Measurement of the Concentration and the Brightness for Samples Containing Multiple Molecules with Different Brightness Using Fluorescence Correlation Spectroscopy

In this study, the concentration and brightness measured by fluorescence correlation spectroscopy (FCS) in samples containing multiple species with different brightness levels was demonstrated. FCS measurements of such samples are generally difficult. However, the calculation we introduced here can provide the measurement results of the FCS. The effectiveness of the calculation was investigated based on simulations and experiments in the case of a mixture of fluorescent beads with known brightness and other fluorescent beads with unknown brightness. The results show that the concentration of the known brightness agrees well with the expected values. The obtained concentration and brightness of the species with unknown brightness is possible, and it worked well in the simulation; however, the accuracy for the species was lower than that of the species with known brightness. As a result, the calculation is useful in measuring the concentration of species with known brightness in samples containing undesired bright species, such as aggregation. The calculation for the species with unknown brightness may also be useful if good protocols or instruments are established in the future.