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Interactive Java Tutorials
Scanning System Basics
The three basic requirements of a laser scanning confocal microscope system are to bring the laser illumination to a focal point on the specimen, scan a selected area of the surface in a raster pattern, and then gather only the secondary fluorescence that originates from the immediate region being excited by the focused laser beam. During scanning, the focal point size should exhibit even illumination and be maintained as small as possible, two requirements that necessitate the objective rear aperture being completely filled with light throughout the scanning cycle. This interactive tutorial examines how the galvanometer-driven mirrors and optical system of a typical confocal microscope are configured to enable the objective rear aperture to be continuously filled with light during the raster scanning operation.
The tutorial initializes with a schematic diagram of a confocal microscope configuration containing a single galvanometer mirror, scanning lens, tube lens, and the objective rear aperture (illustrated as a single lens). A virtual laser beam is incident on the galvanometer mirror from the right-hand side of the window and is scanned across the specimen surface (only the x direction is shown in the tutorial). In order to operate the tutorial, use the Laser Excitation slider to access the various available laser spectral lines, and the Scan Rate slider to control the scanning speed across the specimen surface. Note that the objective rear aperture is continuously illuminated throughout the scanning cycle with a constant beam position across the aperture.
Confocal microscopy scanning systems are designed around several popular motifs that vary significantly with regard to the actual scanning mechanism. One of the simplest designs employs a pair of closely coupled galvanometer mirrors in which the laser is reflected along a mutual axis from the surface of the rotating mirrors into the microscope optical system. The mirror producing the line scan oscillates back and forth on the optical axis very quickly, while the other mirror moves more slowly in a perpendicular operation to trace the frame scan. This scanning mechanism suffers from artifacts when the beam is stationary on one of the mirrors, resulting in the illumination beam moving towards the edges of the objective aperture at the extreme ends of the scan. As a result, image intensity fluctuates across the field (brightest in the center and lesser at the edges) and resolution can be impaired in the outermost regions of the image.
More advanced scanning system designs incorporate modified mirror assemblies to circumvent the stationary beam problem, including the technique of mounting one of the mirrors on an arm that oscillates around a remote axis from the beam reflection point. Another solution is to mount a single galvanometer-driven mirror on a gimbal mechanism to produce a rotational scan by tilting the mirror in two directions simultaneously. Yet a third, and far more versatile, solution utilizes a pair of concave reflectors to image the first mirror onto the second. These scanning systems all have benefits and drawbacks that should be carefully considered when investigating design parameters for laser scanning confocal microscopy.