Laser Confocal Microscopy Scanning Modes
The wide range of laser scanning modes available in modern confocal microscopy enable investigators to fine-tune acquisition strategies in order to optimize data collection for three-dimensional imaging, time-lapse analysis, and a host of other specialized applications. Among the common scanning modes featured by most commercial microscopes are point, line, free line, parallel plane, and rectangle scanning over one or more dimensions. This interactive tutorial examines various scanning mechanisms in confocal microscopy utilizing a cube-shaped virtual specimen.
The tutorial initializes with the cubic specimen volume appearing in the window, labeled with respect to the x, y, and z axes, and the Line Scanning mode set to the x-t option (scanning the x axis at a single y value as a function of time, t). Scanning modes include x, y, Diagonal, and Free Line as a function of time in the Line option, as well as x-y, x-z, y-z, Rectangular, Diagonal, and Free Line as the two-dimensional choices versus time. Three-dimensional scanning modes are x-y-z and Rectangular-z as a function of time. Any of the scanning options can be selected using the appropriate radio button. Line scanning and two-dimensional scanning modes can be translated across the volume axis (either x, y, or z, as appropriate) using the Axis Translation slider. In cases where translation across the volume axis is not an option, the slider is inactivated.
In order to operate the tutorial, choose a scanning mode using the radio buttons and observe how the specimen volume is scanned by the virtual laser beam. The mouse cursor can be used (click and drag) to rotate the cubic volume around the z axis to enable examination of the scanning mechanism from a variety of angles. Line and two-dimensional scanning modes can take advantage of the Axis Translation slider to move the scanned line or plane across the perpendicular axis and simulate data collection from deep within the specimen volume or across the opposite face. The Scan Rate slider can be used to modulate the speed of the virtual scanning beam.
One of the primary advantages of confocal microscopy is the ability to collect images from relatively thick specimens through optical sectioning across a selected volume. Traditionally, serial sections are collected by raster scanning the x-y lateral dimensions in selected increments along the microscope optical (z) axis to produce a digital rendition of the specimen volume (x-y-z-t scanning). However, in modern commercial instruments, scanning modes are extremely versatile and serial optical sections can also be collected diagonally in horizontal (Rectangular-z-t scanning mode) planes or vertically in single slices (x-z-t and y-z-t) to produce profile views of the specimen.
Point scanning (not illustrated in the tutorial) allows the instrument to collect data from a single point at any location within the scanning range of the specimen volume. In practice, point scanning enables rapid collection of data that can be utilized to monitor fluorescence emission intensity changes in dynamic settings, such as localized ion concentration investigations in living cells. Line scanning along the x, y, or z axis, or diagonally between axes, provides intensity information across a single set of coordinates in the lateral or axial dimension. This scanning mode is useful for obtaining accurate quantitative information about rapid physiological events, such as calcium waves or sparks. The free line scanning mechanism, available in some commercial configurations, can be used to gather intensity changes over a period of time along the length of a freely drawn line, such as the trace of a neural axon or across a cellular junction.