Orientation of cyanine fluorophores terminally attached to DNA via long, flexible tethers

Cyanine fluorophores are commonly used in single-molecule FRET experiments with nucleic acids. We have previously shown that indocarbocyanine fluorophores attached to the 5′-termini of DNA and RNA via three-carbon atom linkers stack on the ends of the helix, orienting their transition moments. We now investigate the orientation of sulfoindocarbocyanine fluorophores tethered to the 5′-termini of DNA via 13-atom linkers. Fluorescence lifetime measurements of sulfoindocarbocyanine 3 attached to double-stranded DNA indicate that the fluorophore is extensively stacked onto the terminal basepair at 15°C, with properties that depend on the terminal sequence. In single molecules of duplex DNA, FRET efficiency between sulfoindocarbocyanine 3 and 5 attached in this manner is modulated with helix length, indicative of fluorophore orientation and consistent with stacked fluorophores that can undergo lateral motion. We conclude that terminal stacking is an intrinsic property of the cyanine fluorophores irrespective of the length of the tether and the presence or absence of sulfonyl groups. However, compared to short-tether indocarbocyanine, the mean rotational relationship between the two fluorophores is changed by ∼60° for the long-tether sulfoindocarbocyanine fluorophores. This is consistent with the transition moments becoming approximately aligned with the long axis of the terminal basepair for the long-linker species.     

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.     

Surface plasmon resonance imaging on a microchip for detection of DNA-modified gold nanoparticles deposited onto the surface in a non-cross-linking configuration

We report theoretical predictions and experimental observations of the reduced detection volume with the use of surface-plasmon-coupled emission (SPCE). The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With direct excitation of the sample (reverse Kretschmann excitation) the detection volume is restricted only by the distance-dependent coupling of the excitation to the surface plasmons. However, with the excitation through the glass prism at surface plasmon resonance angle (Kretschmann configuration), the detection volume is a product of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10nm). The height of the detected volume size is 40-70nm, depending on the orientation of the excited dipoles. We show that, by using the Kretschmann configuration in a microscope with a high-numerical-aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2attoL. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be used to study the small conformational changes of macromolecules.     

Simulation of structure, orientation, and energy transfer between AlexaFluor molecules attached to MscL

Measurements of time-resolved fluorescence anisotropy and fluorescence resonance energy transfer are finding many applications in the study of biological macromolecules as they enable structural properties of the host molecules to be determined in their natural environment. A difficulty in interpreting these experiments is that they both require knowledge of the relative orientation of the fluorophores, a property that is almost impossible to measure. Here we conduct simulations of AlexaFluor488 and AlexaFluor568 attached to two sites on the membrane channel MscL to provide an alternative mechanism for determining the likely configurations and orientational freedom of the fluorophores, as well as the most likely value of the orientation factor κ² for energy transfer between them. The fluorophores are relatively mobile, and are found to be more so when immersed in bulk water than when they interact with the lipid membrane. The fluorophores never insert deeply into the lipid, despite their hydrophobic linkers and aromatic headgroup structures. Properties such as the fluorescence anisotropy decay can be predicted from simulations of the fluorophores in bulk water that closely match experimental data. In contrast, when the fluorophores were attached to the large MscL protein it was difficult to sample all the possible configurations of the fluorophores due to the computational time required. While this approach is likely to provide useful data on solvent-accessible fluorophores attached to small proteins, simulations lasting >50 ns or the use of biasing forces are required to accurately predict orientation factors for use in energy transfer experiments on larger membrane-bound proteins.     

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07–0.2 (on a scale of 0–1). Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.     

Polarization sensing of fluorophores in tissues for drug compliance monitoring.

We used a new method, polarization sensing, to monitor the concentration of the fluorophore rhodamine 800 in an intralipid suspension and in chicken tissue. Rhodamine 800 (Rh800) could be excited at 648 nm using a laser pointer. We developed a simple device for measuring the combined emission from a highly polarized reference film and the unpolarized or orthogonally polarized emission of Rh800 from the scattering intralipid or tissue. The concentration of Rh800 in this medium was revealed by large changes in the polarization (P) with values of P ranging from 0.8 to -0.9. It is possible to vary the sensitive Rh800 concentration range by variation of the detected emission wavelengths, orientation of the excitation polarizer, or fluorophore concentration in the reference film. Polarization sensing of fluorophores in tissue requires only steady-state detection, and can be accomplished with simple and/or portable electronics. Such devices may find use in electronic detection of ingested medicines based on transdermal detection of nontoxic long-wavelength fluorophores.     

