Two Photon Absorption Cross Sections
Overview
Two-photon and multiphoton microscopy are two of most recent innovations in optical microscopy. They offer many advantages over traditional light microscopy, especially the ability to reveal three-dimensional information about a specimen. The efficiency of multiphoton microscopy depends on the efficiency of a fluorophore in absorbing two or more photons simultaneously. The most common fluorophores used currently are ones that have been optimised for strong one-photon absorption (1PA). Measuring this efficiency - the absorption coefficient - is easy for 1PA, but the process of absorbing many photons is completely different from absorbing one photon and it is likely that these good one-photon dyes might not be the best for multiphoton applications.
Objectives
To develop a technique for measuring two-photon absorption (2PA) cross-sections directly and routinely;
To explore the photophysical and photochemical properties of molecules subjected to two-photon illumination.
How the Experiment is Done
The amount of light absorbed by a substance under normal single photon conditions is given by Beer’s Law, in which the amount of absorbed light is (in the weak absorption limit) proportional to the absorption cross-section of the molecule, s, the pathlength, l, and the concentration of absorbing species, C. In 2PA, the absorption is proportional to the square of the light intensity. As a consequence, 2PA occurs only for very intense light, which, in common application, occurs at the focus of a laser beam. The photo at right shows fluorescence of a dye following 1PA and 2PA. The laser at the top of the cuvette is exciting the dye by 1PA causing yellow fluorescence emission. The emission can be seen clearly along the whole focussed laser path. The laser at the bottom of the cuvette is exciting the dye by 2PA, which causes the same yellow fluorescence. This time emission only occurs at the focal point of the laser because of the (intensity)2 dependence of the 2PA. There are several important consequences of the effect in the photo for fluorescence microscopy:
- There is no blurring of the image due to fluorescence from out of the microscope focal plane. This both sharpens the image and removes the need for spatial filtering of the fluorescence;
- The fluorescence arises from a specific, well-defined depth in the sample. This means that a sample can be "optically sectioned" and a 3-dimensional image built up by recombining these sections;
- 2PA utilizes red or near-infrared light. The penetration of near-IR light through a sample is significantly further than visible light because very few species absorb near-IR light.