Photophysical properties of 2-Phenylindole in poly (vinyl alcohol) film at room temperature. Enhanced phosphorescence anisotropy with direct triplet state excitation.

We report the spectral properties of 2-Phenylindole (2PI) embedded in rigid poly (vinyl alcohol) (PVA) film. The 2PI in PVA film shows relatively strong and structured fluorescence with a maximum at 370 nm and surprisingly strong room temperature phosphorescence with an emission maximum of about 500 nm. The dye is highly immobilized in the polymer matrix, thus presenting high fluorescence anisotropy in an isotropic film of about 0.3 at room temperature. The 2-Phenylindole phosphorescence excited in the usual way through the electronic singlet state excitation (S0 â†’ S1 absorption) results in a very low, near zero anisotropy. We now report that we can directly excite the dye to the triplet state T1 and observe high phosphorescence anisotropy similar to the fluorescence anisotropy. The extinction coefficient for S0 â†’ T1 absorption in the PVA matrix is unusually high- only about 3 orders of magnitude lower than S0 â†’ S1 absorption. We consider this direct excitation to indole's triplet state a very significant finding that may lead to many practical applications. The unusually long-wavelength of excitation around 400 nm, much above typical UV absorption, results in a high phosphorescence anisotropy. This provides a new way to study rotational motion of larger biological objects in the microsecond time scale not accessible through typical fluorescence studies.

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.

The spatial distribution of actin and mechanical cycle of myosin are different in right and left ventricles of healthy mouse hearts.

The contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. Indeed, differences have been observed in the contractile performance between left and right ventricular myocytes in dilated cardiomyopathy, in congestive heart failure, and in energy usage and speed of contraction at light loads in healthy hearts. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance, or power among the RV and LV in dogs. This report shows that on a mesoscopic scale [when only a few molecules are studied (here three to six molecules of actin) in ex vivo ventricular myofibrils], the two ventricles in rigor differ in the degree of orientational disorder of actin within in filaments and during contraction in the kinetics of the cross-bridge cycle.

Mesoscopic analysis of motion and conformation of cross-bridges.

The orientation of a cross-bridge is widely used as a parameter in determining the state of muscle. The conventional measurements of orientation, such as that made by wide-field fluorescence microscopy, electron paramagnetic resonance (EPR) or X-ray diffraction or scattering, report the average orientation of 1012-109 myosin cross-bridges. Under conditions where all the cross-bridges are immobile and assume the same orientation, for example in normal skeletal muscle in rigor, it is possible to determine the average orientation from such global measurements. But in actively contracting muscle, where a parameter indicating orientation fluctuates in time, the measurements of the average value provide no information about cross-bridge kinetics. To avoid problems associated with averaging information from trillions of cross-bridges, it is necessary to decrease the number of observed cross-bridges to a mesoscopic value (i.e. the value affected by fluctuations around the average). In such mesoscopic regimes, the averaging of the signal is minimal and dynamic behavior can be examined in great detail. Examples of mesoscopic analysis on skeletal and cardiac muscle are provided.

Cross-bridge kinetics in myofibrils containing familial hypertrophic cardiomyopathy R58Q mutation in the regulatory light chain of myosin.

Familial hypertrophic cardiomyopathy (FHC) is a heritable form of cardiac hypertrophy caused by single-point mutations in genes encoding sarcomeric proteins including ventricular myosin regulatory light chain (RLC). FHC often leads to malignant outcomes and sudden cardiac death. The FHC mutations are believed to alter the kinetics of the interaction between actin and myosin resulting in inefficient energy utilization and compromised function of the heart. We studied the effect of the FHC-linked R58Q-RLC mutation on the kinetics of transgenic (Tg)-R58Q cardiac myofibrils. Kinetics was determined from the rate of change of orientation of actin monomers during muscle contraction. Actin monomers change orientation because myosin cross-bridges deliver periodic force impulses to it. An individual impulse (but not time average of impulses) carries the information about the kinetics of actomyosin interaction. To observe individual impulses it was necessary to scale down the experiments to the level of a few molecules. A small population (∼4 molecules) was selected by using (deliberately) inefficient fluorescence labeling and observing fluorescent molecules by a confocal microscope. We show that the kinetic rates are significantly smaller in the contracting cardiac myofibrils from Tg-R58Q mice then in control Tg-wild type (WT). We also demonstrate a lower force per cross-section of muscle fiber in Tg-R58Q versus Tg-WT mice. We conclude that the R58Q mutation-induced decrease in cross-bridge kinetics underlines the mechanism by which Tg-R58Q fibers develop low force and thus compromise the ability of the mutated heart to efficiently pump blood.

