Spatial two-photon fluorescence cross-correlation spectroscopy for controlling molecular transport in microfluidic structures

The increasing availability of microfluidic systems of various geometries and materials for the downscaling of chemical or biochemical processes raises a strong demand for adequate techniques to precisely determine flow parameters and to control fluid and particle manipulation. Of all readout parameters, fluorescence analysis of the fluid or suspended particles is particularly attractive, as it can be employed without mechanical interference and with a sensitivity high enough to detect single molecules in aqueous environments. In this study, we present the determination of flow parameters, such as velocity and direction, in microstructured channels by fluorescence correlation spectroscopy (FCS), a method based on single molecule spectroscopy carried out in confocal optical setups. Different modes of FCS, such as auto- and dual-beam cross-correlation techniques by one- and two-photon excitation, are discussed. Known advantages of two-photon excitation, such as highly restricted detection volumes and low scattering background, are shown to be particularly valuable for measurements in tiny channel systems. Although conventional autocorrelation is sufficient for describing the velocity of single molecules, dual-beam cross-correlation allows the separation of isotropic and anisotropic dynamics, for example, to monitor flow directions or to discriminate against photophysical effects that could be mistaken for mobility parameters. It can be shown that time-gated two-photon excitation in the dual-beam mode significantly lowers the undesired cross-talk between the two measurement volumes. Finally, some applications, such as the calibration of microfluidic sorting units and flow profiling, are demonstrated.     

Excitation saturation in two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) has become a powerful and sensitive research tool for the study of molecular dynamics at the single-molecule level. Because photophysical dynamics often dramatically influence FCS measurements, the role of various photophysical processes in FCS measurements must be understood to accurately interpret FCS data. We describe the role of excitation saturation in two-photon fluorescence correlation measurements. We introduce a physical model that characterizes the effects of excitation saturation on the size and shape of the two-photon fluorescence observation volume and derive a new analytical expression for fluorescence correlation functions that includes the influence of saturation. With this model, we can accurately describe both the temporal decay and the amplitude of measured fluorescence correlation functions over a wide range of illumination powers.     

Calibration of probe volume in fluorescence correlation spectroscopy

In fluorescence correlation spectroscopy (FCS), an accurate evaluation of the probe volume is the basis of correct interpretation of experimental data and solution of an appropriate diffusion model. Poor fitting convergence has been a problem in the determination of the dimensional parameters, the beam radius, omega, and the distance along the optical axis of the probe volume, l. In this work, the instability of fitting during the calibration process is investigated by examining the chi(2) surfaces. We demonstrate that the minimum of chi(2) in the omega dimension is well defined for both converging and diverging data. The difficulty of fitting comes from the l dimension. The uncertainty in l could be significantly larger than that in omega, as determined by F-statistics. A modified calibration process is recommended based on examining two data treatment methods, combining several short data sets into a single long run and averaging the correlation functions of several short data sets. It is found that by using the mean of several converging correlation functions from short data sets instead of a long time correlation, more stable and consistent dimensional parameters are extracted to define the probe volume.     

Two-dimensional fluorescence correlation spectroscopy with modulated excitation

Overlap of multiple states or multiple species in a chemical system often creates a congested fluorescence spectrum that is difficult to interpret. The resolution of component spectra is essential for the understanding of the structure and dynamics of such multicomponent systems. In this paper, two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented for the dissection of component spectra using the time correlation function. In 2D FCS, the time response of fluorescence intensity is collected at various wavelengths upon an external perturbation. The time correlation function is evaluated between wavelengths. A two-dimensional fluorescence correlation spectrum, or a plot of the correlation intensity as a function of two wavelength axes, resolves the overall spectrum into component spectra. The characteristics of the two-dimensional time correlation function are demonstrated in the frequency domain fluorescence spectroscopy in which the sinusoidally modulated excitation provides the external perturbation. Using 2D FCS, fine vibronic structures of the component fluorescence emission spectra were completely resolved from a strongly overlapped one-dimensional mixture spectrum. The existence of multiple microenvironments of a probe molecule in a biological system is evidenced by nonzero asynchronous correlation intensities. The corresponding spectra are retrieved from correlation analysis. Unlike traditional resolution methods in fluorescence spectroscopy based on statistical fitting of fluorescence decays, 2D FCS can resolve species whose fluorescence decays are linked by the rate constants in chemical reactions and species displaying multiexponential decay kinetics.     

A closed form for fluorescence correlation spectroscopy experiments in submicrometer structures

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion coefficients in an open detection volume. However, in several practical cases, when FCS measurements are carried out in small compartments like microchannels, neglecting boundary effects could lead to erroneous results. Here, a close form solution is proposed to explicitly account for the presence of walls located at a distance comparable with the characteristic detection volume lengths. We derive a one-dimensional diffusion constrained model and then generalize the solution to the two- and the three-dimensional constrained cases. We further indicate within which limits the standard autocorrelation function (ACF) model gives reliable results in microconfinement. Our model relies just on the assumption of elastic hits at the system walls and succeeds in describing the ACF of fluorescent probes confined along one direction. Through the analysis of FCS experimental data, we are able to predict the correct shape of the ACF in channels of micrometric and submicrometric width and measure the extent of lateral confinement. In addition, it permits the investigation of microstructured material features such as cages and cavities having dimensions on the micrometric range. On the basis of the proposed model, we also show in which conditions confinement could generate an apparent time dependent probe mobility, thus allowing a proper interpretation of the transport process taking place in submicrometric compartments.     

