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ScanImage controls scanned laser beam(s) to generate images. Those scanned laser beam(s) should be aligned in two senses:

1. Scan Alignment: The scan area should be centered with respect to the optical axis (i.e. the axis of the objective lens).
2. Beam Alignment: The laser beam should be centered with respect to the objective's back focal aperture at the 'stationary point' which is (or should be) located at that plane.

ScanImage 3.5 adds some simple tools related to the tasks of scan and beam alignment. All relevant controls are on the Main Controls window:

Panel
title Main Controls
Center New Controls These controls relate to the scanOffsetX/Y Rig Configuration INI-file variables described below. SET 0: Pressing this button causes the current X/Y Shift values (displayed at top right of Main Controls) to be added to the current Offset X/Y values. X/Y Shift values are reset to 0. The option is provided to the user to save the new values to the scanOffsetX/Y variables in the INI file loaded at startup, saving the new alignment for the future. PARK 0: Pressing this button parks the mirrors, and hence the laser beam(s), at the values specified in scanOffsetX/Y. Additionally, the shutter is opened, and each Pockels Cell (where present) is set to the power level specified in the Power Control window for each 'beam'. The user may then employ mirrors or other means to ensure that each beam is centered with respect to the back focal aperture of the objective lens. When done, the user closes the dialog box as prompted, to close the shutter and return the mirrors (beams) to the parked laser position.

Usage Notes

The scanOffsetX/Y values are considered INI file Rig Configuration variables because an individual rig should only have one correct scan alignment. However, different users could employ separate scanOffsetX/Y values by using distinct INI files, as described in Initialization (INI) File Handling.

For scan alignment, typical approaches involve the use of a pool of fluorescent dye as the specimen. The resulting image of uniform fluorescence should be centered in the acquisition windows. If needed, the Scan Amplitude X/Y can be enlarged somewhat (in the Configuration window, accessbile via Edit >> Configuration... from the Main Controls menubar). This forces the scan area to exceed the objective and/or excitation/detection path's field-of-view. To center the image, the Left/Right/Up/Down and other image shift controls in the Main Controls window are adjusted, affecting the X/Y Shift values. The SET 0 button is then used when the image centering is deemed satisfactory.

For beam alignment, typical approaches involve centering the beam with respect to the back focal aperture by inspection, or alternatively, for cases with an overfilled back focal aperture, by measuring the power throughput through the objective lens (power throughput is maximized when centering is optimal).