Cross talk free fluorescence cross correlation spectroscopy in live cells

Fluorescence correlation spectroscopy (FCS) is now a widely used technique to measure small ensembles of labeled biomolecules with single molecule detection sensitivity (e.g., low endogenous concentrations). Fluorescence cross correlation spectroscopy (FCCS) is a derivative of this technique that detects the synchronous movement of two biomolecules with different fluorescence labels. Both methods can be applied to live cells and, therefore, can be used to address a variety of unsolved questions in cell biology. Applications of FCCS with autofluorescent proteins (AFPs) have been hampered so far by cross talk between the detector channels due to the large spectral overlap of the fluorophores. Here we present a new method that combines advantages of these techniques to analyze binding behavior of proteins in live cells. To achieve this, we have used dual color excitation of a common pair of AFPs, ECFP and EYFP, being discriminated in excitation rather than in emission. This is made possible by pulsed excitation and detection on a shorter timescale compared to the average residence time of particles in the FCS volume element. By this technique we were able to eliminate cross talk in the detector channels and obtain an undisturbed cross correlation signal. The setup was tested with ECFP/EYFP lysates as well as chimeras as negative and positive controls and demonstrated to work in live HeLa cells coexpressing the two fusion proteins ECFP-connexin and EYFP-connexin.     

Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion

A characteristic feature of prion diseases such as bovine spongiform encephalopathy (BSE) is the accumulation of a pathological isoform of the host-encoded prion protein, PrP. In contrast to its cellular isoform PrP(C), the pathological isoform PrP(Sc) forms insoluble aggregates. All commercial BSE tests currently used for routine testing are based on the proteinase K (PK) resistance of PrP, but not all pathological PrP is PK-resistant. In the present study, single prion particles were counted by fluorescence correlation spectroscopy (FCS). The property of PK resistance is not required, i.e., both the PK-resistant and the PK-sensitive parts of the prion particles are detectable. PrP aggregates were prepared from the brains of BSE-infected cattle, as well as from scrapie-infected hamsters, by the NaPTA precipitation method without PK digestion. They were labeled using two different PrP-specific antibodies for FCS measurements in the dual-color mode (2D-FIDA). Within the limited number of samples tested, BSE-infected cattle and scrapie-infected hamsters in the clinical stage of the disease could be distinguished with 100% specificity from a control group. Thus, a diagnostic tool for BSE detection with complete avoidance of PK treatment is presented, which should have particular advantages for testing animals in the preclinical stage.     

A protease inhibitor discovery method using fluorescence correlation spectroscopy with position-specific labeled protein substrates

We developed novel substrates for protease activity evaluation by fluorescence correlation spectroscopy (FCS). Substrates were labeled in a position-specific manner with a fluorophore near the N terminus and included a C-terminal, 30 kDa, highly soluble protein (elongation factor Ts [EF-Ts]). The C-terminal protein enhanced the substrate peptide solubility and increased the molecular weight, enabling sensitive detection by FCS. Using the labeled substrates, caspase-3 and matrix metalloproteinase-9 (MMP-9) activities were confirmed by FCS. To demonstrate the suitability of this FCS-based assay for high-throughput screening, we screened various chemical compounds for MMP-9 inhibitors. The screening results confirmed the inhibitory activity of one compound and also revealed another potential MMP-9 inhibitor. Thus, this combination of position-specific labeled protein substrates and FCS may serve as a useful tool for evaluating activities of various proteases and for protease inhibitor screening.     

Unveiling mRNP composition by fluorescence correlation and cross-correlation spectroscopy using cell lysates

Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic treatments. Moreover, FCCS employing minute amounts of cells closely corroborated previously reported RNA dependent interactions and provided estimates of the relative overlap between factors in the mRNPs, thus depicting their heterogeneity. The described lysate-based FCS and FCCS analysis may not only complement current biochemical approaches but also provide novel opportunities for the quantitative analysis of the molecular composition and dynamics of single mRNPs.     