Superlocalization of single molecules and nanoparticles in high-fidelity optical imaging microfluidic devices

Superlocalization of single molecules and nanoparticles with a precision of subnanometer to a few tens of nanometers is crucial for elucidating nanoscale structures and movements in biological and chemical systems. A novel design of ultraflat and ultrathin glass/polydimethylsiloxane (PDMS) hybrid microdevices is introduced to provide almost uncompromised optical imaging quality for on-chip superlocalization and super-resolution imaging of single molecules and nanoparticles under a variety of microscopy modes. The performance of the high-fidelity (Hi-Fi) optical imaging microfluidic device was validated by precisely mapping micronecklaces made of fluorescent microtubules and 40 nm gold nanoparticles and by demonstrating the activation and excitation cycles of single Alexa Fluor 647 dyes for direct stochastic optical reconstruction microscopy in PDMS-based microchannels for the first time. Furthermore, the microdevice's feasibility for multimodality microscopy imaging was demonstrated by a vertical scan of live cells in epi-fluorescence and differential interference contrast (DIC) microscopy modes simultaneously.     

Fluorescence correlation spectroscopy: a review of biochemical and microfluidic applications

Over the years fluorescence correlation spectroscopy (FCS) has proven to be a useful technique that has been utilized in several fields of study. Although FCS initially suffered from poor signal-to-noise ratios, the incorporation of confocal microscopy has overcome this drawback and transformed FCS into a sensitive technique with high figures of merit. In addition, tandem methods have evolved to include dual-color cross-correlation, total internal reflection fluorescence correlation, and fluorescence lifetime correlation spectroscopy combined with time-correlated single-photon counting. In this review, we discuss several applications of FSC for biochemical, microfluidic, and cellular investigations.     

Afterpulsing and its correction in fluorescence correlation spectroscopy experiments

Afterpulsing arises from feedback in a photon detector. This means that each real signal pulse can be followed by an afterpulse at a later time. This effect is particularly troubling in photon correlation experiments. Few treatments of this effect have appeared in the literature, and few software programs to solve the problem have been written. We demonstrate the afterpulsing effect in fluorescence correlation spectroscopy by using different avalanche photodiodes. We prove theoretically that under simple and reasonable conditions afterpulsing in autocorrelation can be eliminated to the leading order; we have found it easy to program software for the correction. We compare our results with those from cross correlation. We also discuss some experimental parameters that may affect the afterpulsing.     

Quantitative kinetic analysis in a microfluidic device using frequency-domain fluorescence lifetime imaging

A novel microfluidic approach for the quantification of reaction kinetics is presented. A three-dimensional finite difference numerical simulation was developed in order to extract quantitative kinetic information from fluorescence lifetime imaging experimental data. This approach was first utilized for the study of a fluorescence quenching reaction within a microchannel; the lifetime of a fluorophore was used to map the diffusion of a quencher across the microchannel. The approach was then applied to a more complex chemical reaction between a fluorescent amine and an acid chloride, via numerical simulation the bimolecular rate constant for this reaction was obtained.     

Disposable microscope objective lenses for fluorescence correlation spectroscopy using latex microspheres

We explore the combination of a latex microsphere with a low NA lens to form a high performance optical system, and enable the detection of single molecules by fluorescence correlation spectroscopy (FCS). Viable FCS experiments at concentrations 1-1000 nM with different objectives costing less than $40 are demonstrated. This offers a simple and low-cost alternative to the conventional complex microscope objectives.     

Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy

Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.     

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.     

Electron multiplying charge-coupled device camera based fluorescence correlation spectroscopy

A fluorescence correlation spectroscopy (FCS) setup is built with an electron multiplying charge-coupled device camera. Although the instrument has a limited time resolution of 4 ms, compared to 0.1-0.2 mus for common instruments using avalanche photodiodes, it allows multiplexing of FCS measurements, has a software-adjustable pinhole after data collection, performs flow speed as well as flow direction measurements in microchannels and could be used to do spectral FCS. Measurements are performed on fluorescent dyes and polystyrene beads in high-viscosity media and on epidermal growth factor receptors in Chinese hamster ovary cells. Using real measurements on single spots, multiplexing of focal spots and detection elements are simulated and the results are discussed.     

Spectrally resolved fluorescence correlation spectroscopy based on global analysis

Multicolor fluorescence correlation spectroscopy has been recently developed to study chemical interactions of multiple chemical species labeled with spectrally distinct fluorophores. In the presence of spectral overlap, there exists a lower detectability limit for reaction products with multicolor fluorophores. In addition, the ability to separate bound product from reactants allows thermodynamic properties such as dissociation constants to be measured for chemical reactions. In this report, we utilize a spectrally resolved two-photon microscope with single-photon counting sensitivity to acquire spectral and temporal information from multiple chemical species. Further, we have developed a global fitting analysis algorithm that simultaneously analyzes all distinct auto- and cross-correlation functions from 15 independent spectral channels. We have demonstrated that the global analysis approach allows the concentration and diffusion coefficients of fluorescent particles to be resolved despite the presence of overlapping emission spectra.     

