k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

Abstract

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or "blinking". Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of alpha5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection.

Digital Fourier microscopy for soft matter dynamics

“…In particular, the total intensity associated to a point-like fluorescing particle decreases with the distance z of the particle from the focal plane as´d 2 xK(x, z) ⇠ (z/z 0 ) 2 . The existence of a well defined optical section strongly influences the image intermediate scattering function which, for small wave-vectors is typically dominated by the fluctuation along the axial direction [41,33]. An explicit expression for the image intermediate scattering function in a confocal microscope for a system of Brownian particles can be found in Ref.…”

“…An explicit expression for the image intermediate scattering function in a confocal microscope for a system of Brownian particles can be found in Ref. [33].…”

Fluorescence-based transient state monitoring for biomolecular spectroscopy and imaging

“…Moreover, since the fluctuations of the transient states to be investigated typically take place in the microsecond time range, also a relatively high-time resolution of the detection is required. Total internal reflection (TIR)-mediated evanescent field excitation, combined with a highly sensitive fluorescence detection by an electron multiplied charge-coupled device (EM-CCD) camera, has been shown to allow FCS measurements with both autocorrelation (Kannan et al 2006;Kolin et al 2006) and more recently also cross-correlation fluctuation analyses of molecular diffusion and transport in molecular membranes (Sankaran et al 2009). However, while sufficient for relatively slow dynamics, transient state fluctuations typically take place in the microsecond time range and require a time resolution far beyond that of an EM-CCD camera.…”

Visualization of barriers and obstacles to molecular diffusion in live cells by spatial pair-cross-correlation in two dimensions

“…Then, from a conceptual point of view, we need an efficient algorithm able to visualize the connectivity between ~10 5 -10 6 points in one slice of the cell interior, i.e., crosscorrelate the fluctuations between all pairs of points of large images collected for a certain period of time. Although many investigations have focused on the study of the dynamical properties of proteins in live cells, most of the results are local, purely temporal or spatially averaged and in all these cases spatial communication is not visualized [11,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37]. Also, many of the current approaches require an interpretative model and a consequent analytical relationship between the measured properties (usually a change in fluorescence intensity) and the physical properties of the particle motion (e.g., diffusion coefficient).…”