1、UCSF Tomography - User instructionsThe following set of instructions is meant as a guide to the use of our tomographic data collection software. Keep in mind that it has been developed (and is constantly updated and improved) for use on our Tecnai T20 and Gatan CCD. Users with other instruments need
2、 to be aware that not all default values will work on all instruments. One must define experimental conditions etc. appropriately. Before beginning, make sure that the instrument is well aligned. Beam tilt pivot points and rotation center alignments are especially important.Flat Fielding of the CCDT
3、he T20 is equipped with a slow scan CCD camera for digital imaging. The camera works by converting electrons which hit the scintillator into photoelectrons which are transfered along fiber optics onto the chip itself. Unprocessed CCD images are marred by small variations in intensity caused by diffe
4、rential responses of each pixel. Furthermore, there are defects on the chip as well as irregular features (and dirt) on the scintillator, and variations in the fiber optics coupling. An “uncorrected“ image will reflect all of these flaws and so must be “corrected“.The CCD correction is done by deter
5、mining two numbers for every individual pixel of the CCD chip array. These 2 numbers are an offset (in counts, and determined by the dark current of the CCD chip) and a gain (in sensitivity per count, indicating the response of that pixel to the electrons), and a time-dependant dark current (reflect
6、ing the changes in the offset with respect to exposure time).While an initial flat fielding will always be necessary, the correction should be stable for several weeks. If there are discontinuities in an image of the beam (Fig. 1), or if quadrants are visible at reasonable counts (10k) a new correct
7、ion file should be made. Also, changes in the accelerating voltage can necessitate the acquisition of a new flat field correction.Figure 1Should you need to flat field the CCD, proceed by choosing each and every CCD readout sizes/binnings to be used, and run the calibration by simply hitting the cal
8、ibrate botton. After it finishes, an image should look like a fine grain. (Fig. 2) The total counts in an image should reflect imaging conditions which will be used for data collection.Figure 2CalibrationData collection parameters must be calibrated any time the microscope has been realigned. Typica
9、lly the calibrations last for weeks or months if the instrument is stable.Choose an image size of 1k x1k, binning 1, and use the central quarter of the image (Fig. 3). For calibrations it is helpful to use a thin, high contrast sample imaged at true focus.Figure 3Stage ShiftMake the sample eucentric
10、, focus to true focus (minimal contrast0, reset image shift in the image settings of the TUI, and select intensity zoom in the beam settings of the TUI. Set the magnification to 2500X. Use a 1-2 um induced shift and click the start button to run (Fig. 4)Figure 4To verify that the stage calibration h
11、as run correctly, go to Image and select the move stage and show center buttons. Acquire an image and double click on a feature to re-center (Fig 5). The feature should move closer to the center and will more accurately move to the center with a second double click.Figure 5Image ShiftSet the magnifi
12、cation to your data collection magnification. Set to true focus. Use the default value of 300 pixels for the induced shift. Hit start to run (Fig 6).Figure 6Verify the image shift calibration by going back to the Image tab. NOTE: make sure that the stage shift box is unchecked. Acquire an image, dou
13、ble click a feature, aquire another image and the feature should be centered. (Fig 7). After verification, reset the image shifts in the TUI. Complete the remaining calibrations at a magnification of 25K, beginning with image shift (if it has not already been done at this mag).Beam TiltUse the defau
14、lt value of 5 um, and start the calibration. After running, the beam tilt intersection should be close to 90 degrees. (Fig 7)(Generally somewhere between 85-90 degrees). If this is not the case, check beam tilt alignments and repeat.Figure 7FocusClick on the focus botton and start the calibration. A
15、gain, leave the default value at 5 um to start. (Fig 8). Figure 8After running the focus calibration, check it by setting a defocus value in the autofocus field and click autofocus to determine if it is correct. (Fig 9).Figure 9Eucentric Focus AlignmentClick on the Eucentric button and start (Fig 10
16、). After running, one can check by clicking the eucentricity button, then wobble the stage (in the Stage tab of TUI), to determine if it remained wucentric and in focus. In order for this to properly function, the microscopes eucentric focus alignment must be very good.Figure 10Optical AxisSet the e
17、ucentric height, and make sure that you can tilt the specimen +50 degrees tilt. Start the program and allow it to run. This will take about 15 minutes. Once finished, the value in the last column should show a change below 0.1 um (Fig 11).Figure 11TomographySetting up for data collection in the Tomo
18、graphy tab is quite simple (Fig 12).Figure 121. Identify an object of interest.2. Adjust eucentricity and check that the sample can be seen at the full + tilt range.3. Continue as outlined below:Input the values for the minimum and maximum tilt (angular range), and the starting angle.Input the tilt
19、step (increment). A negative value here will tell the program to collect data in the negative direction first. Exposure - if Cos thickness is selected the program will adjust exposure times during data collection using 1/cos angle. If Exp is selected, the program will use the thickness value to esti
20、mate subsequent exposures.Focus If the Measure button is selected, the program will measure and correct the focus at the interval specified by the user.Data If the Cryo box is selected the counts in the image data will be boosted 10 fold to make up for the lack of signal in the raw data. This does n
21、ot effect the electron actual dose on the sample. In the pull-down, if signed is selected each pixel is assigned 15 bits (35,000 max counts). If unsigned is chosen, then the data will be 16 bits (64,000 counts).Unsigned should be used for cry data collection.XCF cross-correlation binning. Generally
22、use 2 or 4, depending upon your speed and signal to noise requirements. (A value of 2 will be pretty accurate for higher dose data, and 4 works well for low contrast images).Manual If this button is chosen, the user is allowed to determine the cross-correlation image in the second window in order to correct any large shifts that may occur during data collection.File Name - A file name must be selected.File Note - File notes can be up to 80 characters.Press the Start button and data collection will begin.