Surface-reactive acrylic copolymer for fabrication of microfluidic devices

A surface-reactive acrylic polymer, poly(glycidyl methacrylate-co-methyl methacrylate) (PGMAMMA), was synthesized and evaluated for suitability as a substrate for fabrication of microfluidic devices for chemical analysis. This polymer has good thermal and optical properties and is mechanically robust for cutting and hot embossing. A key advantage of this polymeric material is that the surface can be easily modified to control inertness and electroosmotic flow using a variety of chemical procedures. In this work, the procedures for aminolysis, photografting of linear polyacrylamide, and atom-transfer radical polymerization on microchannel surfaces in PGMAMMA substrates were developed, and the performance of resultant microfluidic electrophoresis devices was demonstrated for the separation of amino acids, peptides, and proteins. Separation efficiencies as high as 4.6 x 10(4) plates for a 3.5-cm-long separation channel were obtained. The results indicate that PGMAMMA is an excellent substrate for microfabricated fluidic devices, and a broad range of applications should be possible.     

Chemical reaction imaging within microfluidic devices using confocal raman spectroscopy: the case of water and deuterium oxide as a model system

Microfluidic devices face presently a tremendous interest, especially for the development of labs-on-a-chip systems. One of the primary challenges for such applications is the ability to perform local chemical detection and analysis from various species. In this paper, we investigate the use of confocal Raman spectroscopy from both qualitative and quantitative sides, to obtain spatially resolved concentration maps of chemically reactive fluids flowing in different channels networks. As a model chemical reaction, we used the isotopic exchange reaction between D(2)O and H(2)O, which is diffusion-controlled and whose equilibrium states exhibit distinct Raman signatures depending on the composition. Two types of chip technologies were studied, which are typical of those used for chemical kinetics investigations. In the first one, reagent mixing occurs by molecular interdiffusion of the two streams (H(2)O and D(2)O) flowing side by side in the same channel; in the second one, reagents are hosted in droplets moving in winding channels that enhance the mixing. In the first series of experiments, we were able to extract Raman images of H(2)O, D(2)O, and HOD concentrations in the main channel together with an estimate of an interdiffusion coefficient, and in the second one, we evidenced the influence of channel wiggles on mixing efficiency.     

Micropreparative fraction collection in microfluidic devices

Micropreparative fraction collection following microchip-based electrophoretic analysis of biomolecules is of major importance for a variety of biomedical applications. In this paper, we present a microfabricated device-based fraction collection system. Various size DNA fragments were separated and collected by simply redirecting the desired portions of the detected sample zones to corresponding collection wells using appropriate voltage manipulations. The efficiency of sampling and collection of the fractions was enhanced by placing a cross channel at or downstream of the detection point. Following the detection of the band of interest, the potentials were reconfigured to sampling/collection mode, so that the selected sample zone migrated to the appropriate collection well of the microdevice. The potential distribution assured that the rest of the analyte components in the separation column was retarded, stopped, or reversed, increasing in this way the spacing between the sample zone being collected and the immediately following one. By this means, a precise collection of spatially close consecutive bands could be facilitated. Once the target sample fraction reached the corresponding collection well, the potentials were switched back to separation mode. Alternation of the separation/detection and sampling/collection cycles was repeated until all required sample zones were physically isolated. The integrated device consists of a sample introduction, separation, fraction sampling, and fraction collection compartments. The feasibility of the fraction collection technique was tested on a mixture of dsDNA fragments. The amounts of DNA collected in this way were enough for further downstream sample processing, such as conventional PCR-based analysis.     

Numerical fluorescence correlation spectroscopy for the analysis of molecular dynamics under nonstandard conditions

The suitability of mathematical models used to extract kinetic information from correlated data constitutes a significant issue in fluorescence correlation spectroscopy (FCS). Standard FCS equations are derived from a simple Gaussian approximation of the optical detection volume, but some investigations have suggested this traditional practice can lead to inaccurate and misleading conclusions under many experimental circumstances, particularly those encountered in one-photon confocal measurements. Furthermore, analytical models cannot be derived for all measurement scenarios. We describe a novel numerical approach to FCS that circumvents conventional analytical models, enabling meaningful analyses even under extraordinarily unusual measurement conditions. Numerical fluorescence correlation spectroscopy (NFCS) involves quantitatively matching experimental correlation curves with synthetic curves generated via diffusion simulation or direct calculation based on an experimentally determined 3D map of the detection volume. Model parameters are adjusted iteratively to minimize the residual differences between synthetic and experimental correlation curves. In order to reduce analysis time, we distribute calculations across a network of processors. As an example of this new approach, we demonstrate that synthetic autocorrelation curves correspond well with experimental data and that NFCS diffusion measurements of Rhodamine B remain constant, regardless of the distortion present in a confocal detection volume.