Myosin cross-bridges do not form precise rigor bonds in hypertrophic heart muscle carrying troponin T mutations.

Distribution of orientations of myosin was examined in ex-vivo myofibrils from hearts of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C. Humans are heterozygous for sarcomeric FHC mutations and so hypertrophic myocardium contains a mixture of the wild-type (WT) and mutated (MUT) TnT. If mutations are expressed at a low level there may not be a significant change in the global properties of heart muscle. In contrast, measurements from a few molecules avoid averaging inherent in the global measurements. It is thus important to examine the properties of only a few molecules of muscle. To this end, the lever arm of one out of every 60,000 myosin molecules was labeled with a fluorescent dye and a small volume within the A-band (~1 fL) was observed by confocal microscopy. This volume contained on average 5 fluorescent myosin molecules. The lever arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientations by recording polarization of fluorescent light emitted by myosin-bound fluorophore during rigor and contraction. The distribution of orientations of rigor WT and MUT myofibrils was significantly different. There was a large difference in the width and of skewness and kurtosis of rigor distributions. These findings suggest that the hypertrophic phenotype associated with the TnT mutations can be characterized by a significant increase in disorder of rigor cross-bridges.

Evidence for pre- and post-power stroke of cross-bridges of contracting skeletal myofibrils.

We examined the orientational fluctuations of a small number of myosin molecules (approximately three) in working skeletal muscle myofibrils. Myosin light chain 1 (LC1) was labeled with a fluorescent dye and exchanged with the native LC1 of skeletal muscle myofibrils cross-linked with 1-ethyl-3-[3(dimethylamino) propyl] carbodiimide to prevent shortening. We observed a small volume within the A-band (∼10(-15) L) by confocal microscopy, and measured cyclic fluctuations in the orientation of the myosin neck (containing LC1) by recording the parallel and perpendicular components of fluorescent light emitted by the fluorescently labeled myosin LC1. Histograms of orientational fluctuations from fluorescent molecules in rigor were represented by a single Gaussian distribution. In contrast, histograms from contracting muscles were best fit by at least two Gaussians. These results provide direct evidence that cross-bridges in working skeletal muscle assume two distinct conformations, presumably corresponding to the pre- and post-power-stroke states.

Observing cycling of a few cross-bridges during isometric contraction of skeletal muscle.

During muscle contraction a myosin cross-bridge imparts periodic force impulses to actin. It is possible to visualize those impulses by observing a few molecules of actin or myosin. We have followed the time course of orientation change of a few actin molecules during isometric contraction by measuring parallel polarized intensity of its fluorescence. The orientation of actin reflects local bending of a thin filament and is different when a cross-bridge binds to, or is detached from, F-actin. The changes in orientation were characterized by periods of activity during which myosin cross-bridges interacted normally with actin, interspersed with periods of inactivity during which actin and myosin were unable to interact. The periods of activity lasted on average 1.2 +/- 0.4 s and were separated on average by 2.3 +/- 1.0 s. During active period, actin orientation oscillated between the two extreme values with the ON and OFF times of 0.4 +/- 0.2 and 0.7 +/- 0.4 s, respectively. When the contraction was induced by a low concentration of ATP both active and inactive times were longer and approximately equal. These results imply that cross-bridges interact with actin in bursts and suggest that during active period, on average 36% of cross-bridges are involved in force generation.

