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FCS Analysis

Fluorescence Correlation Spectroscopy (FCS) analyzes the temporal fluctuations of fluorescence intensity emitted by molecules diffusing through a tiny confocal volume.

Like DLS, FCS operates as a mathematical overlay on top of the Multi-Tau Correlator (CORM). The C++ background thread dynamically fits the correlation decay curves to standard 3D diffusion and triplet-state models, emitting physical metrics such as molecular concentration and diffusion times.

Function Reference

Function	Parameters (In/Out)	Returns	Description
nexatom_tt_enable_fcs_analysis	[In] nexatom_tt_handle device [In] bool enable	nexatom_error_code_t	Enables host FCS analysis. An available selected result has analysis_type == 2; enabling alone does not guarantee it.
nexatom_tt_set_fcs_fit_range	[In] nexatom_tt_handle device [In] uint32_t start_index [In] uint32_t end_index	nexatom_error_code_t	Restricts the Levenberg-Marquardt solver to a specific temporal region of the 80-bin CORM lag curve, typically excluding the sub-microsecond photophysics (unless analyzing triplet states).
nexatom_tt_set_fcs_confocal_volume	[In] nexatom_tt_handle device [In] double omega_xy_um [In] double omega_z_um	nexatom_error_code_t	Calibrates the theoretical shape of the laser excitation volume. Defines the 1/e² lateral (omega_xy) and axial (omega_z) beam radii in micrometers.
nexatom_tt_set_fcs_experimental_conditions	[In] nexatom_tt_handle device [In] double temperature_celsius [In] double viscosity_mPa_s [In] double wavelength_nm [In] double calibration_diffusion_um2_s	nexatom_error_code_t	Supplies experimental parameters/reference diffusion; this call alone does not establish an experimental confocal-volume calibration.
nexatom_tt_set_fcs_fitting_control	[In] nexatom_tt_handle device [In] double tolerance [In] int max_iterations [In] double initial_n [In] double initial_tau_d	nexatom_error_code_t	Sets tolerance, iteration budget and initial estimates; convergence must be checked on the result.

Data Structures: nexatom_fcs_analysis_result_t

An available FCS result is exported when analysis_type == 2; enabling the solver alone does not guarantee selection or success. General fit quality is in the parent fit_result. The fields below describe the public representation: uncomputed quantities can be unavailable/NaN/zero rather than valid physical measurements.

Field	Type	Description
diffusion_coefficient_um2_s	float	3D translational diffusion coefficient (Dt) in μm²/s.
diffusion_time_us	float	Characteristic diffusion time (τD) representing how long a molecule remains in the focal volume.
concentration_nM	float	Absolute molecular concentration in nanomolar (derived from N and volume).
particles_per_volume	float	Average number of fluorescent particles observed in the volume (N).
confocal_volume_fl	float	Effective focal volume in femtoliters.
structure_parameter_s	float	Focal volume shape parameter s = ωz/ωxy.
molecular_brightness_khz	float	Average photon emission rate per individual molecule.
triplet_fraction	float	Fraction of molecules residing in the dark triplet state.
triplet_lifetime_us	float	Characteristic relaxation time of the triplet state.
anomaly_parameter_alpha	float	α parameter for anomalous sub-diffusion models (1.0 = normal Brownian motion).
flow_velocity_um_s	float	Directed velocity for active transport or flow models.
flow_fraction	float	Fraction of molecules exhibiting directed flow.
component1_fraction	float	Fraction of component 1 (two-component model).
component1_diffusion_time_us	float	τD for component 1.
component2_fraction	float	Fraction of component 2 (two-component model).
component2_diffusion_time_us	float	τD for component 2.

C Example: Configuring an FCS Experiment

// Fragment: illustrative inputs; validate the actual microscope/sample calibration.
// Check every return and preserve the complete acquisition owner's cleanup.
double w_xy = 0.3; // 300nm lateral radius
double w_z = 1.5;  // 1.5um axial radius
nexatom_tt_set_fcs_confocal_volume(my_device, w_xy, w_z);

// 2. Set ambient parameters for a buffer solution at room temp
nexatom_tt_set_fcs_experimental_conditions(
    my_device, 
    22.5,   // 22.5 Celsius
    1.002,  // Viscosity (mPa*s)
    488.0,  // Excitation Laser (nm)
    0.0     // No auto-calibration diffusion reference
);

// 3. Exclude very fast lags to bypass detector afterpulsing
nexatom_tt_set_fcs_fit_range(my_device, 3, 80);

// 4. Activate the FCS physics pipeline
nexatom_tt_enable_fcs_analysis(my_device, true);

Fitting seeds cannot ensure convergence. Configure the confocal volume explicitly; do not assume supplying a calibration diffusion value alone performs an experimental calibration. See analysis validity.