FEI Titan Protocol

Titan Standard Operating Procedure 

version:  January 2015 

 

Note: This procedure is not entirely complete and should be used with discretion or under direct supervision of the facility manager. The procedure is exclusively for the FSU Titan Krios that is interfaced with Leginon acquisition software. 

 

1. Cooling the Microscope down and DED camera 

1. If the Titan is not at cryo temps, it needs to be cooled before anything can be loaded into it. It takes ~ 25-30 min to reach cryo temperatures. While it cools down, skip to steps 4 and 5. 

1. In the ‘stage’ tab 

1. All LN temperatures 

2. Make sure there is enough LN to cool it completely 

2. Also need to make sure that the camera is cooled down  

1. Open the MicroManager (on desktop) 

1. Camera settings 

1. Cool down 

2. Check if its cooling down 

1. Tools 

1. Property Browser 

1. To monitor the temperature, the cold finger is the best metric to determine the current temperature 

3. Wait for it to equilibrate. I noticed it stops around -38.4 C 

 

2. Loading Grids onto the cassette 

1. After successfully clipping the grids into titan rings (not trivial, and not discussed here), fill the loading station with LN and allow to fully equilibrate. 

1. Load the grids so that the ‘cup faces up’. 

2. The side with the c-clip will face away from the foot of the cassette. 

3. Once all of the grids have been loaded, GENTLY check that the grids are properly seated.  

1. GENTLY touch the top of the grid with the loading tweezers to make sure it is as far down into the cassette as possible. 

2. GENTLY press against the grid (without causing damage to the grid, ring, cassette, or springs) on the bottom side to check that the springs have the proper tension and respond to the force applied. 

3. If you drop a grid in the cassette, it is nearly impossible to recover, but it can be done with extreme caution as to not damage any other grids or the cassette. If you find yourself in this predicament, Godspeed. 

2. If there is a problem loading the cassette, or the spring(s) don’t seem to be responding accordingly, get the facility manager. 

 

3. Filling the capsule and docking onto the Titan 

1. Capsule 1 and 2 are used for loading. 

2. Capsule 3 is used ONLY for unloading. 

1. Fill the capsule with LN and allow to equilibrate. Don’t let the LN stand on the rubber matting surface because it will cause premature wear and crack the rubber. Use the lid of the capsule to clear any standing LN. Just put the lid on, and it will boil off. 

2. Securely attach the capsule to the grid loading station. If its not secure, keep trying. It can be a little finicky, but its 100% necessary to have a flush attachment. 

3. Once attached, QUICKLY transfer the cassette into the capsule. Don’t dilly-dally with the cassette once its out of LN 

4. After transferring the cassette into the capsule, detach from the loading station and immediately place the lid on the capsule. This will help prevent unwanted ice contamination. 

5. Remove the capsule lid and place the capsule into the Titan docking bay. 

6. Under the Stage tab, select ‘dock’. 

7. Watch the status of the docking; if a problem occurs, get the facility manager. 

8. Remove the capsule when prompted to.  

3. If there is problem with docking the capsule or the autoloader has a hissy fit, get the facility manager. 

 

4. Beam Alignment  

1. Before loading a grid onto the stage, the direct alignments need to be checked: 

1. 50x mag (low mag). 

2. Insert pointer half way with the screen in. 

3. Spot size 3. 

4. Focus the beam on the center of the tip; a cross should appear (refer to picture below). 

5. To better visualize the ‘cross’ the contrast might need to be changed. Use the roller wheel on the mouse to do this.  

6. If the beam doesn’t look like this, get the facility manager. 

 

5. Gun Shift 

1. Need to have an empty stage. 

1. Go to 11500x – 15000x mag, spot size 3. 

2. Select gun shift under the alignments tab. 

3. Focus the beam to crossover using the intensity knob. 

4. Use the multifunction knobs to center the beam. 

5. Click done and go to spot size 9 

6. Focus the beam using the intensity knob 

7. Use beam shift (or termed image shift) to center the beam (the roller ball is the beam shift by default) 

8. Repeat this 3 times at the current mag 11500x – 15000x 

9. Go to higher mag ~59000x and repeat the above procedure 3 times 

10. By the final alignment, the beam shouldn’t have to move much. 

2. If there are any problems, or the beam is ‘wonky’ get the facility manager 

 

6. Starting Leginon 

1. The Linux box, Zeus, interfaces with the Titan and the camera. 

2. On a new terminal screen, login in with the appropriate credentials (For assistance get the facility manager) 

3. The command start-leginon.py will launch leginon. 

1. Go through the GIU and enter the required information. 

1. Name 

2. Project 

3. Session 

4. Clients 

1. Titan 

2. DED camera 

3. Main (computer) = Zeus 

5. C2 = 70 

6. Anything else 

4. Make sure the client screens have been launched on the Titan Computer and also the DED computer 

1. Weird little icon, but hover over it and it should say ‘leginon client’ 

5. Once the leginon GUI has appeared, select run under the applications tab. 

1. For cryo, use the MSI-T2 3.0 application 

2. For negative stain, use the MSI-Raster application 

6. The necessary nodes should appear in on the left side. If it looks weird or something chocked, get the facility manager. 

7. Import the desired presets. 

8. Take a simulated exposure to ensure all the clients have established communication. Should be a crappy image, but this is good.  

1. Remember to take the screen out 

9. Any problems, get the facility manager 

 

7.  Setting Z-focus 

1. The following will be used for allowing the microscope to determine how far to raise the stage in z-height.  

2. An alternative would be to manually wobble the stage and drive to eucentric height. Either works 

1. Load a grid onto the stage. 

1. Edit the slot state (which slots in the cassette have grids in them). 

2. Select the desired grid and hit ‘load’ 

3. Watch the status to ensure the autoloader doesn’t freak out and drop the grid or some other horrendous event occurs. 

4. When loading a grid, make sure the following have been selected: 

1. Turbo ON 

2. Column valves closed 

3. Screen in 

2. Find a square or an area worth sacrificing.  

3. In the Z-Focus node, select the blue bar icon 

4. Enable the necessary Z-focus settings as desired. The following are the most critical: 

1. Stage-Rough 

2. Stage-Small-Wobble 

5. Simulate a target and see if the correlation is correct.  

3. Any problems, get the facility manager. 

 

8. Align Presets 

1. Find a nice turd or a large piece of ‘crud’ to align on (large piece of hexagonal ice, on the tip of a sharp corner) that has lots of contrast and can be clearly seen at all magnifications. 

2. First select ‘ex’ to ‘hl’ 

1. Select the green cross icon on both screens and the mouse icon on the right screen 

2. Once you have aligned the ‘hl’ screen to where the ‘ex’ cross is located click done. 

3. Repeat for the following presets 

1. ‘hl’ to ‘sq’ 

2. ‘sq’ to ‘gr’ 

 

9. Gain References/Camera normalization 

1. Need to be over a broke square and on the ‘ex’ or high mag (>29000x) 

2. If unsure about which camera dimension and exposure time to use, check in the presets manager (Dimensions, exposure time….) 

3. In the correction node 

1. Select the settings icon 

1. Start with 480 x 480 bin by 8 (for gr and fa presets) 

1. 1250 ms exposure 

2. 3 images to combine 

2. Dark first, then bright; both channels 

3. Once the Dark and Bright images have been collected, take a corrected image. Should look like pure noise 

4. Repeat the above procedure but on the 960 x 960 bin by 4 setting (for sq and hl presets) 

1. 500 ms exposure 

2. 3 images to combine 

5. Repeat the above procedure but on the 960 x 960 bin by 1 setting (for fc preset) 

1. 1000 ms exposure 

2. 3 images to combine 

6. Repeat using the 3840 x 5120 bin by 1 

1. Exposure 1000 ms (check in preset manager!!!) 

2. Combine 10 images 

7. Take a corrected image, and if anything looks weird, redo the above procedure. If it’s still abnormal, get the facility manager. 

