Abstract
Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of fluorescent molecules in cells are not always equivalent. Methods relying on spot measurements are more prone to bleaching and movement artifacts that can lead to underestimation of brightness values. We advocate for the use of multiple FFS techniques to study molecular brightnesses to overcome and compliment limitations of individual techniques.
Highlights
In the past 15 to 20 years, there has been an explosion in the advancement of imaging instrumentation and analytical tools to measure molecular dynamics in live cells
Molecular brightness determination using fluorescence correlation spectroscopy (FCS) data is well suited for homogenous single species, but this method breaks down in the face of more complex heterogeneous samples
The same laser power was used for FCS/photon-counting histogram (PCH) and number and brightness (N&B) measurements, but the N&B values are lower because of the reduced laser exposure time, due to raster scanning, that the enhanced green fluorescent protein (EGFP) molecules experience as the laser scans across the sample
Summary
In the past 15 to 20 years, there has been an explosion in the advancement of imaging instrumentation and analytical tools to measure molecular dynamics in live cells Diverse processes such as chemical kinetics, molecular diffusion, protein transport, protein oligomerization, molecular interactions, and stoichiometries can be followed with single molecule sensitivity and at microsecond timescales in biological systems.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18] These microscopy-based techniques that make up this burgeoning field at the interface of biology and physics are collectively called fluorescence fluctuation spectroscopy (FFS) techniques Measurements of molecular brightness in living systems, such as eukaryotic cells, are not optimal due to a variety of diverse factors that include cellular movement, sample thickness bias, geometric constraints, and slow diffusion of molecules leading to a greater propensity for photobleaching.[29,30,31,32,33] Are all brightness analysis techniques equivalent when studying protein dynamics inside complex living systems under nonideal conditions? The purpose of this tutorial is twofold: (1) provide practical advice for the implementation of three widely used FFS techniques (FCS, PCH, and N&B) in measuring the molecular brightness of proteins in live cells and (2) provide two examples where these techniques are not equivalent in determining molecular brightness due to system instabilities, properties of the protein under investigation, or the mode of acquisition (spot scan versus raster scan)
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