Live-cell imaging applications require the ability to rapidly switch between different wavelengths of light to independently excite a range of fluorophores in the same specimen. This task is generally accomplished with the use of multiple filters, beamsplitters, rotating filter wheels, tunable optical filters, or monochromators. One of the most useful and cost-effective configurations involves placing a polychroic mirror in a standard fluorescence filter optical block and using separate filter wheels under computer control to rotate the proper excitation and emission filters into the optical pathway when necessary. This interactive tutorial examines wavelength switching with an aftermarket filter wheel coupled to an external metal halide lamphouse.
The tutorial initializes with a virtual 4-color specimen being sequentially excited in 200 millisecond intervals with an 8-position filter wheel coupled to a metal halide illuminator. To operate the tutorial, use the Choose Filter Set check boxes to eliminate one or more filters from the excitation sequence. The filter wheel speed can be adjusted with the Sequence Speed slider between a range of 30 and 500 milliseconds. At any point, the tutorial can be frozen by clicking on the Stop Sequence button.
Filter wheels used in fluorescence microscopy are designed to accommodate flat, circular optical glass interference filters, heat filters, neutral density filters, and ultraviolet blocking filters in a rotating frame that houses between 4 and 10 filters having a diameter ranging from 25 to 50 millimeters. The filter frame is mounted in an aluminum housing that provides easy access to the filter slots for interchanging filters, and is driven by a precise stepper motor controlled by an external drive unit. Filters are able to rotate into a single optical port in the wheel housing, which can be equipped with adapters for attachment to the microscope input and output ports or an external light source. High-performance filter wheels are able to rotate a new filter into position in approximately 25-30 milliseconds.