A flexible approach to the calculation of resonance energy transfer efficiency between multiple donors and acceptors in complex geometries

Resonance energy transfer provides a practical way to measure distances in the range of 10-100 A between sites in biological molecules. Although the relationship between the efficiency of energy transfer and the distance between sites is well described for a single pair of fluorophores, the situation is more difficult when more than two fluorophores are present. Using a Monte Carlo calculation scheme, we demonstrate how resonance energy transfer can be used to measure distances between fluorophores in complex geometries. We demonstrate the versatility of the approach by calculating the efficiency of energy transfer for individual fluorophores randomly distributed in two and three dimensions, for linked pairs of donors and acceptors and pentameric structures of five linked fluorophores. This approach can be used to relate the efficiency of energy transfer to the distances between fluorophores, R0, molecular concentrations, laser power, and donor/acceptor ratios in ensembles of molecules or when many fluorophores are attached to a single molecule such as in multimeric proteins.     

Optical properties of Alexa 488 and Cy5 immobilized on a glass surface

The absorption and emission spectra were measured for Cy5 and Alexa 488 fluorophores confined on a glass surface. The data were obtained using fluorometry and spectroscopic ellipsometry. Red shifts of the surface-immobilized fluorophore absorption spectra relative to the fluorophore spectra in aqueous solution were observed using both methods. We interpret these red shifts in terms of a change in the polarizability and polarity of the effective solvent. A formula is given that can be used to estimate expected shifts in absorption and emission maxima for surface-immobilized fluorophores. Spectroscopic ellipsometry measurements provide identification of the fluorophores confined on a glass surface. These results suggest that the design of microarray detection systems should be based on the optical properties of fluorophores attached to the surface and not on the optical properties of fluorophores in solution.     

Radiative decay engineering. 2. Effects of Silver Island films on fluorescence intensity, lifetimes, and resonance energy transfer

Metallic surfaces can have unusual effects on fluorophores such as increasing or decreasing the rates of radiative decay and the rates of resonance energy transfer (RET). In the present article we describe the effects of metallic silver island films on the emission spectra, lifetimes, and energy transfer for several fluorophores. The fluorophores are not covalently coupled to the silver islands so that there are a range of fluorophore-to-metal distances. We show that proximity of fluorophores to the silver islands results in increased fluorescence intensity, with the largest enhancement for the lowest-quantum-yield fluorophores. Importantly, the metal-induced increases in intensity are accompanied by decreased lifetimes and increased photostability. These effects demonstrate that the silver islands have increased the radiative decay rates of the fluorophore. For solvent-sensitive fluorophores the emission spectra shifted to shorted wavelengths in the presence of the silver islands, which is consistent with a decrease of the apparent lifetime for fluorophores near the metal islands. We also observed an increased intensity and blue spectral shift for the protein human glyoxalase, which displays a low quantum yield for its intrinsic tryptophan emission. In this case the blue shift is thought to be due to increased emission from a buried low-quantum-yield tryptophan residue. Increased intensities were also observed for the intrinsic emission of the nucleic acid bases adenine and thymine and for single-stranded 15-mers poly(T) and poly(C). And finally, we observed increased RET for donors and acceptors in solution and when bound to double-helical DNA. These results demonstrate that metallic particles can be used to modify the emission from intrinsic and extrinsic fluorophores in biochemical systems.     

Theory and application of fluorescence homotransfer to melittin oligomerization.

Fluorescence homotransfer (electronic energy transfer between identical fluorophores) has the potential to quantitate the number of subunits in membrane protein oligomers. Homotransfer strongly depolarizes fluorescence emission as a result of intermolecular excitation energy exchange between an initially excited, oriented molecule and a randomly oriented neighbor. We have theoretically treated fluorescein labeled subunits in an oligomer as a cluster of molecules that can exchange excitation energy back and forth among the subunits within that group. We find that the larger the number of subunits, the more depolarized is the emission. The general equations to calculate the expected anisotropy for complexes composed of varying numbers of labeled subunits are presented. Self-quenching of fluorophores, orientation, and changes in lifetime are also discussed and/or considered. To test this theory, we have specifically labeled melittin on its N-terminal with fluorescein and monitored its monomer to tetramer equilibrium both in solution and in lipid bilayers. The calculated anisotropies are close to the experimental values when non-fluorescent fluorescein dimers are taken into account. Our results show that homotransfer may be a promising method to study membrane-protein oligomerization.     

