogen grid is placed onto grid cartridge carbon facing down and clipped with the C-clip ring specimen is then placed in the special grid box Specimen for lamila preparation is loaded to dual beam FIB/SEM microscope and fit with cryo-stage preparation chamber loading pot is properly cooled down and filled with liquid nitrogen sample holder called shuttle is placed into the and cooled down as well grid box with specimen is transferred from the storage dewar to the pot and grids are placed into two slots in the shuttle carbon side with cells facing up in case of use of FIB grid cartridges the cut out of the grid cartridge should be carefully aligned to twelve o'clock in the shuttle grids are tightened by the screw and the shuttle is flipped down to loading position loading pot is placed in transfer station and covered with lid Transfer chamber with loading pot is placed on the top of the lid and pumped through the low vacuum shuttle with grids is fastened on the rod and closed inside of the small transfer chamber chambers collected to the preparation chamber are pumped and shuttle is inserted on cryo-stage in high vacuum SEM chamber stage is tilted to forty five degrees and moved four millimeters on the center position of the grid inserting the needle of gas injection system creates protective layer of organic on biological specimen against radiation damage during focused ion beam milling grids are then sputter coated with thin veil of inorganic metal to create conductive layer on biological material focused ion beam milling of lamella in cell cluster or monolayer is done in few steps stage is moved to milling angle region of interest is found and top and bottom milling patterns are created rough lamella is milled with gradual decrease of FIB current and lamella thickness the final polishing of lamella is performed only with upper pattern and low FIB current to avoid front end damage and curtaining effect on lamella surface electron beam shows the progress during the milling process here we can see the final polished lamella The final thickness of lamella needs to be less than two hundred and fifty nanometers to be transparent for transmission of electron beam the illustration of lamella milled in cell cluster and monolayer with pros and cons of both sample types grids with lamali are very gently transferred through preparation chamber back to the loading pot pot is cooled down and filled with fresh and clean liquid nitrogen to avoid ice contamination of milled lamali grids are removed from the shuttle and returned back to grid box specimen with milled lamali is finally transferred to transmission electron cryo microscope grid has to be ninety degrees rotated before insertion to the TEM autoloader cassette to ensure proper orientation of the lamella with respect to the TEM tilt access Lamili are screened and region of interest for acquisition of cryo-electron tomographic data is localized Lamella main axis is perpendicular to the tilt axis of the microscope to see the right field of view at high tilt angles dosimetric method is used to collect cryo electron tomography data collected tomogram is then processed in Etomo software and monolayer segmented in Amira software In this protocol we showed you how to efficiently prepare a yeast cell sample for a lamella micro machine using cryo focused ion beam milling and how to prepare and process electron tomography data and cell lamali
