Walkthrough on manual marker clicking
Reconstruction of a tomogram implies the availability of a set of alignment parameters that bring the tilt series delivered by the microscopio onto a compatible reference. Fiducial markers can be located using automated methods. In this walkthrough, we describe the tools that Dynamo offers to locate them manually through graphical interaction with the tilt series.
- 1 Data set
- 2 The dmarkers GUI
- 3 Fitting to an alignment model
- 4 The reconstruction GUI
- 5 Reconstruction through the command line
We suggest to follow this tutorial with data set from the public EMPIAR database (entry number 10064, data item VPP_tomo4.mrc), and can be accessed through and ftp command
in linux, or, in Mac:
The dmarkers GUI
Loading the data
Opening the dmarkers GUI on a tilt series:
It is convenient to first read the tilt series:
ts = dread('VPP_tomo4.mrc');
and then pass the variable to the gui dmarkers:
gui = dmarkers('riboVpp.mrc','tiltAngles',-60:2:60);
here, gui is just a handle to the GUI. This can be used on a later point to operate on it. We also pass the tilt angles, as they will be needed for
[[File:ManualAlignmentDmarkers.png dmarkers GUI on an empty ]]
Basic visualization controls
Slide on micrographs
- Use the slide bar to view a transition of the micrographs (lower resolution images will be shown during the transition, full resolution images will be shown )
- left and right arrows move the previous or next micrograph in the tilt series.
- You can zoom into a point by using the wheel.
- The small red window in the tool bar can be used as magnifying-glass to zoom in into a hand-draw a region.
- The tool with the four arrows pointing into the corners zooms out of the scene totally.
[[File:ManualAlignmentControls.png dmarkers GUI on an empty ]]
The scene gets dragged by moving the mouse while keeping pressed the primary mouse button.
Elimination of defective tilts
Some micrographs may be defective because of errors during acquisition. They can be markers as invalid by the key $x$. If you want to undo the marking of a micrograph as invalid, use shift+x.
Initiating a trace
A three-dimensional gold bead is represented by a trace. A trace is a set of observations, where we call observation to the location of the projection of a gold bead in a given micrograph.
The tool with a black point and a red edge starts the trace initiator. The cursor in the GUI becomes a cross; while this cursor is active, clicking on the screen will create a new gold bead.
Following a trace
To select a gold bead, click on it with the mouse, a green box will appear on the observation corresponding to the selected markers in the current micrograph. Important to add observations into a trace that you have just created, you need to select it, otherwise your next clicks on the screen will be assigned to the trace that is currently under selection.
Once you have selected a gold bead, you should switch off the tool for adding gold beads, and switch on the tool for adding observations to the gold bead currently under selection. The icon represents a point being tracked across several micrographs.
You can use the arrows to move to the next or previous micrograph after clicking the gold bead.
Additionally, you can connect the click and right or click and left tools. When they are switched on, after a click on the screen, the GUI will automatically move to the next micrograph.
Key controls during clicking
- d will delete the closest observation (point in currently shown micrograph).
- shift + d will delete a full trace.
- m will move the observation of the currently selected shape to the location of the cursor.
Auxiliar views during clicking
There are two auxiliary GUIs that can be used to control the growth of the traces based as you click the points. The occupancy window updates as a new observation is added into a gold bead, showing the current content of each trace. And additional control shows the content of each trace as new points are clicked inside.
Fitting to an alignment model
When you have enough observations, you can proceed to fitting the location of your gold beads to a projection model. The tool with the icon Fit will compute a set of shifts and a single angle psi<tt> of rotation for the tilt axis.
In the Fit menu on the GUI, you can find the current error, i.e., the mean residual between each observation and the projection estimated by the fitting model.
Extended fitting models
In a coarse fitting, Dynamo only tries to solve for shifts and a single rotation angle for all micrographs. It is possible to refine this fitting allowing a different rotation of the tilt axis for each micrograph. You can select for a richer model that computes for one different rotation angle on each micrograph by checking the option <tt>eachTilt on the parameter psi (default is single).
Although you will get a lower error value, this might be due to overfitting. Thus, richer parametric models should not be used if you don't have enough markers on each micrograph (in the order of the tens.)
The reconstruction GUI
Once you have clicked on your markers, and you have fitted them to an alignment model, you can proceed to compute a reconstruction. The [Reconstruction] tab in dmarkers opens an external GUI that uses the fitting model computed in dmarkers to create an aligned version of the tilt series and then a reconstruction.
This GUI can be followed on a step by step basis. Completed steps will be shown in bold. Results produced by the computing step can be visualized through secondary-clicking of each view panel.