FCSXpert Solutions: Fluorescence Correlation Spectroscopy Simplified!.
## FCS Classroom

#### Autocorrelation vs. Cross-correlation Assays

### Properties of Autocorrelation Assaysback to top

#### General Properties of Autocorrelation Assaysback to top

#### Critical Autocorrelation Assay Design Factorsback to top

#### Autocorrelation Assays of Direct Binding Interactionsback to top

##### Typical Procedure: Autocorrelation Assay of Direct Binding:

#### Autocorrelation Assays of Competition Binding Interactionsback to top

##### Typical Procedure: Autocorrelation Assay of Competition Binding:

### Properties of Cross-correlation Assaysback to top

#### General Properties of Cross-correlation Assaysback to top

#### Cross-correlation Assays of Direct Binding Interactionsback to top

##### Typical Procedure: Cross-correlation Assay of Direct Binding:

#### Cross-correlation Assays of Competition Binding Interactionsback to top

FCS Assays can be designed with one fluorescent molecule in autocorrelation mode (mass-dependent), or with two fluorescent molecules in cross-correlation mode (mass-independent), giving you the flexibility to use a broad range of dyes and study the interaction of molecules with similar molecular weights.

The QuantumXpert FCS Spectrometer has been specifically designed for solution-based FCS assays, and offers the flexibility of two excitation wavelengths (491 nm and 532 nm) and three emission detection channels.

The easiest biological interactions to monitor with FCS are binding interactions. FCS can be used to detect both direct binding and competition binding interactions, which can be used to quantify an analyte of interest.

This section briefly describes the properties of the two correlation assay types when measuring both forms of binding interactions and critical factors to consider when designing and implementing FCS measurements:

The QuantumXpert can be used to obtain autocorrelation functions in three distinct emission channels. The recommended dyes for these three channels are:

- Channel A (510 nm to 525 nm): Alexa-488, GFP, Quantum Dots 525
- Channel B (550 nm to 575 nm): Alexa-532, R6G, Quantum Dots 565
- Channel C (590 to 710 nm): Cy3.5, Cy3.5b, Alexa-568, Texas Red, Quantum Dots 605

These fluorescent dyes have been selected because of:

- Minimal crosstalk between detection channels
- Resistance to irreversible photobleaching
- Resistance to triplet state excitation or intersystem crossing
- Availability in reactive form to label probes

Autocorrelation measurements provide information about diffusion time and aggregation state of the fluorescent molecules being studied. Binding is typically measured by monitoring the formation of bound complexes which result in a shift to slower diffusion times and decrease in average number of particles in the detection volume.

**Quality of Fluorescent Probes:**It is critical that the probes are purified to minimize free dye. Free dye in excess of 3-5% of total fluorescence will affect your ability to quantitatively analyze autocorrelation measurements.**Molecular Weight of the Complex:**It is critcal to consider the difference in molecular weight of the dye-labeled probe relative to the complex that is formed upon binding. In general, the molecular weight should be increased 3 to 5 fold when the freely diffusing probe binds to the target. In the event that the target and probe are of similar molecular weight, it is also possible to immobilize the probe on a larger particle to increase the molecular weight (e.g. carrier protein, bead or Quantum Dot), or to use a target with multiple binding sites for the fluorescently labeled probe.

Additional factors to consider when designing FCS assays can be found at: Optimizing FCS Measurement Conditions

Direct binding of a fluorescently labeled probe to a target will provide
information about the total concentration of probe binding sites (B_{max})
and the binding affinity of the probe (K_{D}, or Equilibrium Binding Dissociation
Constant).

where [L] is the concetration of labeled probe, B_{max} is the total binding sites, and K_{D} is the equilibrium dissociation binding constant.

- Determine diffusion time (τ
_{D}) of the fluorescently labeled probe alone (Free Probe). - Determine if quantum yield for the labeled probe changes when
it binds to the target. If the fluorescence intensity changes for the same
probe concentration after the target is added, then a correction factor must
be calculated in order to accurately calculate B
_{max}. - Choose a single target concentration and incubate with a series of different probe concentrations that span the predicted range of binding affinity.
- Measure the fluorescence correlation data of each sample with The QuantumXpert FCS Spectrometer. Only autocorrelation functions will be used in the analylsis.
- Fit the data with FCSXpert software using either a one- or two-component diffusion model (see Choosing Between FCS Fitting Models). In the two-component fit, the fast diffusion time should be the Free Probe, and the slow diffusion time the Probe-Target complex.
- Plot the Fraction Bound as a function of probe concentration.
- Fraction Bound is calculated as fraction of slow diffusing component (F2) * the total number of diffusing particles (N) in the detection volume.

- Both B
_{max}and K_{D}can then be obtained from this plot by fitting with a hyperbola (saturation binding isotherm).

Competition binding assays allow you to determine the binding properties of an unlabeled analyte.

