For Fluorescence Correlation Spectroscopy Research Articles

"Flexible hinge" dynamics in mismatched DNA revealed by fluorescence correlation spectroscopy.

Altered unwinding/bending fluctuations at DNA lesion sites are implicated as plausible mechanisms for damage sensing by DNA-repair proteins. These dynamics are expected to occur on similar timescales as one-dimensional (1D) diffusion of proteins on DNA if effective in stalling these proteins as they scan DNA. We examined the flexibility and dynamics of DNA oligomers containing 3 base pair (bp) mismatched sites specifically recognized in vitro by nucleotide excision repair protein Rad4 (yeast ortholog of mammalian XPC). A previous Forster resonance energy transfer (FRET) study mapped DNA conformational distributions with cytosine analog FRET pair primarily sensitive to DNA twisting/unwinding deformations (Chakraborty et al. Nucleic Acids Res. 46: 1240-1255 (2018)). These studies revealed B-DNA conformations for nonspecific (matched) constructs but significant unwinding for mismatched constructs specifically recognized by Rad4, even in the absence of Rad4. The timescales of these unwinding fluctuations, however, remained elusive. Here, we labeled DNA with Atto550/Atto647N FRET dyes suitable for fluorescence correlation spectroscopy (FCS). With these probes, we detected higher FRET in specific, mismatched DNA compared with matched DNA, reaffirming unwinding/bending deformations in mismatched DNA. FCS unveiled the dynamics of these spontaneous deformations at ~ 300µs with no fluctuations detected for matched DNA within the ~ 600ns-10ms FCS time window. These studies are the first to visualize anomalous unwinding/bending fluctuations in mismatched DNA on timescales that overlap with the < 500µs "stepping" times of repair proteins on DNA. Such "flexible hinge" dynamics at lesion sites could arrest a diffusing protein to facilitate damage interrogation and recognition.

Multifocus Fluorescence Correlation Spectroscopy with Spatially Separated Excitation Beams

Abstract We report on a new multifocus optical system developed for fluorescence correlation spectroscopy (FCS). This system utilizes a beamsplitter array to spatially separate excitation beams. Fluorescence from each spot is individually detected with a single-photon avalanche photodiode (SPAD) through a fiber bundle. The combined use of beamsplitters and multiple SPADs is beneficial to give a higher detection uniformity among focal spots than the existing multifocus FCS systems. Applications of this setup show that the variation of the optical parameters among seven focal spots are as small as ∼5% and prove its high performance in FCS measurements. High efficiency of this system is also demonstrated in application to an advanced FCS method, two-dimensional fluorescence lifetime correlation spectroscopy. Along with the results of these applications, we describe a method for proper correction of the crosstalk effect in the multifocus setup.

PJ-1 The HIV-1 antisense protein ASP is a structural protein of the viral envelope

