Confined detection volume of fluorescence correlation spectroscopy by bare fiber probes

A fiber-tip-based near-field fluorescence correlation spectroscopy (FCS) has been developed for confining the detection volume to sub-diffraction-limited dimensions. This near-field FCS is based on near-field illumination by coupling a scanning near-field optical microscope (SNOM) to a conventional confocal FCS. Single-molecule FCS analysis at 100 nM Rhodamine 6G has been achieved by using bare chemically etched, tapered fiber tips. The detection volume under control of the SNOM system has been reduced over one order of magnitude compared to that of the conventional confocal FCS. Related factors influencing the near-field FCS performance are investigated and discussed in detail. In this proof-of-principle study, the preliminary experimental results suggest that the fiber-tip-based near-field FCS might be a good alternative to realize localized analysis at the single-molecule level.     

Measuring Mobility in Chromatin by Intensity-Sorted FCS

The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average correlation functions that are associated to different chromatin regions. This sorting and averaging strategy renders the method statistically robust while preserving the observation of intranuclear variations of mobility. Using this approach, we quantified diffusion of monomeric GFP in high versus low chromatin density regions. We found that GFP mobility was reduced in heterochromatin, especially within perinucleolar heterochromatin. Moreover, we found that modulation of chromatin compaction by ATP depletion, or treatment with solutions of different osmolarity, differentially affected the ratio of diffusion in both regions. Then, we used the approach to probe the mobility of estrogen receptor-α in the vicinity of an integrated multicopy prolactin gene array. Finally, we discussed the coupling of this method with stimulated emission depletion FCS for performing FCS at subdiffraction spatial scales.     

High light field confinement for fluorescent correlation spectroscopy using a solid immersion lens

In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 microm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.     

Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation.

Multiphoton excitation (MPE) of fluorescent probes has become an attractive alternative in biological applications of laser scanning microscopy because many problems encountered in spectroscopic measurements of living tissue such as light scattering, autofluorescence, and photodamage can be reduced. The present study investigates the characteristics of two-photon excitation (2PE) in comparison with confocal one-photon excitation (1PE) for intracellular applications of fluorescence correlation spectroscopy (FCS). FCS is an attractive method of measuring molecular concentrations, mobility parameters, chemical kinetics, and fluorescence photophysics. Several FCS applications in mammalian and plant cells are outlined, to illustrate the capabilities of both 1PE and 2PE. Photophysical properties of fluorophores required for quantitative FCS in tissues are analyzed. Measurements in live cells and on cell membranes are feasible with reasonable signal-to-noise ratios, even with fluorophore concentrations as low as the single-molecule level in the sampling volume. Molecular mobilities can be measured over a wide range of characteristic time constants from approximately 10(-3) to 10(3) ms. While both excitation alternatives work well for intracellular FCS in thin preparations, 2PE can substantially improve signal quality in turbid preparations like plant cells and deep cell layers in tissue. At comparable signal levels, 2PE minimizes photobleaching in spatially restrictive cellular compartments, thereby preserving long-term signal acquisition.     

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.     

lambda-Repressor oligomerization kinetics at high concentrations using fluorescence correlation spectroscopy in zero-mode waveguides

Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for measuring transport properties and kinetics at low fluorophore concentrations. In this article, we demonstrate that simple optical nanostructures, known as zero-mode waveguides, can be used to significantly reduce the FCS observation volume. This, in turn, allows FCS to be applied to solutions with significantly higher fluorophore concentrations. We derive an empirical FCS model accounting for one-dimensional diffusion in a finite tube with a simple exponential observation profile. This technique is used to measure the oligomerization of the bacteriophage lambda repressor protein at micromolar concentrations. The results agree with previous studies utilizing conventional techniques. Additionally, we demonstrate that the zero-mode waveguides can be used to assay biological activity by measuring changes in diffusion constant as a result of ligand binding.     

Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) extracts information about molecular dynamics from the tiny fluctuations that can be observed in the emission of small ensembles of fluorescent molecules in thermodynamic equilibrium. Employing a confocal setup in conjunction with highly dilute samples, the average number of fluorescent particles simultaneously within the measurement volume (approximately 1 fl) is minimized. Among the multitude of chemical and physical parameters accessible by FCS are local concentrations, mobility coefficients, rate constants for association and dissociation processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resolution of less than 0.5 microm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local molecular dynamics provides access to environmental parameters such as local oxygen concentrations, pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quantitative assessment of interactions and dynamics of small molecular quantities in biologically relevant systems.     

Measuring rotations of a few cross-bridges in skeletal muscle

The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.     

New fluorescence correlation spectroscopy enabling direct observation of spatiotemporal dependence of diffusion constants as an evidence of anomalous transport in extracellular matrices

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (D(obs)), the size of the effective confocal volume (V(eff)) for FCS sampling (sampling volume) was continuously changed on a scale of 300-500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the D(obs) of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in V(eff) (220-340 nm in the half-axis), because the network structure of HA of 7-33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount ( approximately 0.1 wt %) of HA in ECM. Because the change in D(obs) along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.     

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 μl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.     

