Extracting the number of quantum dots in a microenvironment from ensemble fluorescence intensity fluctuations

We estimate the number of CdSe quantum dots (QDs) in a microenvironment by analyzing the intensity fluctuation of the fluorescence time trace from a collection of QDs in the fluorescence intensity steady-state regime. The steady state is chosen because the aged blinking dynamics of single QDs leads to a higher probability for observing large on and off waiting times relative to the typical time bins chosen to generate fluorescence time traces. In the steady state, the relative standard deviation of the fluorescence intensity from a collection of QDs is bin-size independent, which enables a simple estimation of the absolute number of QDs under observation. This is not possible for most conventional chromophores, whose triplet blinking dynamics are much faster than a typical bin size, or for QDs undergoing blinking dynamics that are fast compared to the bin size, as occurs prior to the steady state. The estimated number of QDs incorporated into a single silica microsphere as determined by our method agrees well with the number obtained using conventional absorption measurements on an ensemble of microspheres.