Introduction: The antisense strand of the HIV-1 genome encodes a 189-aa, highly hydrophobic protein (ASP) with no known homologs. Humoral and cellular immune responses against ASP in HIV-1 infected individuals demonstrate its expression invivo. However, whether ASP plays a role in viral replication is still unknown. We studied the expression and subcellular localization of ASP in various chronically infected cell lines. In addition, we investigated the impact of ASP expression on viral fitness. Methods: For intracellular and nuclear stainings, we used BD Cytofix/Cytoperm and eBioscience FoxP3 kits. Flow cytometry data were acquired on a Millipore Guava and analyzed with FlowJo. Confocal and super-resolution microscopy images were acquired on Zeiss LSM 800 and Nikon N-STORMTi-E microscopes, and analyzed with Zen Blue and Nikon Elements Software Version 3.1. For virion-capture assays, antibodies against ASP and gp120 were immobilized on Protein G-dynabeads. For Fluorescence Correlation Spectroscopy (FCS) we used an ISS Q2 confocal microscope and the data were analyzed with ISS VistaVision. Results/Discussion: We generated an in-house monoclonal antibody 324.6 against an epitope located between 2 putative transmembrane domains in the ASP protein. Using this antibody in flow cytometry and confocal microscopy, we found thatASP is expressed in the nuclei of chronically infected myeloid (U1andOM-10.1), and lymphoid (H9-IIIB, H9-CC, H9-MN, H9-RF, ACH2 and 8E5) celllines, but not their parental uninfected lines. However, ASP was not detectable neither on the cell surface or in the cytoplasm of infected cell lines. ASP expression was higher in cells cultured at low density compared to cells cultured at high density, suggesting that the rate of cell division may influence ASP expression levels. Confocal microscopy showed a polarized nuclear distribution of ASP, preferentially in areas of the nucleus containing actively transcribed chromatin. However, after reactivation of HIV-1 expression with PMA, we found that ASP translocated to the cytoplasm and to the cell membrane of all infected cell lines. In addition, the ability to detect ASP on the cell surface without permeabilization indicates that the ASP epitope recognized by324.6 antibody is extracellular. Confocal microscopy showed that ASP and gp120 co-localize on the cell surface of productively infected cells, and we determined that the Manders overlap coefficient is 78%. Super-resolution microscopy analyses provided further evidence of ASP and gp-120 co-localization. These results suggest that ASP might be incorporated in the membrane of budding virions upon their release from infected cells. Indeed, the anti-ASP 324.6 antibody was able to capture HIV-1 particles with an efficiency similar to the anti-gp120 VRC01 antibody. Moreover, FCS showed that the binding efficiency of 324.6 antibody to single virions in solution was ∼28%. Altogether, these 2 assays demonstrate the presence of ASP on the surface of mature HIV-1 particles released from infected cells. To investigate whether ASP expression impacts viral replication, we mutated single nucleotides in the HIV-1NL4-3 genome thus introducing early stop codons in the ASP open reading frame without affecting the amino acid sequence of Env encoded on the opposite strand. We found that these ASP-deficient viruses displayed a ∼50% reduction in replication rate compared to wildtype virus. Conclusions: Our results indicate that ASP is expressed in the nuclei of non-productively infected cell lines. Upon viral reactivation, ASP translocates to the surface where it co-localizes with gp120. Moreover, ASP is present on the membrane of viral particles, indicating that ASP is a structural protein in the envelope of mature HIV-1 virions. Further, ASP expression promotes viral replication. Overall, our results suggest that ASP may represent a new target for therapeutic or preventive vaccine.

Temperature dependent FCS studies using a long working distance objective: Viscosities of supercooled liquids and particle size.

In this work, we describe new experimental setups for Fluorescence Correlation Spectroscopy (FCS) where a long working distance objective is used. Using these setups, FCS measurements in a broad temperature range for a small sample volume of about 50 μl can be performed. The use of specially designed cells and a dry long working distance objective was essential for avoiding temperature gradients in the sample. The performance of the new setups and a traditional FCS setup with immersion objectives is compared. The FCS data in combination with the Stokes-Einstein (SE) relation were used to obtain the values of the nanoviscosity of a fluid. We show for selected molecular van der Waals supercooled liquids that despite the fact that in these systems, a characteristic length scale can be defined, the nanoviscosity obtained from FCS is in a very good agreement with the macroscopic (rheometric) viscosity of the sample in a broad temperature range. This result corroborates the applicability of the SE relation to supercooled liquids at temperatures above 1.2 Tg. We also show that the temperature dependent size of thermoresponsive microgel particles can be determined by FCS using the designed cells and a long working distance objective in a broader size range without a need to use the correction procedure since the size correction is proportional to the square of the ratio of the hydrodynamic radius to the confocal volume size.

Interaction of the signaling state analog and the apoprotein form of the orange carotenoid protein with the fluorescence recovery protein.

Photoprotection in cyanobacteria relies on the interplay between the orange carotenoid protein (OCP) and the fluorescence recovery protein (FRP) in a process termed non-photochemical quenching, NPQ. Illumination with blue-green light converts OCP from the basic orange state (OCPO) into the red-shifted, active state (OCPR) that quenches phycobilisome (PBs) fluorescence to avoid excessive energy flow to the photosynthetic reaction centers. Upon binding of FRP, OCPR is converted to OCPO and dissociates from PBs; however, the mode and site of OCPR/FRP interactions remain elusive. Recently, we have introduced the purple OCPW288A mutant as a competent model for the signaling state OCPR (Sluchanko et al., Biochim Biophys Acta 1858:1-11, 2017). Here, we have utilized fluorescence labeling of OCP at its native cysteine residues to generate fluorescent OCP proteins for fluorescence correlation spectroscopy (FCS). Our results show that OCPW288A has a 1.6(±0.4)-fold larger hydrodynamic radius than OCPO, supporting the hypothesis of domain separation upon OCP photoactivation. Whereas the addition of FRP did not change the diffusion behavior of OCPO, a substantial compaction of the OCPW288A mutant and of the OCP apoprotein was observed. These results show that sufficiently stable complexes between FRP and OCPW288A or the OCP apoprotein are formed to be detected by FCS. 1:1 complex formation with a micromolar apparent dissociation constant between OCP apoprotein and FRP was confirmed by size-exclusion chromatography. Beyond the established OCP/FRP interaction underlying NPQ cessation, the OCP apoprotein/FRP interaction suggests a more general role of FRP as a scaffold protein for OCP maturation.

