Multiphoton Excitation Fluorescence Microscopy

Methods in Cellular Imaging 
Oxford University Press

Edited by 
Ammasi Periasamy, Ph.D.


Multiphoton Excitation Fluorescence Microscopy




Two-photon absorption was theoretically predicted by Goppert-Mayer in 1931 and was experimentally observed for the first time in 1961 using a ruby laser as the light source. More recently, multiphoton (two- or three-photon) excitation microscopy has become common talk among scientists as a result of the contributions made by Watt Webb’s group (Denk, Strickler and Webb., Science 248: 73-76, 1990) from Cornell University. More importantly, the commercialization of multiphoton imaging systems by Bio-Rad has increased awareness and application of this technology in biomedical imaging. It should also be recognized that laser companies played a key roll in introducing a tunable (700-1100 nm) high-speed femtosecond infrared-pulsed laser system (Ti: sapphire) for multiphoton imaging. Moreover, the future will bring easier-to-use equipment and increased sensitivity, which will allow greater flexibility in the simultaneous imaging of multiple fluorophores while images are collected over time and at greater depths inside tissue.

To describe the multiphoton process in brief, an infrared femtosecond pulsed laser is required to create multiphoton absorption in a biological sample. Multiphoton excitation occurs at only a single, diffraction-limited spot where the photon flux is great enough to allow absorption of more than one photon. For one-photon excitation, a wavelength of 480 nm CW (continuous wave) is selected for the FITC molecule, but for two-photon excitation, a single 960 nm wavelength from a Ti: sapphire pulsed laser is used. But for most of the fluorophore the two-photon absorption cross-section is blue shifted (for example: FITC could be excited using 780 or 920 nm). The multiphoton excitation microscopic images have better signal-to-noise compared to confocal images because of a considerably less amount of light scattering. In addition autofluorescence, and photobleaching are minimized because there is not absorption throughout the specimen due to illumination (for more details, see chapters 9-14).

The basic principles of multiphoton absorption, absorption cross-section calculation, and demonstration of deep tissue imaging are described in Chapter 9. Chapter 9 also includes instructions on building a multiphoton system using a wide-field fluorescence microscope. Chapter 10 describes the conversion of an existing confocal system to a multiphoton system, and compares the one- and multiphoton microscopy systems. Chapter 11 addresses many aspects of multiphoton imaging including depth penetration for different wavelengths, Monte Carlo simulation demonstrating the excitation and emission, point spread functions at different depths, and the decrease in fluorescence signal with increase in excitation pulse width.

Chapter 12 focuses on the use of multiphoton microscopy in developmental biology.  Additionally, Chapter 12 reviews the implementation of the multiphoton system to monitor the dynamic aspects of development of frog, sea urchin, and mammalian embryos, and the optimization of the fluorescent probes for labeling embryonic cells. Also described is the use of the two-photon system for photo uncaging for sea urchin embryogenesis. Chapter 13 describes an in situ measurement of the diffusion mobility of biologically relevant molecules using multiphoton fluorescence photobleaching recovery and fluorescence correlation spectroscopy (FCS) methods. The theory behind these two methods, instrumentation, data collection and comparison of this methodology with conventional FRAP techniques (Chapter 7) are provided. Chapter 14 focuses primarily on the interaction between excitation radiation and single living cells in laser microscopes. In particular, the influence of continuous wave ultraviolet (UV) and near infrared (NIR) microbeams, as well as that of femtosecond and picosecond NIR laser pulses on cellular metabolism, ultrastructure, and viability is described.