Fluorescence correlation spectroscopy in living cells

Fluorescence correlation spectroscopy (FCS) is an ideal analytical tool for studying concentrations, propagation, interactions and internal dynamics of molecules at nanomolar concentrations in living cells. FCS analyzes minute fluorescence-intensity fluctuations about the equilibrium of a small ensemble (<10(3)) of molecules. These fluctuations act like a 'fingerprint' of a molecular species detected when entering and leaving a femtoliter-sized optically defined observation volume created by a focused laser beam. In FCS the fluorescence fluctuations are recorded as a function of time and then statistically analyzed by autocorrelation analysis. The resulting autocorrelation curve yields a measure of self-similarity of the system after a certain time delay, and its amplitude describes the normalized variance of the fluorescence fluctuations. By fitting the curves to an appropriate physical model, this method provides precise information about a multitude of measurement parameters, including diffusion coefficients, local concentration, states of aggregation and molecular interactions. FCS operates in real time with diffraction-limited spatial and sub-microsecond temporal resolution. Assessing diverse molecular dynamics within the living cell is a challenge well met by FCS because of its single-molecule sensitivity and high dynamic resolution. For these same reasons, however, intracellular FCS measurements also harbor the large risk of collecting artifacts and thus producing erroneous data. Here we provide a step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell. A discussion of advantages and disadvantages of one-photon versus two-photon excitation for FCS is available in Supplementary Methods online.     

Fluorescence correlation spectroscopy and quantitative cell biology

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules. If the FCS observation volume is positioned inside a living cell, these parameters can be measured in vivo. FCS can provide the requisite quantitative data for analysis of molecular interaction networks underlying complex cell biological processes.     

Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated with correlated molecular dynamics there is another source of noise that appears at very large lag times. We call this the "particle noise," as its behavior is governed by the number of particles that have entered and left the laser beam sample volume during large dwell times. The standard deviations of all the points on the correlation function are calculated analytically and shown to be in good agreement with experiments. We have also investigated the bias associated with experimental correlation function measurements. A "phase diagram" for FCS experiments is constructed that demonstrates the significance of the bias for any given experiment. We demonstrate that the value of the bias can be calculated and added back as a first-order correction to the experimental correlation function.     

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.     

Near-Field Optical Imaging of a Porous Au Film: Influences of Topographic Artifacts and Surface Plasmons

In this work, near-field scanning optical microscopy is employed to study a porous Au film and the direct observation of topographic artifacts and surface plasmon influences is revealed. Under tip illumination, topographic artifacts are found to be present in a reflection mode optical image but not in a transmission mode image. A simple algorithm is used for filtering the topographic artifacts and extracting a correct near-field optical image approximately. On the other hand, surface plasmon influences are present in both modes. By using three exciting wavelengths of 488, 647.1, and 520.8 nm, it is confirmed that a suitable wavelength should be chosen for avoiding the surface plasmon interference in a near-field optical investigation of morphological or material dielectric contrast. Finally, plasmonic or nonplasmonic regions on the porous Au film can be identified from the observed optical intensity variation in the optical images obtained at incident polarizations of 0°, 90°, and 45°.     

On the statistics of fluorescence correlation spectroscopy

I present a detailed statistical analysis of fluorescence correlation spectroscopy (FCS) which is a natural extension of an early work. This analysis more realistically takes account of the following issues. (1) A spatial Gaussian laser excitation of fluorescence, (2) the effect of a small number of fluorescent molecules in the observation volume, (3) the shot noise effect due to random emission of fluorescent photons, and (4) a hyperbolic form for the fluorescence autocorrelation function obtained in the case of diffusion. Based on these assumptions, the results differ from the earlier work in several respects, in particular, the dependence of the signal-to-noise ratio on sample concentration and the understanding of shot noise in fluorescence fluctuation moments.     

Fluorescence correlation spectroscopy in small cytosolic compartments depends critically on the diffusion model used

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results. In this paper, we derive a modified FCS model that takes into account the confinement of the detection volume. Using this model, we have carried out the first FCS measurements in dendrites of cultured neurons. We further derive, for the case of confined diffusion, the limits within which the standard two- and three-dimensional diffusion models give reliable results.     

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets¹, monitor dynamics in living cells^2-4, and visualize the three dimensional evolution of entire organisms^5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo⁷ and are currently being applied to disease imaging and diagnosis in clinical settings^8,9.Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole¹, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.Download video file.^{(90M, mov)}     

Optical single-channel recording by imaging Ca2+ flux through individual ion channels: theoretical considerations and limits to resolution

Recent developments in microscopy and fluorescent indicators now make it possible to monitor the activity and localization of membrane ion channels by imaging Ca(2+) flux through individual channels. Such optical approaches have advantages over electrophysiological single-channel techniques in that they are less invasive, provide spatial information and can simultaneously and independently monitor hundreds of channels. However, their kinetic resolution does not yet approach that of patch-clamp recordings. To help understand the processes that determine the temporal resolution and noise level of single-channel Ca(2+) fluorescence signals (SCCaFTs), we simulated the microdomains of Ca(2+) ions and Ca(2+)-bound indicator dye that exist around the mouth of an open channel. Further, as an aid to development of improved optical techniques, we modeled the dependence of the amplitude and kinetics of SCCaFTs on parameters such as the imaging volume, the indicator concentration, affinity and mobility, and the presence of endogenous and exogenous Ca(2+) buffers. The results indicate that under optimal conditions, including the use of confocal or total-internal reflection microscopy to image from sub-femtolitre volumes, SCCaFTs should resolve channel openings as brief as 1ms with a signal-to-noise ratio >10.     

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.