Measuring membrane association and protein diffusion within membranes with supercritical angle fluorescence microscopy.

Supercritical angle fluorescence (SAF) detection combines the axial discrimination and exquisite signal-to-noise ratio of total internal reflection fluorescence (TIRF) with the lateral discrimination and convenience of confocal excitation. This combination makes SAF ideal for fluorescence correlation spectroscopy (FCS) on membranes and other structures in close proximity to the coverslip. Here we report a straightforward modification of a commercial microscope to implement SAF FCS and demonstrate in both model supported lipid bilayers and cellular systems that this approach shows an increase in signal from membrane-bound fluorophores relative to fluorophores in solution, benchmarked against line-scanning FCS. SAF FCS allowed us to demonstrate that activation of the T cell receptor resulted in the recruitment of the kinase Lck to the plasma membrane as well as a reduction in Lck mobility within the membrane.

Self-Calibrated Line-Scan STED-FCS to Quantify Lipid Dynamics in Model and Cell Membranes

Only a limited number of noninvasive techniques are available to directly measure the dynamic behavior of lipids in model and cell membranes. Here, we explored whether a commercial instrument could be used for fluorescence correlation spectroscopy (FCS) under pulsed stimulated emission depletion (STED). To overcome issues with photobleaching and poor distinction between confocal and STED signals, we implemented resonant line-scan STED with filtered FCS, which has the additional benefit of autocalibrating the dimensions of the point-spread function and obtaining spatially resolved molecular mobility at subdiffraction resolution. With supported lipid bilayers, we achieved a detection spot radius of 40 nm, although at the expense of decreased molecular brightness. We also used this approach to map the dynamics of Atto646N-labeled sphingomyelin and phosphatidylethanolamine in the plasma membrane. Despite the reliability of the method and the demonstration that photobleaching and the photophysical properties of the dye did not influence diffusion measurements, we found great heterogeneities even within one cell. For both lipids, regions of high local density correlated with slow molecular diffusion, indicating trapping of Atto646N-labeled lipids. Future studies with new dyes are needed to reveal the origin of the trapping.

Strengths and Weaknesses of Recently Engineered Red Fluorescent Proteins Evaluated in Live Cells Using Fluorescence Correlation Spectroscopy

The scientific community is still looking for a bright, stable red fluorescent protein (FP) as functional as the current best derivatives of green fluorescent protein (GFP). The red FPs exploit the reduced background of cells imaged in the red region of the visible spectrum, but photophysical short comings have limited their use for some spectroscopic approaches. Introduced nearly a decade ago, mCherry remains the most often used red FP for fluorescence correlation spectroscopy (FCS) and other single molecule techniques, despite the advent of many newer red FPs. All red FPs suffer from complex photophysics involving reversible conversions to a dark state (flickering), a property that results in fairly low red FP quantum yields and potential interference with spectroscopic analyses including FCS. The current report describes assays developed to determine the best working conditions for, and to uncover the shortcoming of, four recently engineered red FPs for use in FCS and other diffusion and spectroscopic studies. All five red FPs assayed had potential shortcomings leading to the conclusion that the current best red FP for FCS is still mCherry. The assays developed here aim to enable the rapid evaluation of new red FPs and their smooth adaptation to live cell spectroscopic microscopy and nanoscopy.

Single Cancer Cell Detection by Near Infrared Microspectroscopy, Infrared Chemical Imaging and Fluorescence Microspectroscopy

AbstractNovel techniques are currently being developed and established for the accurate chemical analysis and detection of single cancer cells, single embryos and single seeds by Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fourier Transform Infrared (FT-IR), Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1micron resolution are here reported. 400 and 500 MHz, H-1 NMR analyses were carried out that allowed the selection of mutagenized embryos. Detailed chemical analyses are being demonstrated to be also possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cancer cells. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in Functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allowed not only single molecule detection, but also molecular dynamics observations and high resolution, submicron imaging of sub-femtoliter volumes inside living cells with 0.25 micron spatial resolution, in both normal and cancer cells, as well as neoplastic tissues. These novel, ultra-sensitive and rapid FT-NIR/FCS analyses have, therefore, substantial potential for numerous applications in important research areas, such as: medicine, medical/cancer research, pharmacology, agricultural biotechnology, food safety, as well as clinical diagnosis of viral diseases and cancers.