Detection of ligand-induced CNTF receptor dimers in living cells by fluorescence cross correlation spectroscopy

Ciliary neurotrophic factor (CNTF) signals via a receptor complex consisting of the specific CNTF receptor (CNTFR) and two promiscuous signal transducers, gp130 and leukemia inhibitory factor receptor (LIFR). Whereas earlier studies suggested that the signaling complex is a hexamer, more recent analyses strongly support a tetrameric structure. However, all studies so far analyzed the stoichiometry of the CNTF receptor complex in vitro and not in the context of living cells. We generated and expressed in mammalian cells acyl carrier protein-tagged versions of both CNTF and CNTFR. After labeling CNTF and CNTFR with different dyes we analyzed their diffusion behavior at the cell surface. Fluorescence (cross) correlation spectroscopy (FCS/FCCS) measurements reveal that CNTFR diffuses with a diffusion constant of about 2 × 10^− 9 cm² s^− 1 independent of whether CNTF is bound or not. FCS and FCCS measurements detect the formation of receptor complexes containing at least two CNTFs and CNTFRs. In addition, we measured Förster-type fluorescence resonance energy transfer between two differently labeled CNTFs within a receptor complex indicating a distance of 5-7 nm between the two. These findings are not consistent with a tetrameric structure of the CNTFR complex suggesting that either hexamers and or even higher-order structures (e.g. an octamer containing two tetramers) are formed.     

DNA length evaluation using cyanine dye and fluorescence correlation spectroscopy

To develop a high-performance method for measuring the length of double-stranded DNA (dsDNA) fragments, the capability of fluorescence correlation spectroscopy (FCS) was examined. To omit troublesome and time-consuming labeling operations such as PCR with fluorescently labeled mononucleotides or primers, intercalation of dimeric cyanine dye YOYO-1 iodide (YOYO) to dsDNA was utilized as a simple labeling method. Various lengths of dsDNA fragments were prepared and mixed with YOYO prior to FCS, and the dependence of the diffusion time of a dsDNA-YOYO complex on the length of dsDNA fragment and the dsDNA/YOYO ratio was investigated. It was successfully demonstrated that the dsDNA length can be measured using YOYO and FCS, and the calibration curve was developed taking into account the rewinding and expansion of the dsDNA fragment caused by YOYO intercalation.     

Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.     

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.     

Detection of specific DNA sequences using dual-color two-photon fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a biomedical research tool. FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. They can also serve as a diagnostic, detecting particular molecular species with high sensitivity and specificity. We here demonstrate that dual-color FCS measurements can be applied to detect and quantify the concentration of specific non-fluorescent molecular species without requiring any modifications to the molecule of interest. We demonstrate this capability by applying dual-color two-photon fluorescence cross-correlation spectroscopy to detect single stranded gamma tubulin DNA in solution with high sensitivity. This quantification is independent of molecular size, and the methods introduced can be extended to measurements in complex environments such as within living cells.     

Simultaneous membrane interaction of amphipathic peptide monomers,self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane.     

Accessing molecular dynamics in cells by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuations in the fluorescence emission of small molecular ensembles, thus providing information about a multitude of parameters, such as concentrations, molecular mobility and dynamics of fluorescently labeled molecules. Performed within diffraction-limited confocal volume elements, FCS provides an attractive alternative to photobleaching recovery methods for determining intracellular mobility parameters of very low quantities of fluorophores. Due to its high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescence flickering. Furthermore, if molecular mobility is to be probed, nonspecific interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as certain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likely to nonspecifically interact with cellular substructures.     

Cross-talk-free fluorescence cross-correlation spectroscopy by the switching method

Fluorescence cross-correlation spectroscopy (FCCS) is used as a powerful technique to analyze molecular interactions both in vitro and in vivo. This method basically requires two laser excitations for two target molecules labeled with fluorophores of different colors. Their coincidence in a microscopic detection volume is analyzed using two detectors. Any overlap of emission spectra of the two fluorophores, however, gives rise to false-positive data about their interaction. To overcome this problem, we have developed a new FCCS system, in which two excitation lasers are switched alternately by modulation using an acousto-optic tunable filter (AOTF). In this report, we demonstrate the feasibility of switching FCCS for enzymatic cleavage of proteins in living cells. A fusion protein of two fluorophores (EGFP and mRFP) with a cleavage site of caspase-3 inserted was expressed in HeLa cells, and proteolysis assay was performed during apoptotic cell death. Due to the absence of cross-talk signals, the FCCS measurement with the switching function gave a large change in relative cross-correlation amplitude after protein cleavage. Hence, switching FCCS enables more reliable measurement of molecular interactions than conventional FCCS.     

