تسجيل الدخول

تسجيل

Multiple-Photon Microscopes

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P400-401

2026-06-15

84

Many instruments have been further advanced, but still use the same scanning system as the laser-scanning confocal microscope or the spinning-disc systems. In the multiple-photon microscope, the light source is a high-energy pulsed laser with tunable wavelengths, and the fluorophores are excited by multiple rather than single photons. Optical sections are produced simply by focussing the laser beam onto the specimen, since multiple-photon excitation of a fluorophore only occurs where energy levels are high enough – statistically confined to the point of focus of the objective lens (Figure 1).

Fig1. Illumination in wide fi eld, confocal and multiple-photon microscopes. The diagram shows a schematic of a side view of a fluorescently-labelled cell on a coverslip. The shaded areas in each cell represents the volume of fluorescent excitation produced by each of the different microscopes in the cell. Conventional epifluorescence microscopy illuminates throughout the cell. In the laser-scanning confocal microscope, fluorescence illumination is throughout the cell but the pinhole in front of the detector excludes the out-of-focus light from the image. In the multiple-photon microscope, excitation only occurs at the point of focus where the light flux is high enough.

Since red light is used in multiple-photon microscopes, optical sections can be collected from deeper within the specimen than those collected with the laser-scanning confocal microscope. Multiple-photon imaging is generally chosen for imaging fluorescently labelled living cells, because red light (= longer wavelengths, lower energy) is less damaging to living cells than light with shorter wavelengths usually employed by confocal microscopes. In addition, since the excitation of the fluorophore is restricted to the point of focus in the specimen, there is less chance of photobleaching the fluorescent probe and causing damage to the specimen itself (Figure 1).