Picking particles from micrographs can be done in several ways: "by hand"; i.e., interactively using Web particle picking . Usually at this stage, the micrograph is normally presented on the screen in a decimated form: for instance, only every second pixel is displayed, so that the entire micrograph can be done in one round. The "hand" procedure results in center coordinates of the selected particles stored in a document file. The automated procedure (which works best with globular particles) starts with a low-pass filtered version of the micrograph or, what amounts to the same thing, the result of cross-correlating the micrograph with a disk that has the approximate size of the particle. Peak search then yields, again, a list of center coordinates of particle candidates stored in a document file.
The document file can be subsequently used to window particle candidates from the large, undecimated micrograph file into many small files. This is done by using a small SPIDER procedure that has a Do-Loop over all particles and selects windows at coordinates that are calculated from the stored coordinates, taking into account the offset (since top left coos are needed by the windowing operation) and the decimation factor.
Sample SPIDER procedure file (for the case of no decimation):
; ----- Parameters -----
[image-dim] = 64 ; Image dimension
[part] = 1 ; First-particle number
; ------ Inputs -------
FR L
[micrograph]mic001 ; Micrograph
FR L
[coords_doc]doccoords001 ; Coordinates document file
; ------- Output -------
FR L
-
[win]win{***[part]} ; Windowed-image template
; -- END BATCH HEADER --
[half-dim] = [image-dim]/2
; Get number of keys
UD N [num-keys]
[coords_doc]
; Do-loop running over index = key
DO [key] = 1,[num-keys]
; Unsave document, in-core, key, x-coord, y-coord
UD IC, [key],[xcoord],[ycoord]
[coords_doc]
; Compute top left coordinates for window operation (WI)
[topleft-x] = [xcoord] - [half-dim]
[topleft-y] = [ycoord] - [half-dim]
; Window
WI
[micrograph] ; INPUT: micrograph
[win] ; OUTPUT: windowed-image template
[image-dim],[image-dim] ; image dimensions, in x,y
[topleft-x],[topleft-y] ; top-left coordinates
; Increment particle number
[part] = [part] + 1
ENDDO ; End loop
; Close in-core document file
UD ICE
[coords_doc]
EN ; End of SPIDER procedure file
Examples for more sophisticated procedures, taking into account decimation, checking margins, and applying a density ramp corrections, can be found in Techniques - 3D Reconstruction Using Projection Matching
The next step is to apply a screening procedure, which again can be done interactively or in an automated way. Screening is necessary since the decimated raw file does not allow a final assessment of the quality. It is definitely needed if the particles were picked automatically, since they were only selected by the computer on account of their overall features (diameter).
Screening by hand is done by using the "categorize" option of "Web catagorize" command. Choose the selected particles to be displayed, and each particle to be marked or categorized according to its quality. Most of the time, "accept" and "reject" are the only categories used. According to this classification, a flag is set in the output document file.
Another way of screening follows the method introduced by Lata and coworkers (Ultramicroscopy 58 (1995) 381-391): particles are automatically classified according to texture descriptors. This is however not straightforward since the parameters of the texture descriptors vary with the data. A training procedure has to be followed first, in which, with the help of a small particle subset and the "categorize option" in "Web categorize" operation. program in the use of the categories "noise", "good particle", and "junk". On this basis, the texture descriptors are computed, which are then used by the program to screen the data. This obviously only makes sense if the data set to be screened is large.
Bimal Rath has developed a SPIDER procedure pickparticle.spi to automatically pick particles from a micrograph (B.K. Rath and J. Frank, Journal of Structural Biology, Volume 145, Issues 1-2, January 2004, Pages 84-90). The procedure uses local cross-correlation function calculated using Fourier methods outlined by Alan Roseman (Ultramicroscopy, Vol 94, Issues 3-4, (2003), 225-236). The local normalization optimizes the numerical scaling of the search image and hence search results are very accurate. Since most of the computations are done in Fourier space the computation time is of the order of few minutes. Currently, the procedure needs a 3D volume of the search image(as input) to generate projections at desired Eulerian angles for searching different orientations of the search image in the micrograph. Different input parameters can be used, to apply desired interpolation, to choose types of mask (symmetric or asymmetric), to use SPIDER selection doc files (as Eulerian angles input) and to use average of a number of projections as template image. The procedure can be accessed by typing @pickparticle at the SPIDER prompt or inside any SPIDER procedure file. Two sets of input parameters follow as examples. Note: For picking ribosomes from micrographs one may use a zero degree projection, a symmetric mask and an interpolation factor of 2. It works quite well.
SET 1 : Takes a set of projections as template, interpolates down to half and uses a symmetric mask.
micro001 ; Micrograph refvol001 ; 3d volume of the particle noise ; A noise image used to normalize the images as per histogram particle ; Windowed particle template 101 ; Starting particle number for windowed particles annotate ; Document file with particle co-ordinate 0 ; Selection file is NOT used 0 ; PHI start value 10 ; PHI end value 2 ; PHI step size 20 ; THETA start value 50 ; THETA end value 4 ; THETA step size 30 ; PSI start value 40 ; PSI end value 5 ; PSI step size 2 ; Interpolation factor 20000 ; No of peaks to be searched (arbitrary; see Notes (I)) 130 ; Peak separation distance (size of the paticle; see Notes (II)) 1 ; Symmetric mask is used
SET 2 : Takes a selection doc file to create projections as template, interpolates down to half and uses an asymmetric mask.
micro001 ; Micrograph refvol001 ; 3d volume of the particle noise ; A noise image used to normalize the images as per histogram particle ; Windowed particle template 101 ; Starting particle number for windowed particles annotate ; Document file with particle co-ordinate 1 ; Selection file is used 0 ; Each projection is used, average image is NOT used 2 ; Interpolation factor 20000 ; No of peaks to be searched (arbitrary; see Notes (I)) 130 ; Peak separation distance (size of the particle; see Notes (II)) 0 ; Asymmetric mask is used 50 ; Masking threshold pixel value seldoc ; Selection file name
NOTES: (I) Decrease this # if too many picked-particles correspond to noise;
Increase this number if the last dozen of picked-particles correspond
to correct particles)
(II) Only one particle will be picked from an area of a square with sides
equal to these many pixels (130, in this case) around a
cross-correlation coefficient peak.
Source: partpick.html Updated: 02/08/08