Chapter 2: Autoindexing and Cell Refinement

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Step 7: Display Your Image

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Step 7: Display Your Image

Click on the display button to display the first frame (Figure 17).

Figure 17. The Display image button 

Click peak sear and more peaks (if necessary) to select peaks for autoindexing. Then click ok (Figure 18). Occasionally, you may notice that number of peaks that are picked by the program is low. In that case adjust the expected size of the spot by clicking the size peak up or size peak down buttons.

 

Figure 18. The Display window with activated “Peak Search" option

Wow, my crystal diffracts great! Is that the correct resolution in the green box?

Sorry, I wish it was, but most likely it is not. The display program does not know the crystal-to-film distance yet, and so the Distance and Wavelength are set to defaults, which are likely to be different from your actual conditions. Once you have indexed the first frame, the display program knows the correct distance and the numbers in the green box of the image display are accurate.

How many peaks do I want to use?

The main object is to pick peaks for autoindexing. If the program picked around 100 good peaks (clearly belonging to the major lattice, and not coming from ice rings, etc.) then you are ok. If lots of the peaks are very weak, you can increase the chances of getting some good ones by clicking on more peaks. Alternatively, you can click on the frame button to display the next frame, and the peak search will be repeated on this frame, using the same criteria used for the first frame (e.g. more peaks, fever peaks, etc.). These will be added to the peak search from the first frame. This is a way to index poorly diffracting crystals. If you have a double lattice, the best way is to increase weak level to, let’s say, 20, to select only the stronger peaks – this way you may index only one lattice.

What if the Peak Search picks lots of peaks in the ice ring, or picks peaks from a second lattice?

Since Denzo assesses the quality of each peak before using it in autoindexing, most spurious peaks, such as those coming from ice rings, can be ignored by the user. However, if you have a second lattice contaminating the diffraction pattern, you will have to make some effort to assure that the selected peaks come mainly from the primary lattice. The first way is to hope that Denzo can automatically distinguish between the primary lattice and any secondary lattices. If the secondary lattice is weak or at a significantly different orientation from the primary lattice, this is not unreasonable. Alternatively, you can try indexing on a different frame, where the secondary lattice may not be so pronounced. However, if this does not work, you can assist the program by editing the peak search peaks in the Image Display window.

Can I get rid of some peaks I know shouldn’t be included?

Yes, you can assist the program by editing the peak search peaks in Image Display window. If you click on the pick (add) button, the cursor will change to a green cross, and this can be used to select peaks from the peak search for removal (Figure 19). Also, you may change the resolution to, let’s say, 4 Ĺ to remove higher resolution peaks from the autoindexing.

Can I add some peaks that I think the autoindex should use?

Yes. Simply click on the pick (add) button (Figure 19); the cursor should turn into a green cross. Use this cross to select the peaks you want to add by clicking on them. A little green circle will be left over your selections. Note that this does not work in the Zoom window, even though clicking on peaks leaves little green circles in here as well – but they’re not added to the peak list.

*  The pick (add) button should be used in extreme cases when you have very few (less than 10) very week spots per frame.

 

  

Figure 19. The Pick (Add) and Pick (Remove) buttons from Display window

How can I flip through my frames at this stage to see if they all look ok?

In the Image Display window, there is a button labeled frame, with an up or down arrow in it. Clicking on the middle-mouse-button causes the displayed frame to increment in the direction of the arrow. Up arrow means a higher numbered frame, and vice versa. To change the up or down arrow, use the right and left mouse button, respectively, to click on the frame button (Figure 20). The frame button will only increment one frame at a time. If you want to quickly go to a frame that is far away from the current one, you’ll have to close the Image Display window and enter the frame number in the box by Frame Number in the Main page (Figure 17).

 

  

Figure 20. The Frame button from the Display window

Can I index on another frame? Can I index on peaks collected from several contiguous frames?

You can select which frame to display by changing the Frame Number box. Most of the time it's best to start with the first frame of the series, because when Denzo starts autoindexing it will assume you are working with the first frame of a series unless you split the data set. If you're not working with the first frame and you haven't split the data set, then the program will not be able to find the peaks you selected for autoindexing, because while you may have picked peaks from frame 10, Denzo is checking for these peaks on frame 1. You’ll get an error message that says, “Error – Peaks none.” So the rule is that the peak search has to be done on the first frame(s) of a set, and if you want to index starting from the middle you have to split the set so that one series starts there in the middle.

*  One nice feature of HKL-2000 is that you can index on peaks collected from several contiguous frames. Why is this helpful? Well, suppose you have a real turkey of a crystal that doesn’t diffract well and only gives a few low resolution spots. Alternatively, you might have a very sparse reciprocal lattice that only results in a few spots per image. Ordinarily this would be hard or impossible to index. However, if you could combine peaks from several consecutive frames, this might yield enough good reflections so that autoindexing could work.

To accumulate peaks from consecutive images for autoindexing, first display the first image of the series and run your normal peak search. If you need to, you can click on more peaks or fewer peaks, but don’t hit the ok button (Figure 21). Instead, use the middle mouse button to click on the frame button. The next frame will be displayed, and the peak search will be run on the next image. Notice that if you clicked more peaks or Fewer Peaks the search on the second frame will behave as if you have already clicked this. You can select the number of frames and the new peaks are appended to the peaks.file file in the directory from which you launched HKL-2000. Note that for autoindexing to make use of the peaks coming from the second and subsequent frames that the 3D Window (found under the Index tab) must be set equal to or larger than the number of frames on which the peak search was performed. In other words, if you did the peak search on 4 consecutive frames but your 3D Window is set to 3 frames, then the peak search peaks from only the first 3 frames will be used in the autoindexing. Similarly, if your 3D Window is set to 5 but you only did a peak search on the first 2 frames, then only the first two frames will be used for the initial indexing.

 

   

Figure 21. The More Peaks and Fewer Peaks buttons

Adjusting the display: How can I make the diffraction image look “better”? You know, darker peaks, lower background.

The displayed image can be made darker by clicking on the bright button in the Image Display window. Conversely, the image can be lightened by clicking on the dim button. Small increments in either are made by clicking with the left mouse button. Larger jumps are made by clicking with the middle or right mouse buttons. To change the displayed dynamic range, use the floor up or floor down buttons. floor up narrows the dynamic range, while floor down increases it. If you have fairly weak diffraction you can enhance the contrast (i.e. make it look “better” than it actually is) by alternately clicking the bright and floor up buttons (Figure 22).

 

         

Figure 22. The buttons for adjusting the image

 


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