8. Need to do 480 x 640 bin by 8 because this is what is used by the preset alignment camera setting 

1. Exposure = 800ms 

2. Combine 3 images 

10.  Center the objective aperture 

1. sq preset 

1. Go to the center of the grid (can drive there manually using the joy-stick) 

2. Go to z-focus node and simulate a target 

1. Don’t be directly on a bar 

3. Put screen in, change preset to FA and hit the eucentric button 

4. Reset defocus 

5. Normalize all lenses  

6. Make sure that the screen is in…..IN!!! 

7. Screen is in, right?  

8. Hit the diffraction button. 

9. Focus the beam and put into the center using the roller ball. 

10. Spread the beam out and make sure its not cropped or obstructed. 

2. Go to apertures 

1. Objective 

2. Put to 100 

1. The right objective button needs to be yellow; this is when it can be moved. If its grey, then it is inactive. 

2. Select ‘Adjust’ 

3. Center the objective using the multifunction knobs 

1. It might be difficult to see the objective, so increase the contrast using the roller wheel on the mouse 

4. Turn off adjust, and disable the objective. This will lock the objective in place. 

5. TURN OFF DIFFRACTION MODE!!!1 

3. Go to tune tab 

1. Select ‘Beam tilt ppx’ 

1. The beam should be bouncing around in two spots; use the multifunction knobs to make into one spot. 

2. Once its one spot, turn off 

2. Select ‘Beam tilt ppy’ 

1. Use multifunction to make into one spot. 

3. Repeat at higher mag (75000x) 

4. Repeat again at even higher mag (155000x) 

5. If there is anything funky, get the facility manager 

4. After the objective has been inserted and centered, need to check the direct alignments again. See steps 4 and 5 above. 

5. Coma Free Alignment 

1. Center the beam over carbon 

2. Select Coma-free alignment in the Drop Down menu 

3. Go to micromanager on DDD computer (need to close the leginon client on the DDD computer) 

1. Live Mode 

1. Exposure = 200ms 

2. Bin By 4 

3. Plugins 

1. Live FFT 

4. Change the defocus so that there are lots of Thon Rings 

5. Use the MF knobs to adjust the alignment so that the Thon rings are moving in the same direction  

 

11.  Setting up the automatic hole and exposure picker 

1. This is not trivial, and takes some practice.  

2. Need to collect an atlas first. 

1. Grid targeting 

1. Publish targets 

2. Submit targets 

3. Select a bunch of usable squares on the grid and submit (if running overnight, about 10 squares with ~100-150 holes is enough for the titan to chew on). This will also depend on how many particles per hole and a few other factors. Need to be determined at the time of operation, and more holes/targets then necessary is better then running short. 

1. In the ‘hole targeting node’ 

1. Start on template 

1. Cross correlation 

2. Need peaks, not blurry smears 

3. Change the final hole template to get better quality peaks 

1. Essential want to maximize the peaks 

4. Hit ‘test settings’ to see how it looks 

2. Threshold 

1. Change the ‘A’ value until the threshold looks good (most holes are accounted for without including unnecessary amounts of noise) 

2. Test settings 

3. Blobs 

1. Make sure that the blobs are actually in the center of the hole 

2. Test settings 

4. Lattice 

1. Change the reference intensity to be greater than the intensity of the hole 

2. Test settings 

3. Use the minimum to eliminate empty holes, use the maximum to eliminate too thick holes. 

5. Acquisition 

1. Change the mean thickness until all usable holes have been selected. 

2. Test settings 

2. Move on to exposure targeting 

1. Repeat the above tasks on the exposure setting. 

1. Select desired amount of exposure targets. 

2. Move there position by adjust coordinates 

2. Decrease the std. dev. until the ideal focus target is acquired.  

3. Need to also fiddle with the angle offset to make sure it won’t cook a good hole. 

4. Need to make sure that only one focus target is selected 

 

12. Setting up the DED camera and determining the optimal dose rate and exposure time 

1. Facility manager 

 

13.  Coma Free and Rotation center optimization 

1. Facility manager for the most part 

1. ROTATION CENTER 

1. Select rotation center on the right side tab, under direct alignments (?) 

1. The beam should be going in and out of focus with a processive motion; not going side to side 

2.  

2. Some things I’ve learned 

1. In order to do live acquisition, need to bin by 4 

2. Mag = 155000 or greater 

3. Exposure time 250ms or 400ms 

4. Plugins 

1. Live FFT 

2. Change defocus until there is a LARGE tone ring 

1. Want to be as eucentric height as possible 

3. Coma free x, use multifunction x to so that the first tone ring is not changing in width 

4. Coma free y, use multifunction x for the same thing  

5. For astigmatism, hit the stigmator button and get the beam to be as circular as possible. 

1. Use the circle tool to make sure that the tone rings are as circular as possible. 

3. After moving back and forth between the micromanager and leginon WAIT A FEW MINUTES!! For it to chill 

 

14.  If leaving the Titan during any part of the data collection, make sure of the following 

1. Turbo ON 

2. Close column valves 

3. Insert screen 

4. Send ‘ex’ to scope, or make sure that the mag is > 2200  

 

15.  Check list for automatic collection 

1. Defocus Range (set this in the presets manager in the ‘ex’ preset. 

2. User verification deselected (it will stop at the next node) 

3. Turbo pump is off (if its on, all the images will be blurry and essentially unusable) 

4. Objective aperture is in and centered (the beam could be cropped out or if its not in, then the images are garbage). 

5. Reset the field compensator (large white thing in the Titan computer room/station; hit ‘reset’ and it will ding three times then its all good) 

1. Do this after the final tinkering on the exposure settings  

6. Frame Stuff: 

1. This is a little fuzzy for me, but make sure that if frames are being collected, that they’re being saved.  

 

16.  “Quick and dirty” procedure to see what’s ‘down there’ (Grid Screening) 

1. Select grid 

2. Close column valves 

3. Z-focus 

4. Simulate square 

5. Choose holes to target and include a focus spot 

6. After the holes are selected, choose exposure targets and include a focus spot 

 

17.  Troubleshooting 

1. If at any point, there is a problem or operations have gone awry, get the facility manager. Don’t be a hero. 

2. This list could be infinitely long, but here are some things I’ve encountered: 

1. If there is not a dose readout 

1. Clear frames 

2. Take a new dose image and match to set value for e/pix 

1. Take the dose image over a broken square 

2. If there is still no dose readout, take a new correction measurement for gain references (usually just the 3840 x 5200 setting).  

3. If communication between any of the clients and leginon stops; usually the camera has a conniption and will stop working. 

1. Close all the clients down (titan and DED camera). 

2. Close leginon down 

3. Wait ~1 min before reinitializing clients 

4. Reload leginon 

5. Try to simulate an exposure target to see if there is communication between the camera, scope, and leginon. 

6. If the problem persists, close all clients again, and this time reset the DED camera server (on the computer for the camera) and wait ~ 5 min before reinitializing the server. After that, connect the clients and try to simulate and exposure. 

18. ComaFree with Gold grid 

1. 120kX 

2. 400ms exposure 

3. bin by 4 

4. over all gold 

5. find an area that has thon rings, or look for bright diffraction ring in live FFT 

 

presets : gains : atlas

 

 

 

Protocol for data collection (applied for FSU’s titan only):

Before starting let us discuss the networks for the data collection.

1. How many components are involved during data collection/ acquisition?

- There are three main components, namely:

1) Main scope (Titan Krios)

2) Camera / detector (DE 64)

3) Automatic data acquiring system (Leginon)

 

2) What are these components?

The main scope (Titan Krios) is controlled /operated by the Titan computer (scope computer). The Titan computer runs a program called Leginon client scope.

The DE computer controls the camera/detector. The DE computer runs two programs, DE server and Leginon client camera. In addition, the DE computer has access to the High Performance computing (HPC) Cluster.

The automatic data acquiring system (Leginon) is controlled/operated by the Hermes computer. Hermes also has access to the HPC Cluster.

 

3) How do these computers work?

As mentioned earlier, the Hermes computer contains a system called Leginon. Leginon plays the pivotal role during data acquisition, i.e. it connects both the camera and scope. However, Leginon cannot directly connect the camera and scope. Leginon connects to Titan (scope) and DE-64 (camera) via Leginon client scope (present in Titan computer) and Leginon client camera (present in DE computer) respectively.

 

4) Why do we need all these networks/ computers?

Practically, even without Leginon, we can take images but it is not handy for a large data set. For cryo-EM, we need several hundred thousand particles to get atomic to sub-atomic resolution structure. It is not possible to get those large numbers of particles in one or two images. Therefore, we need an automated system and Leginon is the answer. With the help of Leginon, we can automatically acquire thousands of high-quality images in a day. In short, Leginon is an automated system to acquire data from Titan and camera based on phyton-side programs (providing the framework for building applications for image acquisition and analysis), the MySQL database and server (keeping track of

all information) and the PhP-based image and data viewers on a webserver (displaying raw information and organize report)

 

Procedure for image acquisition:

1. Check the temperature of the microscope (scope). Check there are no errors or red marks on viewing screen of the scope computer. If the scope and camera’s temperature is cooling down, then check every 15 min. If the temperature remains same even after half an hour then contact faculty manager. If you see any kind of error messages, contact faculty manager. Do not try to fix it yourself. You should always close the column valve, insert a screen and switch on the turbo at this point.

2. Check the clients are set up or not. If not, then start two clients (Leginon client scope and Leginon client camera).

3. Cool down the capsule for docking of the sample.

4. You should always use chilled capsule (write down the slot numbers containing grids) to load the cassette. Check the coil/spring on capsule whether it recoils properly or not. If it not, then that capsule cannot be used for docking.

5. Docking of the cassette- After checking the condition of the capsule, dock the cassette. For docking, put the capsule with cassette in the docking position. Then, go to scope computer, and press ‘dock’ (check column valve should be closed, a screen should be inserted and turbo should be on during this procedure) button. After the docking of the cassette, check inside of the capsule. Be sure that there is no cassette present after docking. It is normal that microscope temperature will rise up slightly during docking. Wait until it is properly cool down. The temperature should be at least -175˚ C.

6. After the scope is cooled down, press ‘edit slot’ button on scope computer. This will allow you to mark the position of samples in the slot. Once, you marked the slot, hit ‘edit slot’ again button and move to loading procedure.

7. Loading procedure - Click the slot you want to load and then hit ‘load’ button. Wait until the load and unload buttons are highlighted. Once, the buttons are highlighted, open the column value but keep the screen inserted and proceed to direct alignment (at low magnification and high magnification).

8. Direct alignment - can also be done before docking the capsule into the microscope. It is up to you at which magnification you want to do a direct alignment (but at least higher or equal than the exposure magnification). Let us do at 12k and 96 k magnification. Using magnification knob, put

magnification at 12k. Press ‘Tune’ tab on the scope computer, then gun shift. Put ‘3’ on stage and press gun shift. Using the intensity knob, make the beam smaller as much as possible and using the multifunction knob, place the beam at the center of the screen. Then put ‘9’ on stag1e. However, do not press gun shift. Check whether the multifunction knob corresponds to beam shift or not. In this case, multi-function knob should correspond to beam shift. Like previously, make the beam smaller as much as possible and place it at the center of the screen. Repeat it at least 3 times or until the beam is placed at the center. Repeat at stage 9. Once you centered the beam at a magnification at 12k and repeat exactly same procedure at a magnification at 96 k. Once you are once with direct alignment then start Leginon.

