ABSTRACT
As reducing blurring and improving contrast through point-to-point illumination and detection, laser scanning confocal microscope (LSCM) got rapid development in the past few decades. Nowadays, the fluorescent laser scanning confocal microscope (FLSCM) that combines the fluorescent labels and LSCM has been one of the most powerful and versatile methods for biomedical research. In this chapter, the basic principle beneath FLSCM will be illustrated. Techniques based on microscopic imaging and spectral scanning through FLSCM will be introduced. Based on microscopic imaging of FLSCM, three super resolution microscopes, i.e., stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOLFT), and ground state depletion (GSD), will be presented. Photodamage and photobleaching induced by FLSCM have made label-free laser scanning confocal microscope (LLSCM) shows powerful capability for imaging and dynamic tracing in biology. Two types of LLSCMs: laser scanning coherent Raman scattering microscope (LSCRSM) and second harmonic generation microscope (SHGM) will be discussed with their fundamentals, features and recent progresses. Also, the mechanism on improving detection sensitivity, image resolution, and speed of image acquisition will be discussed. In addition, another powerful method based on microscopic spectroscopy of FLSCM, i.e. fluorescence resonance energy transfer (FRET), will also be introduced. Finally, the applications of FLSCM on individual DNA-Protein detection and single-molecule fluorescence resonance energy transfer (smFRET) measurement in live cells will be depicted.