The Covalent Trimethoprim Chemical Tag Facilitates Single Molecule Imaging with Organic Fluorophores

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.     

Polarization-Sensitive Two-Photon Microscopy Study of the Organization of Liquid-Crystalline DNA

Highly concentrated DNA solutions exhibit self-ordering properties such as the generation of liquid-crystalline phases. Such organized domains may play an important role in the global chromatin topology but can also be used as a simple model for the study of more complex 3D DNA structures. In this work, using polarized two-photon fluorescence microscopy, we report on the orientation of DNA molecules in liquid-crystalline phases. For this purpose, we analyze the signal emitted by fluorophores that are noncovalently bound to DNA strands. In nonlinear processes, excitation occurs exclusively in the focal volume, which offers advantages such as the reduction of photobleaching of out-of-focus molecules and intrinsic 3D sectioning capability. Propidium iodide and Hoechst, two fluorophores with different DNA binding modes, have been considered. Polarimetric measurements show that the dyes follow the alignment with respect to the DNA strands and allow the determination of the angles between the emission dipoles and the longitudinal axis of the DNA double strand. These results provide a useful starting point toward the application of two-photon polarimetry techniques to determine the local orientation of condensed DNA in physiological conditions.     

The Covalent Trimethoprim Chemical Tag Facilitates Single Molecule Imaging with Organic Fluorophores

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.     

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)

We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.     

Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.

Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification in a fluorescent microscope. Emitted fluorescence is split by a birefringent crystal giving rise to two images created by light rays polarized orthogonally with respect to each other. The two images are recorded by a high-sensitivity video camera, and polarization of fluorescence at any point is calculated from the relative intensity of both images at this point. From the value of polarization, the orientation of the absorption dipole of the dye, and thus orientation of F-actin, can be calculated. To illustrate the utility of the method, we measured orientation of actin cores in microvilli of chicken intestinal epithelial cells. F-actin in microvillar cores was labeled with rhodamine-phalloidin; measurements showed that the orientation was the same when microvillus formed a part of a brush border and when it was separated from it suggesting that "shaving" of brush borders did not distort microvillar structure. In the absence of nucleotide, polarization of fluorescence of actin cores in isolated microvilli was best fitted by assuming that a majority of fluorophores were arranged with a perfect helical symmetry along the axis of microvillus and that the absorption dipoles of fluorophores were inclined at 52 degrees with respect to the axis. When ATP was added, the shape of isolated microvilli did not change but polarization of fluorescence decreased, indicating statistically significant increase in disorder and a change of average angle to 54 degrees. We argue that these changes were due to mechanochemical interactions between actin and myosin-I.     

Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates.

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are soley due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6-MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.     

Fluorescence polarization studies of squid giant axons stained with N-methylanilinonaphthalenesulfonates

The polarized components of the extrinsic fluorescence of squid giant axons stained with 2,6-MANS or 1,8-MANS were studied. The polarization properties of the fluorescence changes associated with voltage-clamp pulses were found to be very different from those of the static fluorescence, supporting the notion that the optical changes involve highly oriented membrane adsorbed fluorophores. The theoretical expectations according to this hypothesis are discussed in detail. The experimental results are in good agreement with the theory assuming that possible probes reorientations are solely due to the action of the applied electric field upon the probes electric dipole. The quantitative analysis of the data for 2,6 MANS provides a fairly accurate determination of the orientation of the membrane bound 2,6-MANS molecules responsible for the fluorescence changes. Such orientation appears to be independent of the membrane face exposed to staining. The data for 1,8-MANS indicate a very different orientation of this isomer. The results suggest a profitable use of extrinsic fluorophores for studies of the structural organization of nerve membranes.     

Applications of combined spectral lifetime microscopy for biology

Live cell imaging has been greatly advanced by the recent development of new fluorescence microscopy-based methods such as multiphoton laser-scanning microscopy, which can noninvasively image deep into live specimens and generate images of extrinsic and intrinsic signals. Of recent interest has been the development of techniques that can harness properties of fluorescence, other than intensity, such as the emission spectrum and excited state lifetime of a fluorophore. Spectra can be used to discriminate between fluorophores, and lifetime can be used to report on the microenvironment of fluorophores. We describe a novel technique-combined spectral and lifetime imaging-which combines the benefits of multiphoton microscopy, spectral discrimination, and lifetime analysis and allows for the simultaneous collection of all three dimensions of data along with spatial and temporal information.     

Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-''light-sheet'', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-''light-sheet'' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated.