Temporal investigation of events in living cells requires the ability to capture successive images on a wide spectrum of timescales, often ranging from microseconds to minutes. The popular filter wheels commonly used for live-cell imaging applications are limited to switching times on the order of 20 and 50 milliseconds, which preclude the use of these accessories for investigations on a faster time frame. A number of other devices, such as grating monochromators, digital light processing instruments, and acousto-optical filters or modulators are capable of rapid wavelength switching, but they often suffer from lower power output and harmonic contamination. The Sutter Lambda DG-4 featured in this tutorial is a complete interference filter-based xenon-powered illumination system that exhibits switching speeds of less than 2 milliseconds.
The tutorial initializes with the DG-4 operating through four excitation filters at a switching speed of 100 milliseconds. As the galvanometers transition through the filters, the resulting images are displayed in the Specimen Image window. To operate the tutorial, use the filter check boxes to engage or remove filters, and the Sequence Speed slider to transition between switching speeds of 1 to 500 milliseconds. The Choose Specimen pull-down menu can be used to select a new specimen.
The optical design of the DG-4 (and the newer DG-5, which holds five filters) is based on matching dual-scanning galvanometers that first pass the focused excitation light through the interference filters and then to the liquid light guide focusing lens system. Light is directed through parabolic mirrors before entering and after leaving the interference filter channels. A cold mirror placed near the xenon lamp eliminates infrared radiation to remove heat from the optical system of the unit. An integral shutter enables reduction of light intensity over six orders of magnitude, but for mechanical shuttering, an auxiliary unit must be attached.