Two-photon microscopy to spatially resolve and quantify fluorophores in single-bead chemistry.

Abstract

Solid-phase chemistry on polymer beads is widely used in the production and biological screening of combinatorial libraries of compounds.1 Since a lot of analytical methods used in solution chemistry cannot be applied to solid-phase chemistry, monitoring chemical processes on beads has been difficult. To understand solid-phase chemistry, it is desirable to describe the distribution of chemical groups within the solid-phase supports. Previous studies have described the use of autoradiography,2 scanning secondary ion mass spectrometry,3 fluorescence,4 and several microscopy methods, such as confocal fluorescence,5 confocal Raman,6,5d infrared,7 and more recently, two-photon microscopy (TPM).8 However, the spatial quantification of the chemical groups present has not been described before. Here, we show that TPM allows for the direct quantification of fluorophore distributions within single PEGA1900 (poly(ethylene glycol)acrylamide) beads. In TPM,9 the sample is irradiated with a laser with a wavelength approximately twice that of the normal excitation wavelength of the fluorophore. As a result, excitation can occur only when two photons are absorbed simultaneously. Such two-photon events occur at a very high photon density that is reached only at the focal point of the laser beam. Hence, the fluorescence detected originates only in the part of the sample that is in focus. Away from the focal point, there is essentially no absorption of the exciting beam, because there is no chromophore able to absorb single photons of this wavelength. Hence, TPM avoids the artifacts caused by excitation attenuation due to absorption that have been described for confocal fluorescence microscopy.7 In addition, the risk of photodamage to the sample is reduced by application of a pulsed laser permitting recovery of the fluorophore.