9. Starting of Leginon- Open terminal at Leginon computer, load module myamiss and unload module myamidev. Type newgrp cryo_seecm4. You should be in the group called cryo_secm4. Then type start-leginon.py and hit enter. This will open up the Leginon GUI. Then start a new session. Make sure Leginon is connected to Leginon client scope and Leginon client camera. We should able to choose those clients from Leginon GUI. Once everything is set up, inputs all the presets. The best way to input all the presets is by importing preset from the previous session. Press icon ‘import preset' button from the previous session.

10. Then send square ‘sq’ to scope. Then, make sure turbo is off. Then look for the desired square. Once, the desired square is found, place it on a center of the screen and from a square node, and stimulate the target. You will see the image of the target square. On the square targeting node, the symbol '?' appears for user target verification. For a time being, leave it as it. Moreover, proceed to Z height estimation.

11. Z- height estimation- let’s estimate the Z height before taking any exposure image. Put the screen from the scope computer. Select any square that not worth collecting images but should not be broken. Then from preset manager send ex to scope. At the Leginon GUI, look for ‘z focus’ node, select the blue bar icon and then stimulate target and check the correlation. If the correlation is correct then Z height is successful. Then proceed to dose match.

12. Dose match - send ex to scope. Click the ‘acquire dose image’ icon. An image will appear with dose parameter. Check whether the dose is matched or not. If not then put the desired dose, and click the match button. Wait until you will get new dose image. Repeat until you will get the right dose image. Once you get the right dose image then click yes. Moreover, move to gain correction.

13. Gain correction – send ‘sq’ to scope. Search for broken square. Put the broken square at the center of the stage. Then send ‘ex’ to scope. Make sure that microscope is focusing on broken square.

Then lift the screen (note down the parameters such as image dimensions and exposure time). Then go to ‘correction’ node on Leginon computer. Edit the correct parameters such as exposure time, image dimensions and how many images to combine (Since, this is the preliminary gains correction procedure, let use 3 images; we need to correct gain later as well). On a dark, bright reference tab, put dark first then bright for both channels. Once you collect the dark and bright images, take a corrected image. The corrected image should look like pure noise. Once, you correct the gains proceed to grid screening.

14. Grid screening – Remember on step 10, there is a symbol ‘?’ appear at ‘square targeting’ node Go to that node and with the ‘acquisition’ icon which is a green plus (+), select the desired target. Select the desired focus with the ‘focus’ icon which is blue cross (+). Submit the targets. Then at the ‘hole targeting node’, ‘?’ seems to appear. Target the hole with ‘acquisition’ icon that is a green plus (+). Then target the focus with ‘focus’ icon which is blue cross (+) and then submit target. Careful that focus target area should not be at a hole. Usually, an area between the holes is suitable for the focus area. Once you get the desired exposure images and decide to take a large number of images, then start aligning preset.

15. Aligning preset- send ‘sq’ to scope. Then, you should look for a dirt or any kind of easily distinguishable features. You should be able to see these selected dirt/features at all magnifications. Place it at the center of the screen and then send ‘ex’ to scope. Check whether you can see preselected dirt/feature or not. Then from ‘align preset’ icon, get the preset images. Starting from 'ex' to 'hl' then 'hl' to ‘sq’ then 'sq' to 'gr'. After aligning the preset, move to atlas collection.

16. Collecting atlas- go to ‘grid targeting’ node at Leginon GUI. Click the ‘hand’ icon and set radius 0.001m and should give 23 stage. Label ‘a’ and click ‘calculator’ icon and click submit. This will take a while to publish atlas. Once the atlas is published, go to ‘square targeting’, node and press ‘acquisition’ tab and submit target. This will take some time. After square are submitted, ‘?' the icon will appear at ‘hole targeting’ node. Leave it for a while as it is and for a time being move to Z height and objective centering.

17. Z- height- send ‘sq’ to scope. Then lift the screen in. Then manually go to the center of the grid but not on the grid bar. Then send ‘ex’ to scope. Press ‘L2’ or ‘A wobble’. At this point, stage should be least move or wobble. If the stage is wobbling too much then adjust it with z-axis joystick. Once you are satisfied with wobbling, press ‘eucentric’ button followed by ‘reset defocus’ button and then normalize all lenses.

