In particular, N&B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures.
N&B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time.
The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&B) analysis. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. Lastly, we consider the practical limitations of the technique gleaned from our studies and offer suggestions for utilizing the platform to quantitatively analyze nonideal colloidal systems with distributed or heterogeneous system parameters.Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Statistical trends in the data collected from a nominal system of 80 nm gold nanoparticles correspond to theoretical predictions as we vary key design parameters such as particle size, surface charge, and solution ionic strength. We develop and model the device physics to understand the physical underpinnings of the raw device data and then use these models to design proof-of-concept experiments to verify device function. In this work, we present a unique tin dioxide (SnO 2) nanofiber-based total internal reflection microscopy (TIRM) method to efficiently characterize colloidal behavior as a function of particle-level properties in complex fluidic conditions. Comprehensive characterization typically requires disparate techniques that can observe correlations between microscale particle–surface interactions and physical properties of the particles. However, for many applications it remains challenging to predict system-level colloidal behavior.
Bulk colloidal interactions are dictated by the physical properties of individual particles dispersed in solution.
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