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Laser Scanning Confocal Microscopy
Interactive Java Tutorials

Galvanometer-Based Confocal Scanning Systems

In order to generate a digital image from an extended specimen in laser scanning confocal microscopy, the focused beam is scanned laterally (in the x-y plane) across the specimen surface in a rectangular raster pattern. Modern instruments utilize a scanning mechanism based on two high-speed vibrating mirrors driven by galvanometer motors to produce the scanning pattern. This interactive tutorial explores how the scanning mirrors are coordinated to direct the laser beam into the objective, and then to reflect secondary fluorescence gathered from the specimen back through the optical train to the emission filter.

Interactive Java Tutorial
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The tutorial initializes with the major components of a scanning confocal laser microscope optical train positioned in the left-hand side of the window. A virtual argon-ion laser beam (default excitation wavelength of 457 nanometers) passes through an excitation filter and dichromatic mirror before reflecting from the surface of scanning mirror number two, which controls the y scan direction. After leaving mirror number two, the beam is reflected by scanning mirror number one (controlling the x scan direction) through the objective and onto the specimen. Secondary fluorescence emitted by the specimen is arbitrarily shifted to higher wavelengths (50 nanometers) in the tutorial, and passes back through the scan mirrors in a process known as descanning before being transmitted through the dichromatic mirror and the barrier filter on to the photomultiplier.

In order to operate the tutorial, use the Laser Excitation slider to toggle through the available laser line wavelength range of 322 (ultraviolet) to 647 (red) nanometers. As the slider is translated, the color of the laser excitation and secondary fluorescence emission beams changes, as does the color of the corresponding bandpass filters and dichromatic mirror. The Scan Rate slider can be utilized to increase or decrease the scan rate, both on the virtual microscope optical train and across the Specimen Image in the right-hand side of the window. The entire microscope assembly can be rotated around the z-axis (through 360-degrees) for a different view by clicking and dragging with the mouse cursor.

Commercial laser scanning confocal microscopes feature a variety of scanning mirror configurations that often include a relay optical system to assist in generation of the raster pattern. In most cases, the galvanometer-driven scan mirrors are positioned to rotate in mutually perpendicular axes to create a set of telecentric planes. One of the mirrors controls scanning across the x axis, while the other translates the beam along the y axis. The motion of each mirror is coordinated to form the raster pattern, and the scanning speed is regulated by the speed and angular extend of mirror deflection.

Secondary fluorescence emission gathered from the excited specimen by the objective travels back through the optical system and scanning assembly along the same pathway (coaxial) as the excitation illumination in the descanning process. The emission beam position at the pinhole aperture does not follow the raster scanning pattern (as does the excitation illumination), but remains steady and fluctuates only in intensity as the scanning beam excites fluorophores at varying concentrations in the specimen.