K2 acquire image » History » Revision 5
Revision 4 (Anchi Cheng, 07/11/2014 07:06 PM) → Revision 5/11 (Anchi Cheng, 07/11/2014 07:08 PM)
h1. Acquiring K2 images h2. Preparation in DM Hardware dark and gain correction need to be done by DM to count electrons. You should therefore obtain dark and gain references and upload them to the hardware (HW). You can do these in [[Leginon image orientation]] in 2.21 and above versions of DM. * Gatan recommends frequent hardware dark reference update. This does not require the beam. You can do it everyday while getting the scope ready. # Make sure the temperature of the camera is stable at where it should be cooled to. # Set the Direct Detection mode to Counted (just in case of DM gui bug). # Click on "Update HW Dark Reference". This takes about 4 minutes. * Once a while, but not very often (such as once a week or longer), all references should be retaken. This takes at least 15 minutes. h2. Operation # Typical frame saving preset parameters for counted/super-resolution: 0.2 s frame exposure time, 4-5 s total exposure time at 8-10 e/physical pixel/s (Some argues that 5 e/physical pixel/s is better). # /myami/rawtransfer.py should be run on the file server to transfer the raw frames and give them names that are synced with the integrated image as it is produced. See [[DDD_raw_frame_file_transfer]]. K2 frames are saved as mrc stacks. # See [[appion:Direct_Detector_Frame_Processing]] for tools used for processing the frames. # To gain correct and align the frames, start parallel instances of Appion makeDDRawFrameStack.py See [[appion:GainDark correction of the raw frame with or without drift correction]]. h2. Recommended dose rate and exposure time for different K2 modes This recommendation is based on our experience. As a general rule, the dose rate corresponds to the value used during DM gain reference acquisition. You can put down the small viewing screen once the intensity is adjusted at the beginning of DM procedure to get the values. You will notice that Linear mode calibration uses much higher beam intensity than the calibration for counted/super-resolution modes. For our Tecnai F20 at 200 kV, the former reading of exposure time by the small viewing screen is 0.25 s while the value for latter is 1.6 s. You should use similar intensity during Leginon calibration and data collection. At 1.2 A/pixel, we use the following for final exposure that has binning of 1: |mode|small viewing screen exp time (s)|detector dose rate (e/physical pixel)|acquisition exposure time for ~ 20 e/A^2 specimen dose with 1.2 A/pixel pixel size (s)| |Linear|0.25|64|0.5| |Counted/Super-resolution*|1.6|10|5.0| *Most literature recommend at even lower dose in the range of 6-8 e/s/pixel. This is just a number we have had success with. You can use DM's reading of dose rate while it is in counted mode and *UNBINNed* to get a reasonable estimate. h2. Preset Recommendation Because of its small size, we have a modified [[MSI_Quick-start_for_DD_only|Quick-start procedure]] we use regularly at NRAMM FEI F20. See [[Pre-MSI_Set-up#Preset-Design-Example-for-small-pixel-Gatan-K2-camera-alone]] specific for K2 camera using counted/super-resolution mode.