Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

The overall goal of this procedure is to prepare a sample for cryo TEM by extracting a focused ion beam lamella from a cryogenically frozen bulk sample. This is accomplished by first plunge, freezing the sample in liquid nitrogen and mounting it inside the cryo focused ion beam SEM. The second step is to cut out a thin LA Melo with the ion beam and transfer it onto a TEM grid by means of a cooled nano manipulator.

Next, the TEM grid is transferred from the SEM holder to the TEM holder in a transfer station. The final step is transfer of the TEM holder to the cryo TEM for further analysis. Ultimately, the cryo TEM is used to show high resolution images, tomo Grahams, or x-ray maps of the extracted lamella.

This technique will enable us to analyze soft matter with ultimate resolution in the transmission electron microscope by pre-selecting the place of analysis by other microscopies such as light microscopy or scanning electro microscopy. The main advantage of this technique over existing methods like Mac cryo Microtomy, is that preparation artifacts such as compression and def formation of the sample are avoided. First mount two TEM grids for focused ion beam samples.

On the SEM transfer holder, secure the grids by tightening the corresponding screws with a screwdriver and close the shutters mount a sample stub appropriate for the specimen and add a portion of the specimen. For example, use a pipette to deposit droplets on a flat stub. Then mount the SEM transfer holder onto the vacuum transfer device or VTD.

After adding liquid nitrogen into the slushing station and pumping down, open the station and plunge freeze the SEM transfer holder pump down again until boiling is complete and the slush is obtained again. Next, retract the SEM transfer holder into the vacuum chamber of the VTD and seal it. Vent the slushing station and the cryo preparation chamber Airlock vent the outer airlock.

Then match the VTD seal with the airlock of the cryo preparation chamber and pump. When a good vacuum level is reached, gauge the airlock pin to open the seal of the VTD and the outer airlock. Then insert the SEM transfer holder.

Following this, use a cold knife to open the protective lids of the TEM grid slots. Switch off the high tension on the focused ion beam SEM and open the inner airlock. Then use the VTD to transfer the SEM holder into the sample chamber.

Once the SEM holder is in the cold stage, disengage the VTD by pushing and rotating. Retract the VTD rod all the way into the VTD vacuum chamber and close the inner airlock, the outer airlock and the VTD seal. At this point, heat the precursor gas to 24 to 26 degrees Celsius and position the gas injection system or GIS needle to a height of about one millimeter above the sample surface.

While imaging with the electron beam, open the gas valve for a few seconds. Then cure the deposit over the region of interest, or ROI by using a 1000 pico ampire ion beam at low magnification. After curing, tilt the sample to 52 degrees so that the surface is perpendicular to the ion beam.

Mill away. Two terrorist trenches. On either side of the ROI tilt the sample back to zero degrees and use the ion beam to cut away the sides and underside of the lamella, making sure the cut marks go through the entire lamella.

Once the GIS needle has been inserted, maneuver the nano manipulator until its tip is in physical contact with the lamella, preferably on the side. Open the GIS valve for a few seconds and monitor the cryo deposition by continuous imaging with the electron beam. When an additional one to two micrometer layer of platinum has been cryo deposited, close the valve following this cure the platinum only in the few micrometers around the point where the nano manipulator is in contact with the lamella.

Then use a high ion beam current to cut the lamella free. Carefully maneuver the nano manipulator to extract the lamella from the trenches and move it at least 500 micrometers above the sample surface. After retracting the needle, lower the sample stage a few millimeters and move it until one of the TEM grids is in view.

Move the attachment area on the grid into the working position and insert the GIS needle. Next, carefully maneuver the nano manipulator to bring the attached lamella into physical contact with the attachment area on the TEM grid, open the gas valve for a few seconds and cryo deposit an additional one to two micrometer layer of platinum cure the platinum only in a few micrometers around the point of contact between the lamella and the TEM grid. Then use a high ion beam current to cut the lamella free of the nano manipulator.

Tilt the sample to 52 degrees and use the ion beam to thin it to electron transparency. After flushing the cryo transfer station with dry nitrogen gas, insert the cryo transfer TEM holder in the appropriate slot of the cryo transfer station and fill with liquid nitrogen. Following this immerse a screwdriver, the TEM sample clamping tool and tweezers in a cryogenic cup filled with liquid nitrogen in order to cool their tips to the desired temperature.

Once the VTD is matched to the outer airlock, bring the cold stage to the transfer height of 16 millimeters. After turning off the high tension, open the VTD seal, the outer airlock, and the inner airlock. Then use the VTD rod to lock into the SEM transfer holder by pushing and rotating clockwise.

At this point, retract the SEM transfer holder into the cryo preparation chamber. Use the cold knife to close the protective lids of the TEM grids. Then use the VTD rod to move the sample into the vacuum chamber of the VTD close and seal the airlock.

Then vent the outer airlock and detach the VTD. Match the VTD to the SEM port of the cryo transfer station. While flushing with dry nitrogen, use the pin on the station to open the seal of the VTD and slide the SEM transfer holder into the doer of the cryo transfer station.

Using the cooled screwdriver, open one of the lids and loosen the corresponding screw, keeping the TEM grid in place. Then pick up the TEM grid and place it into the TEM holder using the cooled tweezers. Following this, fasten the TEM grid onto the TEM holder.

Using the cooled hex ring, close the shutter of the cryo transfer TEM holder and disconnect the cryo transfer station from the pumping system. Transport the station and heater controller of the TEM holder near the TEM and reconnect them. Set the TEM sample stage to a tilt of negative 70 degrees.

Next, set the shortest pumping time for the airlock with only one cycle of purging with dry nitrogen gas. Remove the TEM holder from the cryo transfer station and insert it into the first stage of the tilted goniometer, which will start the pumping cycle. Once the cycle is completed, set the goniometer to tilt back to zero degrees while holding the TEM holder so that it does not rotate with the goniometer.

Then insert the holder through the goniometer fully inside the TEM. Finally refill the cryo transfer TEM holder doer with liquid nitrogen. In this method, aspergillus Niger spores are first imaged by SEM to identify the extraction site.

A cross section of any spore is sufficient, but it is possible to position the ROI for extraction with sub micrometer precision to slice a specific cell at a specific distance from the cell membrane. Once the feature of interest has been identified, the first step of the cryo deposition is implemented To protect the sample from beam damage during ion milling, the sample is tilted to 52 degrees, followed by sputtering of two trenches on both sides of the mella. The sample is then tilted back and further milled, leaving only two small bridges connecting it to the bulk.

The cooled nano manipulator is brought into contact with the mella and another cryo deposition of platinum solders them together. The small connecting bridges are milled away, and the nano manipulator moves the lamella near the TEM grade attachment area, where it is soldered with a final cryo deposition of platinum. The nano manipulator is then separated from the lamella, which is thin down to electron transparency.

With the ion beam, the lamella is transferred to the TEM where high resolution imaging spectroscopy, tomography, and other techniques can be employed. This method will contribute to understand heart soft matter interfaces in systems such as neuro probes or bone implants. After watching this video, you should have a good understanding of how to prepare cryo TM samples that are artifact three, and that can be extracted from a specific region of a bug sample with sub micrometer precision.