18. Objective centering- IMPORTANT!!!! Make sure screen is in. Once you are 100% sure that the screen is in, hit the ‘diffraction’ button. Focus the beam and put into the center using ‘multifunction’ knob. Make sure that nothing is obstructing the beam at this point. Then go to ‘apertures’ followed by ‘objective’. Put 100 and hit the ‘objective’ button. This allows you to move the objective aperture (objective). Center the objective using ‘multifunction’ knob. If you cannot see the objective aperture, please increase the contrast using the roller wheel on the mouse. Once, you are sure that objective is center, then hit adjust button, which will lock the objective in place. Hit the ‘diffraction’ button again and now, you are out of diffraction mode. Be sure that you are out of ‘diffraction’ mode. Once, the objective is in and center, move to pivot point.

19) Pivot points- send ‘ex’ to scope. Lift the screen in. Then, click the ‘tune’ tab on the scope computer. Click the ‘beam tilt ppx’. The beam will start wobbling in two spots. Make the beam smaller with ‘intensity’ knob. Make beam in one spot. Once it is done, then click ‘beam tilt ppy’ and repeat same as before. Once you are satisfied with pivot points correction, hit done button and move to Dose match

20) Dose match: send ‘ex’ to scope. Click the ´acquire dose image ´icon. An image will appear with dose parameter. Check whether rise match or not. If not then put the desired dose, and click the match button. Wait until you will get new dose image. Repeat until you will get the right dose image. Once you get the right dose image then click yes. Moreover, move to gain correction.

21) Gains correction- send ‘recoils’ to scope. Search for broken hole. Put the broken hole at the center of the stage. Then send ‘ex’ to scope. Make sure that microscope is focusing on broken square. Then lift the screen (note down parameters such as image dimensions and exposure time). Then go to correction node. Edit the correct parameters such as exposure time, image dimensions and how many images to combine (this time, put at least 15 images). On dark, bright reference tab, put dark first then bright for both channels. Once you collect the dark and bright images, take a corrected image. The corrected image should look like pure noise. Once, you correct the gains, move on to dose match.

22) Dose match: send ‘ex’ to scope. Click the ‘acquire dose image’ icon. An image will appear with dose parameter. Check whether the dose is matched or not. If not then put the desired dose, and click the match button. Wait until you will get new dose image. Repeat until you will get the right dose image. Once you get the right dose image then click ‘yes’. Now, we can move to automatic data collection.

24) Automatic data collection-Remember after atlas collection, there is a ‘?’ the icon at ‘hole targeting’ node. Click on ‘hole targeting’ node. Here we need to play around many parameters.

Start with on template – hit test setting- check how is the cross correlation. We need clear peak inside the whole. Try to play around parameters such as final hole template to get pick. To see the change, hit test setting button.

Threshold- hit test setting button. Make sure that threshold looks good and follow the pattern. If not then change ‘A’ value until the threshold looks good. To see the change, hit ‘test setting’ button.

Blobs- hit test setting button. Make sure blob lies in the center of the hole.

Lattice – hit test setting button. Make sure lattice and blobs concise. If then, play around reference intensity. To see the changes, hit test setting button.

Acquisition –Hit test setting button. Make sure you select all holes (esp. good ones). If not then change, the mean thickness and hit test setting button.

After hole targeting is done, hit submit target buttons. Check whether you select all usable holes. If not then play around with the parameter. If you happy with the number of holes selection and hit the submit queue button. And ‘?’ will appear at exposure targeting.

25) For exposure targeting – Repeat exactly as described in hole targeting procedure. Select the desired number of exposure targets. You can also move position by adjusting coordinates. Play around with std. dev until you acquire the desired focus target. Hit test setting. Once the desired exposure target is set up hit, submit target buttons. And finger cross.

At last, go through the checklist on the titan room. Note: if you want to change the defocus value or fresh gains: go to exposure targeting node and click allow user verification box. This will pause the process on exposure targeting node