Image Formation in Multiphoton Fluorescence Microscopy∗

Abstract

Abstract This chapter is dedicated to image formation in multiphoton fluorescence microscopy. In particular, the comparison of image formation is based on the three-dimensional intensity point spread function (IPSF), the three-dimensional optical transfer function (OTF), and axial and transverse image resolution for thin and thick objects. Since its inception, confocal scanning microscopy has become a widely used and important tool in many fields, including biology, biochemistry, chemistry, physics, and industrial inspection (1–6). One of the main advantages in confocal microscopy is its ability of three-dimensional (3-D) imaging of a thick object. Because of the 3-D imaging property in confocal scanning microscopy, confocal fluorescence microscopy was achieved in the same period as confocal bright-field microscopy. Under the illumination of intermediate power, one incident photon can be absorbed in the sample under inspection to excite the electron transition from the ground state to an excited state. The excited electron returns to the ground state by radiating fluorescence light. The energy of the radiated fluorescence photon is slightly less than the incident one due to the nonradiation relaxation during the downward transition, and the corresponding microscopy in which the fluorescence light is imaged is termed single-photon (1-p) fluorescence microscopy.