Detection, descanned

Laser-scanning microscopes can be classified by the way they excite and detect fluorescence in the sample. One-photon microscopes use a NUV or visible CW laser to excite the sample. Two-photon, or Multiphoton , microscopes use a femtosecond laser of high repetition rate. The fluorescence light can be detected by feeding it back through the scanner and through a confocal pinhole. The principle is termed confocal or descanned detection. A second detection method is to divert the fluorescence directly behind the microscope objective. The principle is termed direct or nondescaimed detection. 

For thin samples, such as single cells, scattering is negligible. In these cases two-photon excitation is also used in conjunction with descanned detection. The pinhole is usually opened wide and used principally to suppress daylight leaking into the objective lens. 

A relatively simple way to build a lifetime microscope with descanned detection is to use a fibre output from the scanning head. Fibre outputs are available for the Biorad Radianee 2100, and for the Zeiss LSM 510 NLO and LSM 510 Meta systems. The required connections are shown in Fig. 5.73. 

Figure 5.71, left, shows the principle of a laser scanning microscope with one-photon excitation. The laser is fed into the optical path via a dichroic mirror. It passes the optical scanner, and is focused into the sample by the microscope objective. The focused laser excites fluorescence inside a double cone throughout the complete depth of the sample. The fluorescence light is collected by the objective lens. Detection of the fluorescence light can be accomplished by either descanned or nondescanned detection. 

Commercial laser scanning microscopes use the same microscope body and the same scan optics for one-photon and two-photon excitation. Most two-photon microscopes have lasers for one-photon excitation as well. They can switch between both modes, and between descanned and nondescanned detection. Moreover, in both the descanned and the nondescanned detection path, the light is split spectrally by additional dichroic mirrors or dispersion prisms and several detectors are used to record images in selectable wavelength ranges. The dichroic mirrors and filters are assembled on motor-driven wheels and are changed on command. The laser power... 

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