Near Infrared Microspectroscopy, Fluorescence Microspectroscopy, Infrared Chemical Imaging and High-Resolution Nuclear Magnetic Resonance Analysis of Soybean Seeds, Embryos and Single Cells

AbstractChemical analysis of soybean seeds, somatic embryos and single cells were carried out by Fourier Transform Infrared (FT-IR), Fourier Transform Near Infrared (FT-NIR) Microspectroscopy, Fluorescence and High-Resolution NMR (HR-NMR). The first FT-NIR chemical images of biological systems approaching 1 micron (1μ) resolution are presented here. Chemical images obtained by FT-NIR and FT-IR Microspectroscopy are presented for oil in soybean seeds and somatic embryos under physiological conditions. FT-NIR spectra of oil and proteins were obtained for volumes as small as 2μ3. Related, HR-NMR analyses of oil contents in somatic embryos are also presented here with nanoliter precision. Such 400 MHz 1H NMR analyses allowed the selection of mutagenized embryos with higher oil content (e.g. ~20%) compared to non-mutagenized control embryos. Moreover, developmental changes in single soybean seeds and/or somatic embryos may be monitored by FT-NIR with a precision approaching the picogram level. Indeed, detailed chemical analyses of oils and phytochemicals are now becoming possible by FT-NIR Chemical Imaging/ Microspectroscopy of single cells. The cost, speed and analytical requirements of plant breeding and genetic selection programs are fully satisfied by FT-NIR spectroscopy and Microspectroscopy for soybeans and soybean embryos. FT-NIR Microspectroscopy and Chemical Imaging are also shown to be potentially important in functional Genomics and Proteomics research through the rapid and accurate detection of high-content microarrays (HCMA). Multi-photon (MP), pulsed femtosecond laser NIR Fluorescence Excitation techniques were shown to be capable of Single Molecule Detection (SMD). Therefore, such powerful techniques allow for the most sensitive and reliable quantitative analyses to be carried out both in vitro and in vivo. Thus, MP NIR excitation for Fluorescence Correlation Spectroscopy (FCS) allows not only single molecule detection, but also molecular dynamics and high resolution, submicron imaging of femtoliter volumes inside living cells and tissues. These novel, ultra-sensitive and rapid NIR/FCS analyses have numerous applications in important research areas, such as: agricultural biotechnology, food safety, pharmacology, medical research and clinical diagnosis of viral diseases and cancers.

New cholesterol-specific antibodies remodel HIV-1 target cells’ surface and inhibit their in vitro virus production

The importance of membrane rafts in HIV-1 infection is still in the focus of interest. Here, we report that new monoclonal anticholesterol IgG antibodies (ACHAs), recognizing clustered membrane cholesterol (e.g., in lipid rafts), rearrange the lateral molecular organization of HIV-1 receptors and coreceptors in the plasma membrane of HIV-1 permissive human T-cells and macrophages. This remodeling is accompanied with a substantial inhibition of their infection and HIV-1 production in vitro. ACHAs promote the association of CXCR4 with both CD4 and lipid rafts, consistent with the decreased lateral mobility of CXCR4, while Fab fragments of ACHAs do not show these effects. ACHAs do not directly mask the extracellular domains of either CD4 or CXCR4 nor do they affect CXCR4 internalization. No significant inhibition of HIV production is seen when the virus is preincubated with the antibodies prior to infection. Thus, we propose that the observed inhibition is mainly due to the membrane remodeling induced by cholesterol-specific antibodies on the target cells. This, in turn, may prevent the proper spatio-temporal juxtaposition of HIV-1 glycoproteins with CD4 and chemokine receptors, thus negatively interfering with virus attachment/entry.

Near-field optical probes provide subdiffraction-limited excitation areas for fluorescence correlation spectroscopy on membranes

Abstract Near-field optical probes have been used to produce a subdiffraction-limited observation area for fluorescence correlation spectroscopy (FCS) experiments on supported membranes. The design of a bent, etched fiber probe that is compatible with biological imaging in an aqueous environment is described. This probe design is used for proof of principle experiments to measure lipid diffusion in a fluid-supported bilayer. A reduction in excitation area of approximately one order of magnitude (relative to a confocal FCS experiment) is obtained with a probe aperture diameter of 140 nm. We also demonstrate a simple approach for modeling the autocorrelation decay due to diffusion within the excitation profile at the near-field scanning optical microscopy (NSOM) probe aperture. The use of probes with smaller apertures is expected to provide an additional order of magnitude reduction in the observation area, thus enabling the study of cellular membranes with higher concentrations of fluorophores than is currently possible with diffraction-limited techniques.