These assays are conducted by pre-incubating
the labeled probe and target, then adding a series of unlabeled analyte
preparations at different concentrations. The unlabeled probe will displace
fluorescently labeled probe from the probe-target complex, providing
information on binding affinity (K_{i}) and the fraction displaced (or fraction
of labeled probe remaining in the complex).

- Determine diffusion time (τ
_{D}) of the fluorescently labeled probe alone (Free Probe). - Conduct a Direct Binding Interaction Assay for probe and target, as described
above, in order to obtain a K
_{D}for this interaction. - Pre-incubate labeled probe and target until equilibrium is
reached. This will form the Competition Complex that is used to quantify
unlabeled analyte. It is assumed that the unlabeled analyte being characterized
is a competitive inhibitor of the labeled probe used to form the Competition
Complex.
- NOTE: In order to achieve maximal sensitivity when binding
unlabeled analyte, concentrations should be selected so that ~50% binding
is achieved (i.e. near the K
_{D})

- NOTE: In order to achieve maximal sensitivity when binding
unlabeled analyte, concentrations should be selected so that ~50% binding
is achieved (i.e. near the K
- Incubate the Competition Complex with a series sample preparations that contain the unlabeled analyte.
- Measure the fluorescence correlation data of each sample with The QuantumXpert FCS Spectrometer. Only autocorrelation functions will be used in the analylsis.
- Fit the data with FCSXpert software using either a one- or two-component diffusion model (see Choosing Between FCS Fitting Models). In the two-component fit, the fast diffusion time should be the Free Probe, and the slow diffusion time the Probe-Target complex.
- Plot Fraction Bound as a function of unlabeled sample concentration.
- Fraction Bound is calculated as fraction of slow diffusing component (F2) * the total number of diffusing particles (N) in the detection volume.

- IC
_{50}and Fraction Bound can be obtained by analyzing the Fraction Bound plot with the the equation below: where [I] is the concentration of inhibitor, IC_{50}is the 50% inhibitory concentration, and p is the Hill Coefficient (slope factor) which should be 1 for simple binding. - K
_{i}for the unlabeled analyte can be calculated from IC_{50}using the Cheng-Prusoff Relationship: where K_{D}is the equilibrium dissociation binding constant for the labeled probe, [L] is the concentration of the labeled probe, and IC_{50}is the 50% inhibitory concentration.

The QuantumXpert can be used to obtain three cross-correlation functions. In cross-correlation, two different fluorescent labels with distinct excitation and emission properties are detected in two emission channels. Coincidence of these fluorescent labels on the same diffusing particle results in a cross-correlation signal. The QuantumXpert calculates the cross-correlation functions for:

- Channels AxB
- Channels AxC
- Channels BxC

The QuantumXpert also acquires cross-correlation data for BxA, CxA and CxB, and the FCSXpert Software uses this additional data to improve the counting statistics for the cross-correlation functions reported.

Direct binding of a fluorescently labeled probe to a target will provide
information about the total concentration of probe binding sites (B_{max})
and the binding affinity of the probe (K_{D}, or Equilibrium Binding Dissociation
Constant).

where [L] is the concetration of labeled probe, B_{max} is the total binding sites, and K_{D} is the equilibrium dissociation binding constant.

- Determine diffusion times (τ
_{D}) of the two fluorescently labeled particles being studied. This may be a labeled probe and a labeled target, or two labeled probes that recognize and bind to the same target particle. - Determine if quantum yield for either of the labeled probes
changes when it binds to the target. If the fluorescence intensity changes
for the same probe concentration after target is added, then a correction
factor must be calculated in order to accurately calculate B
_{max}. - Choose a single labeled target concentration and incubate with a series of different labeled probe concentrations that span the predicted range of binding affinity.
- Measure the fluorescence correlation data of each sample with The QuantumXpert FCS Spectrometer. Both autocorrelation and cross-correlation functions will be used in the analysis.
- Fit the data with FCSXpert software using either a one- or two-component diffusion model (see Choosing Between FCS Fitting Models). In the two-component fit, the fast diffusion time should be the Free Probe, and the slow diffusion time the Probe-Target complex.
- Plot fraction bound as a function of probe concentration.
- Fraction Bound is the number of diffusing particles reported in the cross-correlation analysis panel.

- Both B
_{max}and K_{D}can then be obtained from this plot by fitting to a saturation binding isotherm.

Competition binding assays allow one to determine the binding properties of an unlabeled analyte.

These assays are conducted by pre-incubating
the labeled probe and target, then adding a series of unlabeled analyte
preparations at different concentrations. The unlabeled probe will displace
fluorescently labeled probe from the probe-target complex and will provide
information on binding affinity (K_{i}) and the fraction displaced (or fraction
of labeled probe remaining in the complex).

Cross-correlation assays of competition binding can be developed using the principles outlined above.