Quantitative measurement of fluorescence brightness of single molecules

Single-molecule fluorescence spectroscopy and imaging probe many characteristics of the fluorescence from individual molecules like relative intensity, polarization, lifetime and spectrum. However, such an important and fundamental parameter as absolute fluorescence intensity (or in other words fluorescence brightness), which is proportional to the absorption cross section and fluorescence quantum yield, has not yet been sufficiently exploited in the field. One reason for that is the difficulty of absolute fluorescence brightness measurements. In the present work a detailed description of fluorescence brightness measurements of single molecules is given. We discuss several important factors like the power density and polarization of excitation light, the substrates and the local environment. It is shown that the fluorescence brightness of a single molecule indeed can be measured with sufficient accuracy and used as a powerful parameter for characterization of materials at single molecule/particle level. The brightness of a single object can give similar information as the fluorescence quantum yield that is crucial for understanding the photophysical properties for individual multi-chromophoric systems in inhomogeneous environments.