Tcspc flim with different optical scanning techniques

Abstract

Scanning a sample with a focused laser beam and detecting light from the illuminated spot delivers images of superior quality. The images are largely free of lateral scattering, and data can be obtained from a defined plane inside the sample. Moreover, nonlinear optical techniques and optical near-field techniques like multiphoton microscopy, STED or NSOM can be applied to record images from deep sample layers or at a spatial resolution below the diffraction limit. Multi-dimensional TCSPC combines favourably with these techniques. The result is a FLIM technique with excellent spatial resolution, excellent image contrast, optical sectioning capability, near-ideal photon efficiency, excellent time resolution, and resolution of multi-exponential decay profiles in the individual pixels of the image. Moreover, it can be used to record multi-wavelength FLIM data, FLIM data for several excitation wavelengths, spatial mosaics of FLIM data, FLIM Z stacks, and temporally resolved FLIM data of physiological changes on the millisecond time scale. This chapter describes the implementation of multi-dimensional TCSPC in the optical systems of confocal and multiphoton laser scanning microscopes, the extension of the wavelength range into the near infrared, the combination of TCSPC with galvanometer scanners, piezo scanners and polygon scanners, the combination with endoscopic systems and with optical systems for imaging of millimeter and centimeter-size objects, and the use of TCSPC FLIM in near-field optical scanning (NSOM) and stimulated emission-depletion (STED) microscopy systems.