Fluorescence correlation spectroscopy (FCS) or fluorescence fluctuation spectroscopy (FFS) have emerged as sensitive techniques to monitor a variety of biochemical reactions, such as protein-protein interactions,1-2
and equilibrium concentrations.5-6
FCS was introduced more than 35 years ago to measure diffusion and chemical dynamics of DNA-drug interactions through the analysis of concentration fluctuations about the equilibrium of a small ensemble (~103
) of molecules.7
Recently, FCS has been applied as a tool to screen for protein interactions for both fundamental research and drug development/discovery applications due to its noninvasiveness, single-molecule limits-of-detection, rapid readout, high analytical sensitivity and accessibility to physical and chemical information at the single-molecule level free from ensemble averaging.8-10
The common instrumental setup for a FCS experiment consists of a laser beam with a Gaussian profile, which is directed into a microscope objective possessing a high numerical aperture and focused into an aqueous solution containing molecules under study that are labeled with a fluorescent dye. The focused laser beam creates a small open volume element called the probe volume. The temporal average of the particle number inside the probe volume is typically between 0.1 and 1,000 for FCS or FFS.11
The occupancy number fluctuates about its equilibrium value as molecules diffuse in and out of the probe volume or as fluorescent molecules are chemically transformed to and from non-fluorescent species undergoing enzymatic processing. The temporal autocorrelation of the fluorescence signal fluctuation yields the time scale of such dynamics and its variance provides the average number of independent fluorophores (<N>) in the probe volume.12
Most applications of FCS are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently-labeled biomolecules that undergo temporal changes in their diffusion properties. However, when analyzing an enzymatic reaction, the change in mass between enzyme and enzyme-substrate complex are usually small and thus, not recognizable due to the logarithmic time-scale of the diffusion calculated by the Stokes-Einstein equation.11
To overcome this, a substrate molecule can be labeled with two spectrally distinguishable fluorophores and the molecular changes invoked on this substrate, for example by an enzyme, monitored using Fluorescence Cross Correlation Spectroscopy (FCCS). For example, a double-stranded DNA substrate was labeled with a red (Cy5) and green (Rhodamine green) dye at opposite ends and the restriction endonuclease, E
co RI, was added to clip the DNA at an internal sequence recognition site. Due to the site specific breaks induced by Eco
RI, the number of doubly-labeled DNA substrate molecules decreased successively with the enzyme reaction progress.13
This process is called dual-color FCS (dcFCS) or simply FCCS.12-14
The normalized cross-correlation function, Gc
), is calculated as the time average of the product of the fluorescence fluctuations of two species, i
at times t and j
at t + τ
, normalized by the product of the time-averaged fluorescence signals of the two species i
(see equation 1
The angular brackets (< >) indicate averaged values, I
is the fluorescence signal as a function of time, and τ
is the delay time. Compared to FCS in which a single autocorrelation function where i
, cross-correlation functions use i
as shown in equation (1)
. Other detailed mathematical relationships for FCCS can be found in the literature.4, 12, 16-17
When performing FCCS measurements, special considerations to cross-excitation, cross-emission, and fluorescence resonance energy transfer (FRET) must be provided, which in many cases requires additional mathematical processing to prevent false positive signals arising from spectral leakage and to improve data quality.18
These issues are typically a result of the broad excitation/emission envelopes associated with most molecular dyes. For example, if cross-emission occurs in a FCCS measurement, a series of negative control experiments with the same sample concentrations are required to determine cross-talk parameters, such as the bleed-through ratio used to correct the impaired data.19-20
If there are only cross-excitation and/or FRET, coincidence events can be considered as truly double-labeled molecules whatever the excitation rate or FRET efficiency. However, cross-excitation and/or FRET may cause other problems in FCCS, such as photobleaching.20-21
To solve issues associated with cross-talk in FCCS, new methodologies have been suggested, such as single laser wavelength FCCS (SW-FCCS),22
two alternating pulsed excitation,26
and switching FCCS.21
For example, Hwang et al.
proposed SW-FCCS using a single laser excitation beam at 488 nm to excite a combination of labels emitting at 510 nm and 695 nm.22
Unfortunately, cross-talk was not completely suppressed. While the aforementioned methods were fairly successful at minimizing spectral cross-talk, additional mathematical compensation steps were still required.27
Although cross-talk can be corrected quantitatively,19-20
the corrections are rather provisional, complex, and time-consuming.
Recently, Thews et al.
successfully eliminated cross-talk signals in their FCCS measurements by adopting an acousto-optic modulator (AOM)-based pulse picker system to generate alternating pulsed excitation laser beams of different colors interleaved with a 50 ns spacing.26
The elimination of cross-talk was accomplished through differences in the dye's finite upper state lifetimes. More recently, Takahashi et al.
demonstrated the feasibility of cross-talk-free, switching FCCS system using acousto-optic tunable filters (AOTFs) in the excitation laser to produce precise alternating laser beams for the study of the activity of caspase-3.21
Both of these techniques required modulators and in some cases pulsed lasers, which increased the complexity of the optical system and required post-processing of the data to synchronize the time course of the alternating detected signals.
We present in this work a simple dual-color FCCS system capable of studying bioreactions to provide near real-time results using continuous wave dual excitation that negated the need for mathematical compensations/post-processing steps through the use of a chromophore set with widely divergent excitation/emission maxima (Cy3 and IRD800) to provide cross-talk free dual-color FCCS. Due to the large spectral separation (~250 nm), cross-talk and FRET between the two dyes and color channels was completely suppressed. The system was evaluated using a model system, in this case monitoring the activity of the enzyme, APE1 (also known as Hap1, Apex, and Ref-1), which is responsible for >95% of the nicking activity of the phosphodiester backbone in DNA on the 5′ side of an apurinic/apyrimidinic site.28-29