Cryo Soft X-ray tomography can provide quantitative valuable information at the cellular level that can be used as a standalone or conjunction with other imaging techniques. Sensor image in a frozen hydrated state which are need for staining or sectioning. In addition, it's a high-throughput technique, because each tomogram is collected in only a few minutes.
Soft X-ray cryo-tomography offers a perfect platform for following the process of cell restoration in infected or defective cells at the single-cell level. This technique can report the efficacy of new antiviral drugs or and gene therapy to revert the infection of redacted phenotypes. To load samples into the transmission X-ray microscope, cool the transfer chamber with liquid nitrogen until it reaches less than 100 Kelvin.
Fill the workstation with additional liquid nitrogen and turn on the heater of the workstation rim. When the workstation rim stops boiling, place the cryo box containing grids into the appropriate locations in the workstation under cryo conditions and load the grids onto the previously cooled sample holders. Load the holders onto the shuttle and protect them with the covers.
Load the shuttle into the transfer chamber at less than 100 Kelvin and pump the chamber down to low vacuum. Attach the transfer chamber to the microscope and follow the vacuum procedure on the screen to load the transfer chamber shuttle into the microscope. Once the shuttle is in the microscope with the samples, use the microscope robot arm to transfer one sample holder to the sample stage.
For brightfield imaging of the grid with the online visible light microscope, select the visible light microscope camera and turn on the visible light microscope LED source for brightfield imaging. Click Motion, Control, Sample, and Sample theta to rotate the sample to minus 60 degrees so that it faces the visible light microscope objective, and select Motion Control and Sample and change sample X and sample Y to move the sample to the expected centered positions. Select Microscope, Acquisition, Acquisition Settings, Acquisition Modes, Continuous, and Start and click Motion Control and Sample to select sample Z to refine the focus with smaller steps down to five microns until the cells and/or the holes of the carbon support film are in focus.
Then select Microscope, Acquisition, Acquisition Settings, Acquisition Modes, Mosaic, and Start to start the acquisition of a full mosaic map of the grid in brightfield mode using the default values for the mosaic. For fluorescence mode mosaic imaging, turn off the visible light microscope LED source for brightfield imaging and select the LED light source corresponding to the desired excitation wavelength and the corresponding optical filter manually on the setup. Select Microscope, Acquisition, Acquisition Settings, Acquisition Modes, Continuous, and Start and click Motion Control and Sample to select sample Z to refine the focus on the fluorescence image.
Then, click Microscope, Acquisition, Acquisition Settings, Acquisition Modes, Mosaic, and Start to acquire a mosaic map retaining the positional X and Y parameters from the brightfield mosaic. Then, switch off the LED light source. For x-ray mosaic acquisition, change the exit slit to five microns and use a one-second exposure at MISTRAL.
Select Microscope, Acquisition, Acquisition Settings, Camera Settings, and Binning to adjust the focus using the sample Z translation. Select Motion Control and Sample to select sample Z.Starting in steps of five microns, refine the focus down to steps of 0.5 microns until the cell or the carbon foil holes are well in focus. To acquire a mosaic map of the mesh square, click Microscope, Acquisition, Acquisition Settings, Acquisition Mode, Mosaic, and Start.
When the map has been acquired, click Motion Control and Sample and change sample X and sample Y to move the sample to a flat field position. Set the exposure time to one second at MISTRAL. Then, normalize the acquired mosaic by the flat field image to obtain the transmission and save the normalized mosaic.
To align the sample on the rotation axis with the rotation at zero degrees, select Motion Control and Sample and change the sample X, sample Y, and sample Z to focus on the feature of the cell to put on the rotation axis. To rotate the sample to a positive theta position, select Motion Control and Sample and change Sample theta to positive theta. Use the line tool to draw a line on the feature of the cell to put on the rotation axis.
To rotate the sample to a negative theta position, select Motion Control and Sample and change the Sample theta to negative theta. Use the line tool to draw a line on the feature of the cell to put on the rotation axis. While at positive or negative theta position, use the sample Z translation to move the selected feature to the center position between both lines and repeat the sample rotation until a minimum line-to-line distance is obtained.
When the sample theta equals zero, move the sample X twice the distance needed to put the selected feature at the center of the field-of-view, and move the zone plate X to bring the feature back to the center of the field-of-view. To re-optimize the zone plate Z position with respect to the new rotation axis, select Microscope, Acquisition, Acquisition Settings, Acquisition Modes, and Focal Series and click Start to record a zone plate Z focal series. Then, click Motion Control and Zone Plate and select zone plate Z to move the zone plate Z to the position at which the sample is in focus.
To acquire a tomogram, click Motion Control and Sample and select Sample theta to move the negative maximum angle to plus 0.1. Click Microscope, Acquisition, Acquisition Settings, Acquisition Modes and Tomography to set the number of images as the total number of angles, taking into account the image at angle zero and the angular range, and set the number of images. Select Angle Start and Angle End, click Microscope, Acquisition, Acquisition Settings, Camera Settings, and set the exposure time.
Click Start to start the acquisition of the tomography tilt series. The ideal sample should have single cells at the center of a square mesh embedded in a thin layer of ice and surrounded by well dispersed gold fiducial markers. Many different organelles, such as mitochondria, endoplasmic reticula, vacuoles, and the nucleus should be able to be distinguished in the final reconstructed tomogram, thanks to the quantitative reconstruction of the linear absorption coefficients.
In this image, a square with higher cell density can be observed. In this example, the blotting was less efficient, leading to a thicker ice layer with cracks. Even though some larger structures can be recognized in this preparation, fine details were lost within the noise and grainy texture due to the poor vitrification quality of the thick ice.
The cryo-preserved sample should be minimally handled, as most of the artifacts are induced when the samples in this stage. Usually, correlative cryo-visible light photomicroscopy, you use a prior to cryo substrate tomography. In addition, spectral microscopy can be done at relevant chemical elements.
This protocol fills a niche by operating in a specimen and resolution regime, which are not readily accessible by any other direct imaging technique, allowing few microns and 30-nanometer half-pitch resolution.
