Particle Selection from Tilt-Pair Images

Access: CP::Particle::Tilt Pair (Tab)

Image pairs with known relative tilt are utilized in the random-conical-tilt (RCT) method for model reconstruction, and they are especially useful for model evaluation as well.  In tilt-pair data acquisition, the same ROI of the specimen is imaged twice at different, yet controlled tilting geometry.  The first exposure produces the "master-image", and the second one produces the "pair-image".  Due to beam radiation damage, the master-image generally contains more information than the pair-image (particularly in cryo-EM).  In the following, the image pair will be named as "A" and "B" respectively, with the exact order of the exposures at the user's discretion (as long as being consistent in the spreadsheet).

Load an Image Pair

An image pair can be displayed in two ways:

• When both images are already in the CP::DataView spreadsheet, set the imaging property to TiltPair (from the pull-down menu below the spreadsheet).  The image pair can then be opened simultaneously via the Display button.  The selected image (LeftClick to high-light) will be displayed as Image-A, and its pair as Image-B.  Note that the pair images should be entered into the spreadsheet back-to-back, and Image-B could be either before or after Image-A, depending on their serial order in the spreadsheet.

• For a quick inspection, the images can also be opened manually from the respective boxes (A & B) in the CP::Particle::Tilt Pair::Image Pair group (on the left of the page).

When opened, Image-A will be displayed on the left of the DP::MainDisplay window, and Image-B on the right.  To navigate the pair display by the computer mouse: LeftClick+Drag to pan, and RightClick+Drag to zoom.  Pre-selected particles in each image (e.g., from CP::Particle::Single Image) can be imported by the respective PTK button, with the total particles shown in the Count value-box.  Note that, when the pair images are newly loaded, the A/B sets of coordinates may not be properly paired until a registration between the images is established.

Manual Pair Annotation

PARTICLE provides functions (in the Image Pair group) for both manual and automatic tilt-pair particle annotation.  Manual selection can be used extensively to identify all particle pairs in the images, or to establish a set of fiducial markers for the subsequent tilt-pair registration and automatic pair identification.  To select particle pairs manually,

1. Choose the Manual function in the Particle Pairing pull-down menu;
2. Select one particle in Image-A (CTRL+LeftClick), the ROI will be displayed in CP::Monitor as well;
3. Identify its correspondence in Image-B (CTRL+LeftClick), use the button A/B to toggle the particle-pair display in CP::Monitor whenever necessary;
4. The pair coordinates will appear in the PairList table on the right;
5. Annotate the next pair by repeating the step #2 to #4, interleaving the selection between Image-A and Image-B;
6. Upon completion, Save (the button below PairList) all the particle pairs in the list.

To facilitate the targeting of manual annotation, PARTICLE is equipped with an auto-centering function that works best for globular particles.  To use this function, set the off-center tolerance Delta (in pixels) above zero, the approximate particle Diameter (in pixels), and the Frame size (in pixels) for particle boxing.  Upon activating the square buttons (adjacent to Delta, for image A/B or both), PARTICLE will prompt to re-center the existing particles in the respective image, and then automatically center the subsequent particle selections in real-time, till this function is deactivated.

Tilt-Pair Registration

During tilt-pair data acquisition, the nominal value of the tilting geometry for each image should be carefully recorded, which later will be used to initialize the refinement of the image tilt parameters.  Because mechanical error in the microscope specimen-stage is inevitable, the nominal reading is only approximate and needs to be refined via fiducial-marker registration.  Otherwise, a significant discrepancy might occur in the subsequent computational pairing.  To refine the tilting geometry,

1. Load the image pair and clear any existing particle selection (PairList becomes empty);
2. Enter the nominal values at the Tilt Angle and Tilt Axis value-boxes for each image;
3. Manually identify at least six particle pairs (or landmarks), preferably distributed evenly in both images;
4. Set refinement restraints by "linking" (green buttons upon activation, by the respective value-boxes) or "locking" (red buttons) the tilting parameters, whenever and wherever deemed appropriate (at user's discretion);
5. Activate the Register button, the geometry refinement will only take a few seconds;
6. Upon completion, the relative rotation and translation between Image-A and Image-B will be displayed in the designated value-boxes, and the value of the registration error will be displayed in CP::MessageBoard.  Normally an error value (in pixels) smaller than the particle size indicates a correct registration.
7. After refinement, activate the Save button to record the tilt parameters into a data file, and/or the Stack button to build a pair of particle stacks (both are internally named and managed).

When exporting particle pairs into stacks, the defocus parameter in the CTF for each particle will be calculated based on the image tilt geometry recorded at Image Property of CP::DataView.  There are two methods to set the tilt geometry of the image: either according to the image tilt determined here via pair registration, or at the stage of image CTF determination.  If you decide to use the image tilt parameters obtained from the pair registration for the particle defocus setup, the tilt geometry of both images can be entered into CP::DataView by the R (record) button.

The C (clear) button will clear all tilt parameter display in the page to zero (but not to reset the recorded values at CP::DataView).

Automated Pair Selection

After an image pair has been properly registered, more particle pairs can be added either semi-manually or automatically:

• Automatic : when activated from the Particle Pairing pull-down menu, a manual selection in Image-A will automatically produce a correspondence in Image-B according to the tilt registration.  This function can also be used to visually verify the registration parameters.

• Match : when the button (in the lower region of the Image Pair group) is activated, all particles in Image-A (for instance, imported via the PTK button from a pre-selection) will be mapped to Image-B according to the established image-pair registration.

In automatic particle pairing, depending on the accuracy of the registration and the fitting model, the particle annotation might not be properly centered.  The auto-centering function (introduced above) can be used to improve the result.  Note that the centering function works best for globular-shaped particles.

Particle Pair Inspection

Particle pairs in PairList can be examined by activating the Inspect button.  The selected pair (mouse LeftClick to high-light a pair entry in the list) will be displayed exclusively, with the particle in Image-A being centered in its display window.  The images of the particle pair will appear in CP::Monitor as well, and can be switched by the A/B button under the monitor window.  The registration error of the selected pair will be displayed in both CP::MessageBoard and DP::MessageBoard simultaneously.  If the pairing is found incorrect, activate the Delete button to remove the entry from PairList.

The two arrows above the PairList can sort the particle pairs by their registration error, in either ascending or descending order.  A sorted list will help identify miss-matched pairs that often contain a large registration error.  The Save button under PairList will export the paired particle coordinates to a data file, and the Stack button will export the pairs into two separate stacks, one from Image-A, and the other from Image-B.  All these files are named and managed internally by the program.  The button Clear removes all particle pairs in PairList and reset the particle counter.