Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/43660

Title

Image formation in multiphoton fluorescence microscopy

Author(s)

Gu, Min

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. 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 fluorescent light is imaged is termed single-photon (1-p) fluorescence microscopy. The ultrashort pulsed laser technology has recently been combined with confocal microscopy for novel imaging modes. Such a combination also allows many novel applications to be possible. One of the emerging areas is nonlinear optical microscopy, which uses the nonlinear radiation generated from the sample by the high peak power of an ultrashort pulse. A multiphoton fluorescence process is one of the nonlinear processes caused by the simultaneous absorption of two or more incident photons under the illumination of an ultrashort pulsed beam. The energy of the resulting fluorescence photon is slightly less than the sum of the energy of the absorbed incident photons. The original idea of two-photon (2-p) fluorescence scanning microscopy was proposed by Sheppard and colleagues along with other nonlinear scanning microscope modes. 2-p fluorescence microscopy was first demonstrated by Denk in 1990. It has been reported that strong three-photon (3-p) fluorescence can also be generated in some organic solutions, where three incident photons are absorbed simultaneously and the radiating photon has energy approximately three times as large as the incident one. It was Hell and his colleagues who measured the first 3-p fluorescence microscopic image using BBO. With multiphoton absorption, one can have access to ultraviolet (UV) photon excitation without using a UV source. Due to the cooperative multiphoton excitation, the photobleaching associated with 1-p fluorescence is confined only to the vicinity of the focal region, and 3-D imaging becomes possible without the necessity for a confocal pinhole mask. However, axial resoltuion in multiphoton fluorescence microscopy can be improved if a confocal mask is used. These imaging properties can be well understood in terms of the method based on the concept of the 3-D transfer function. With this method, the relationship between different optical arrangements can be gained and the image quality can be improved in image processing. This chapter is organized as follows: Section 11.2 presents a general description of image formation in multiphoton fluorescence microscopy with a finite-sized detector. In Sections 11.3 and 11.4, 3-D IPSF and the 3-D OTF are used to understand the performance of the multiphoton fluorescence imaging systems. Resolution in the transverse and axial directions is discussed in Section 11.5 in terms of the calculated images of layer and edge objects. The effect of the fluorescence wavelength on image resolution is presented in Section 11.6.

Publication type

Book chapter

Research centre

Swinburne University of Technology. Faculty of Engineering and Industrial Sciences. Centre for Micro-Photonics

Source

Handbook of biomedical nonlinear optical microscopy / Barry R. Masters and Peter T. C. So (eds.), Part III, chapter 11, pp. 266-282

Publication year

2008

Keyword(s)

Fluorescence microscopy;  Multiphoton excitation microscopy

Publisher

Oxford University Press

ISBN

9780195162608, 0195162609

Publisher URL

http://www.oup.com.au/titles/academic/science/biological_sciences/9780195162608

Publisher URL

http://books.google.com/books?id=4mki1ThMgGYC

Copyright

Copyright © 2008 by Oxford University Press, Inc. All rights reserved.

Peer reviewed