Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement

Abstract

Fluorescence correlation spectroscopy (FCS) is an important technique for studying biochemical interactions dynamically that may be used in vitro and in cell-based studies. It is generally claimed that FCS may only be used at nM concentrations. We show that this general consensus is incorrect and that the limitation to nM concentrations is not fundamental but due to detector limits as well as laser fluctuations. With a high count rate detector system and applying laser fluctuation corrections, we demonstrate FCS measurements up to 38 μM with the same signal-to-noise as at lower concentrations. Optical nanoconfinement approaches previously used to increase the concentration range of FCS are not necessary, and further increases above 38 μM may be expected using detectors and detector arrays with higher saturation rates and better laser fluctuation corrections. This approach greatly widens the possibilities of dynamic measurements of biochemical interactions using FCS at physiological concentrations.

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy

“…Most importantly, we could follow a linear increase of photon count rate with dye concentration and laser excitation power ( Figure 1C Figure 1D) were to be expected due to dye photobleaching and saturation of excited state population and consequently fluorescence emission (i.e. not due to detector saturation, compare Figure 1C at same count rate levels), while slight saturation effects at very high dye concentrations may result from photon re-absorption and dye self-quenching, as indicated previously 33,43 . In due consideration of acquisition count rate being the limiting factor in conventional equipment, we present the rest of the data as a function of this parameter.…”

“…This has posed a severe limitation to the accuracy and flexibility in FCS experiments at high fluorophore concentrations, which are however unavoidable for many applications -for example when measuring binding dynamics of low affinity, or diffusion dynamics and concentrations of cellular proteins at different expression levels. Several approaches have been developed to enable FCS measurements even in such cases: labelling of only a fraction of the molecules, reduction of the simultaneously visible fluorophores via fluorescence photoswitching 31,32 , splitting-up of the signal onto several detectors such as on custom-built detector banks 33 , or reduction of the effective observation volume 34,35 using for example small sample containers 36 , near-field structures 37,38 , plasmonic near-field optics 39-41 , or super-resolution STED microscopy 5,42 . Unfortunately, all of these techniques introduce more complexity and possible bias, for example due to required controls to check whether the fraction of labelled or photoswitched molecules truly reflect the entire population, influence on the sample and fluorescent molecules by surface or small volume effects, setup complexity, or perplexing photophysics of the fluorescent label.…”

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery

“…18 As with any FCS experiment, fluctuations resulting from individual molecules diffusing into the focal volume must be discernable, mandating relatively low concentrations, bright analyte molecules, stable lasers, sensitive detectors, and very low obscuring background. 16–17, 22 In the limit of zero background, the number of molecules in the excitation volume is determined using an autocorrelation fit to a standard diffusing molecule model: $$G_2(\tau )=1+\frac{1}{N}·\frac{1}{\left(1+\frac{4D\tau }{{{\mathrm{w}}_{xy}}^2}\right)}·\frac{1}{\sqrt{1+\frac{4D\tau }{{{\mathrm{w}}_z}^2}}}$$ in which τ is the delay, N is the average number of molecules in the focal volume, D is the diffusion coefficient and w xy , w z are the excitation volume dimensions. 19, 23 Underlying dynamics that lead to fluorescence fluctuations are not synchronized, leading to increased contrast in the autocorrelation for decreasing numbers of molecules contributing to the overall signal.…”

“…15 It is this background emission that typically precludes performing FCS in many biologically relevant systems. 6 Although the correlation fitting model can be modified to include background emission, 16 account for laser fluctuations, 17 or extract photophysics, 18 low analyte of interest concentrations and relatively low background conditions must be maintained. 19 Even with weak background present, the true number of molecules inside the focal volume is not directly recoverable as intensity fluctuations arise from both signal and background emitters.…”

Single molecule data under scrutiny

“…Originally FCS was restricted to the measurements of strong biomolecular interactions in open solutions or in vivo settings as only concentrations in the nanomolar range were accessible. However, it has been recently shown by Laurence et al that the limitations to small concentration volumes in FCS is not a matter of principle but of technical limitations of the used lasers and detectors [8]. By stabilizing the lasers and correcting for detector artefacts they were able to measure concentrations up to 40 µM without the need to engineer the observation volume as happens, e.g., in zero mode waveguides.…”