The tutorial initializes with a schematic diagram of a resonant scanning confocal microscope zoom optical system illustrated on the left-hand side of the window, as well as a Specimen Window and Ronchi Grating shown on the right-hand side. Light emitted by the pixel clock laser is reflected from the rear surface of the resonant galvanometer mirror and focused on the Ronchi grating as the mirror oscillates. Light scattered by the grating is gathered by a beam expanding lens and directed to a photodiode detector. To operate the tutorial, use the Zoom Magnification slider to rotate the Ronchi Grating Disk to higher zoom values and observe how the specimen size changes.

Invoking the confocal zoom function requires changes to the spacing interval of the lines in the Ronchi grating used for the variable pixel clock. The disk in Figure 1 contains Ronchi gratings of four sizes, each of which can be rotated into the pixel clock optical train in order to change the zoom factor. When the line size and spacing dimensions in the Ronchi grating are reduced by half (maintaining the same total number of lines), the zoom factor increases by a factor of two. Likewise, reducing the grating line size dimensions by a factor of four increases the zoom factor to 4x. The number of possible zoom settings depends upon the number of Ronchi grating sizes available to the variable pixel clock. The Nikon A1R HD25 confocal microscope is capable of 7 zoom steps ranging from 1x to 8x. Interchanging gratings requires reducing the sweep angle of the resonant galvanometer, and this is accomplished using pre-stored information in the microscope control electronics that coordinates galvanometer sweep angles with the selected grating.

Illustrated in Figure 1 is a schematic diagram outlining the components of the optical train for a Ronchi grating-based variable pixel clock in a resonant scanning confocal microscope. The primary optical and electronic elements in the pixel clock are shown in the left-hand side of the diagram and include a low-power semiconductor near-infrared laser source, a rotatable disk containing Ronchi gratings of varying spacing, intermediate lenses to focus the reflected laser beam onto the Ronchi grating, and a photodiode sensor that detects light pulses passing through the grating. Components involved in the excitation and imaging optical trains are presented in the right-hand side of the figure, but for simplicity, the number of imaging components is reduced to a minimum. Note that the mirror attached to the resonant galvanometer in Figure 1 has highly reflective surfaces on both sides in order to accommodate the imaging and clocking lasers.

In operation, light emitted by the clocking diode laser is directed to the reflecting backside of the resonant scanner mirror while light from lasers involved in the imaging system are reflected from the frontside of the same mirror. Reflected light from the clocking laser is focused by a condensing lens to sweep across the surface of the Ronchi grating in step with the oscillating motion of the galvanometer mirror. At the grating, the laser beam is scanned across a sequence of alternating opaque lines and transparent spaces of equal width. The condensing lens focuses the clocking beam to a size smaller than the line spacing in the grating to ensure that light is either completely passed or partially blocked by the grating, in an alternating manner dependent upon the position of the galvanometer mirror. A critical point is that the laser beam spot at the grating surface level should be significantly smaller than the grating line width in order to eliminate diffraction. Fluctuations in light intensity induced by the oscillating galvanometer are detected by the photodiode, which is positioned immediately beneath the grating.

During the horizontal scan period of the resonant galvanometer, the clocking photodiode registers pulses of laser light scattered by the Ronchi grating that can be electronically converted to a pixel clock having variable frequency. These light pulses will be more frequent in time when the galvanometer mirror is in the central portion of its oscillatory period, but they will slow down as the mirror reaches the end of a scan and reverses direction. Among the requirements of the photodiode detector are the ability to handle the variable pulse frequency (in terms of bandwidth) and it must be large enough to detect the entire line sweep. The detector is connected to a transconductance amplifier that transforms photodiode current pulses into an amplified voltage, which is then fed to a frequency doubler circuit that doubles the number of pixels per line. The photodiode and associated electronics deliver 256 pulses per line and the frequency doubling circuit transforms this output into 512 pulses per line that are acquired by a frame grabber and used to construct an image.