Today, multicolor fluorescence imaging is a standard in life science research and usually the first step towards multiparameter microscopy. It is all the more important to make sure that the spectral separation of emitted fluorescence light into different detection channels is efficient and flexible.
Confocal microscopes by Leica Microsystems rely on an arrangement similar to a spectrophotometer. Light emitted from the sample is dispersed by a highly transparent prism. Unlike grating designs, this efficiently prevents loss of photons. Each channel is recorded with an individual point detector. This is a concept permitting a large dynamic range for all kinds of samples because each detector can be set to an individual gain and different types of detectors, such as PMTs and HyDs, can be combined. Since different dyes and different colors of fluorescent proteins have non-equal molecular brightness the system can thus optimally adjust to the sample (see Figure 1). For further information on Leica’s spectral (SP) detector please refer to the Leica Science Lab.
Figure 1: Adaptive dynamic range of the SP detector. The SP detection design using individual point detectors avoids two inherent drawbacks of multianode arrays: Loss of dynamics and spectral gaps. All elements of a multianode array are typically controlled with the same gain. Thus the gain setting must make a compromise between underexposure and saturation in case different channels have non-equal molecular brightness. Spectral gaps between array elements can lead to up to 20 % light loss or else channel cross-talk may become severe. Individual point detectors adapt dynamically to the changing needs of a diverse range of samples.