Detection of oxidative stress-induced mitochondrial DNA damage using fluorescence correlation spectroscopy

Using fluorescence correlation spectroscopy (FCS), we tested the feasibility of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small volume. The complete mtDNA genome was amplified by long polymerase chain reaction (LPCR), and the product was fluorescently labeled with an intercalating dye, YOYO-1. The fluorescence autocorrelation function was analyzed using a simple two-component model with the diffusion time of 0.21 ms for the LPCR primer and 18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased significantly. In contrast, the fraction of the nuclear-encoded beta-globin LPCR product remained unchanged. The analysis time of FCS measurement was very short (5 min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the rapid detection of the vulnerability of mtDNA to oxidative stress within a small volume element at the subfemtoliter level in solution. These results suggest that the LPCR-FCS method can be used for epidemiological studies of diseases caused by mtDNA damage.     

Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection

Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative intensities of multiple fluorescent species in FCS measurements and, thus, the amplitude of the intensity autocorrelation function. Here we demonstrate this strategy using lifetime gating in FCS experiments. Using pulsed laser excitation and laser-synchronized gating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating technique are presented: suppression of background fluorescence, simplification of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for measuring forward and backward rates of protein fluctuations in equilibrium and for distinguishing between static and dynamic heterogeneity makes it a promising tool in the investigation of chemical reactions and conformational fluctuations in biomolecules.     

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between preformed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform.     

Distinct immunohistochemical localization in Kuru plaques using novel anti-prion protein antibodies

By immunizing Prnp-knockout mice with synthetic polypeptides, a panel of mAbs directed to bovine PrP(C) was obtained. The mAb panel was characterized by the ELISA method, where synthetic polypeptides were used for epitope mapping. Different reactivity patterns were identified. The ability of these mAbs to detect abnormal PrP(Sc) in CJD cases was studied by immunohistochemistry. All mAbs were tested for PrP(Sc) in murine, bovine, monkey and human brain tissues. Three mAbs recognized the fragmented PrP epitope in our ELISA. Antibody 1D12 was strongly reactive to ovine and squirrel monkey tissues infected with a scrapie agent, although non-reactive to scrapie-infected mouse tissues. Antibody 2D8 was clearly reactive to type-2 but not type-1 CJD human tissues. Of particular interest was the reactivity of mAb 6C4 with the inner structure of Kuru plaques (peripheral pattern) in a type-2 CJD case and mAb T2, 1D12, 2B11, 2D8, 4B5 and 6G3-2 with the central area (central pattern). The fact that different anti-PrP mAbs possess distinct staining properties suggests that the PrP(c) to PrP(Sc) conversion might involve a multiple-step process.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

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.     

Specific detection of prion antigenic determinants retained in bovine meat and bone meal by flow microbead immunoassay

AIMS: The purpose of this study was to develop an effective method for detecting prion (PrP) antigenic determinants remaining in bovine meat and bone meal (MBM) using pressurized fluid extraction (PSE) equipment and flow microbead immunoassay (FMI). METHODS AND RESULTS: Using the FMI, bovine recombinant PrP could be determined quantitatively in the 7 pmol-7 nmol range using anti-PrP peptide polyclonal antibody-coupled microbeads and anti-PrP monoclonal antibody (SAF61) as a detection antibody. PSE extraction at 120 degrees C for 5 min under high pressure was most effective for eluting PrP determinants from bovine MBMs. The FMI was capable of detecting PrP determinants in bovine MBM extracts with high specificity and indicated that the MBMs contained high levels of PrP determinants. This assay was also applied to the detection of PrP(Sc) determinants in bovine MBM spiked with a scrapie-infected brain at a weight ratio of 50 : 1. CONCLUSIONS: These data indicate that this assay was effective for the specific detection of PrP determinants contained in bovine MBM extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report detailing the detection of PrP determinants in bovine MBM. The assay could be applied to securing the safety of bovine MBM.