Near-Field Fluorescence Correlation Spectroscopy Using a Fiber Probe

We have developed a new technique for fluorescence correlation spectroscopy (FCS) using an optical fiber probe, as is employed in near-field scanning optical microscopy under the illumination-collection hybrid mode. A small observation volume beyond the diffraction limit makes it possible to apply FCS to concentrated solutions with high spatial resolution. Owing to the large transmission efficiency of the probe, correlation traces with an adequate signal-to-noise ratio resulted. We performed FCS measurements for fluorescent particles to demonstrate the validity of the method.

Supercritical Angle Fluorescence Correlation Spectroscopy

We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.

Fluorescence correlation spectroscopy at 100 nM concentrations using near‐field scanning optical microscopic (NSOM) geometries and highly diffracting force sensing fiber probes

AbstractA tapered cantilevered optical fiber probe is introduced for fluorescence correlation spectroscopy (FCS). The probe has an aperture that is coated with metal and its dimension can be very well defined. Its design is based on cantilevered near‐field optical fiber probes that allow for on‐line atomic force feedback. This permits unprecedented stability in FCS measurements. Even such probes that have large diameters are below the cutoff wavelength of light propagation in optical fibers. Such large diameter probes produce bright spots of light that are highly diffracting. Therefore, only the fluence of light very close to the surface of the glass probe aperture is sufficient to exhibit a high probability of fluorescence excitation. From the results presented in this paper the z extent of such probes seems comparable to what can be obtained from prism‐based evanescent wave methods, but with much more flexibility and including force sensing capability. In agreement with theoretical results on prism evanescent field‐based excitation, it is shown that even with large xy dimensions for the fiber probes in this paper, fluorescence correlation spectra from 100 nM fluorophore concentrations can be recorded. This is 10 times larger than the concentrations generally required for conventional confocal FCS. The results suggest that such an approach has considerable potential for applications of FCS in membrane, near‐membrane, solution, and even within‐cell environments. The latter is achieved through atomic force feedback controlled penetration of tapered cantilevered metal‐coated glass fibers into cells, and this is also reported in this paper.

Strategies to Improve Photostabilities in Ultrasensitive Fluorescence Spectroscopy

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy.

Both Rotor and Stator Subunits Are Necessary for Efficient Binding of F1 to F0 in Functionally Assembled Escherichia coli ATP Synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.

Four-color fluorescence correlation spectroscopy realized in a grating-based detection platform

We have developed a filterless multicolor detection unit for fluorescence correlation spectroscopy (FCS). This grating-based setup is continuously tunable for multicolor separation and is thus a powerful alternative to the classical cascade of dichroic mirrors and filters. Our tailored platform allows for accommodation of up to 15 detection channels covering the entire visible spectral range. As a proof of principle, we successfully demonstrate simultaneous FCS of four distinct fluorescent quantum dot species being mixed in aqueous solution. Grating-based detection allows for spectral high-resolution FCS in a stable and compact setup and is a feasible tool for quantitative investigation of complexbiomolecular dynamics on a single molecule level.

A new approach for fluorescence correlation spectroscopy (FCS) based immunoassays

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring physicochemical properties, such as concentration and diffusion constant, of bio-molecules in complex mixtures. Although, as such, FCS is well suited for development of homogeneous immunoassays, a major obstacle lies in the relatively high molecular weight of antibodies. This is because in FCS discrimination between unbound fluorescently-labelled antibodies and the same antibodies bound to immune complexes is based on the difference of their respective diffusion coefficients. To overcome this limitation we here propose to use a fluorescently-labelled tag which has two crucial properties: (a) its molecular weight is significantly lower than that of an antibody and (b) it is capable to discriminate between free antibodies and immune complexes. We have evaluated the feasibility of this approach in a model system consisting of mouse monoclonal IgG directed against the Lewis X antigen, and Protein A as a low molecular weight tag.

High-Pressure Fluorescence Correlation Spectroscopy

We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300MPa. The fluctuation amplitude and the diffusion coefficient show a small pressure dependence. The changes of these parameters, which are on the order of 10%, are due to the pressure changes of the viscosity and the density of the aqueous medium.