In many quantitative investigations of biological systems, including, e.g., the study of biomolecular interactions, assembly and disassembly, aggregation, micelle and vesicle formation, or drug encapsulation, accurate determination of particle sizes is of key interest. Fluorescence correlation spectroscopy (FCS), with its exceptional sensitivity for molecular diffusion properties, has long been proposed as a valuable method to size small, freely diffusible particles with superior precision. It is conceptually related to the more widespread particle sizing technique dynamic light scattering (DLS) but offers greater selectivity and sensitivity due to the use of fluorescence rather than scattered light. However, in spite of these apparent advantages, FCS has never become established as a biophysical routine for particle sizing. This is due to the fact that sensitivity can, under certain conditions, indeed be disadvantageous, as it renders the technique error prone and overly susceptible to signal disturbances. Here, we discuss the systematic challenges, as well as the advances made over the past decades, to employing FCS in polydisperse samples. The problematic role of large particles, a common issue in DLS and FCS, and the effect of fluorescent labeling are discussed in detail, along with strategies for respective error mitigation in experiments and data analysis. We expect this overview to guide future users in successfully applying FCS to their particle sizing problems in the hope of fostering a more widespread and routine use of FCS-based technology.
In many quantitative investigations of biological systems, including, e.g., the study of biomolecular interactions, assembly and disassembly, aggregation, micelle and vesicle formation, or drug encapsulation, accurate determination of particle sizes is of key interest. Fluorescence correlation spectroscopy (FCS), with its exceptional sensitivity for molecular diffusion properties, has long been proposed as a valuable method to size small, freely diffusible particles with superior precision. It is conceptually related to the more widespread particle sizing technique dynamic light scattering (DLS) but offers greater selectivity and sensitivity due to the use of fluorescence rather than scattered light. However, in spite of these apparent advantages, FCS has never become established as a biophysical routine for particle sizing. This is due to the fact that sensitivity can, under certain conditions, indeed be disadvantageous, as it renders the technique error prone and overly susceptible to signal disturbances. Here, we discuss the systematic challenges, as well as the advances made over the past decades, to employing FCS in polydisperse samples. The problematic role of large particles, a common issue in DLS and FCS, and the effect of fluorescent labeling are discussed in detail, along with strategies for respective error mitigation in experiments and data analysis. We expect this overview to guide future users in successfully applying FCS to their particle sizing problems in the hope of fostering a more widespread and routine use of FCS-based technology.
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