In spectral imaging and linear unmixing, lambda stack acquisition speed is greatly enhanced by using a multichannel photomultiplier to gather bands of fluorescence emission light that have been separated into component colors using a diffraction grating. By employing a linear array of detection channels, multiple emission bands are imaged in parallel, thus enabling a selected spectral region to be obtained in a single scan across the specimen (minimizing phototoxicity). In commercial microscope implementations, the diffraction grating disperses the emitted fluorescence, which is then directed onto an array of precisely defined bandwidth channels in a specialized multi-anode photomultiplier (either 9.7-nanometer or 10.7-nanometer channels in a 32 channel detector) to generate a separate image from each channel.
The tutorial initializes with emission light (entering the window from the right) being diffracted at the surface of a grating and subsequently entering a lens that directs all spectral components to a central photomultiplier in the ZEISS QUASAR detector. In order to operate the tutorial, use the Left Slider and Right Slider controls to translate the sliding light stops back and forth in the light path to narrow the number of wavelengths reaching the multichannel photomultiplier. The Left Prism and Right Prism sliders can be used to directed a selected band of wavelengths to the auxiliary photomultipliers on the left and right hand side of the central photomultiplier.
The ZEISS QUASAR multichannel photomultiplier detection technology is based on a filter-free system that guides the desired wavelength range to the target detector using adjustable optical wedges and slider light stops. In detail, the detector operates such that the tips of the wedges serve as one detection border while the light stop sliders act as the other. By configuring these elements to create custom spectra, any longpass, bandpass, or shortpass filter strategy can be achieved without the need for traditional dichromatic mirrors or emission filters.
Contributing Authors
Tadja Dragoo and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.