Familial hypertrophic cardiomyopathy can be characterized by a specific pattern of orientation fluctuations of actin molecules .

A single-point mutation in the gene encoding the ventricular myosin regulatory light chain (RLC) is sufficient to cause familial hypertrophic cardiomyopathy (FHC). Most likely, the underlying cause of this disease is an inefficient energy utilization by the mutated cardiac muscle. We set out to devise a simple method to characterize two FHC phenotypes caused by the R58Q and D166V mutations in RLC. The method is based on the ability to observe a few molecules of actin in working ex vivo heart myofibril. Actin is labeled with extremely diluted fluorescent dye, and a small volume within the I-band ( approximately 10(-16) L), containing on average three actin molecules, is observed by confocal microscopy. During muscle contraction, myosin cross-bridges deliver cyclic impulses to actin. As a result, actin molecules undergo periodic fluctuations of orientation. We measured these fluctuations by recording the parallel and perpendicular components of fluorescent light emitted by an actin-bound fluorophore. The histograms of fluctuations of fluorescent actin molecules in wild-type (WT) hearts in rigor were represented by perfect Gaussian curves. In contrast, histograms of contracting heart muscle were peaked and asymmetric, suggesting that contraction occurred in at least two steps. Furthermore, the differences between histograms of contracting FHC R58Q and D166V hearts versus corresponding contracting WT hearts were statistically significant. On the basis of our results, we suggest a simple new method of distinguishing between healthy and FHC R58Q and D166V hearts by analyzing the probability distribution of polarized fluorescence intensity fluctuations of sparsely labeled actin molecules.

Single molecule immunoassay on plasmonic platforms.

We examined the photophysical properties of the new near infrared (NIR) fluorescent label SeTau-665 on a plasmonic platform of self- assembled colloidal structures (SACS) of silver prepared on a semitransparent silver film. A SeTau-665 immunoassay was performed on this platform and a control glass slide. The fluorescence properties of this label substantially change due to plasmonic interactions. While the average brightness increase of SeTau 665 in ensemble measurements was about 70-fold, fluorescence enhancements up to four-hundred times were observed on certain "hot spots" for single molecule measurements. The intensity increase is strongly correlated with a simultaneous decrease in fluorescence lifetime in these "hot spots". The large increase in brightness allows the reduction of the excitation power resulting in a reduced background and increased photostability. The remarkable fluorescence enhancements observed for SeTau 665 on our plasmonic platform should allow to substantially improve single molecule detection and to reduce the detection limits in sensing devices.

Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart.

One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s(-1) in myofibrils from Tg-WT to 1.4 s(-1) in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s(-1) (Tg-WT) to 1.2 s(-1) (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

Novel gene C17orf37 in 17q12 amplicon promotes migration and invasion of prostate cancer cells.

C17orf37/MGC14832, a novel gene located on human chromosome 17q12 in the ERBB2 amplicon, is abundantly expressed in breast cancer. C17orf37 expression has been reported to positively correlate with grade and stage of cancer progression; however the functional significance of C17orf37 overexpression in cancer biology is not known. Here, we show that C17orf37 is highly expressed in prostate cancer cell lines and tumors, compared to minimal expression in normal prostate cells and tissues. Cellular localization studies by confocal and total internal reflection fluorescence microscopy revealed predominant expression of C17orf37 in the cytosol with intense staining in the membrane of prostate cancer cells. RNA-interference-mediated downregulation of C17orf37 resulted in decreased migration and invasion of DU-145 prostate cancer cells, and suppressed the DNA-binding activity of nuclear factor-kappaB (NF-kappaB) transcription factor resulting in reduced expression of downstream target genes matrix metalloproteinase 9, urokinase plasminogen activator and vascular endothelial growth factor. Phosphorylation of PKB/Akt was also reduced upon C17orf37 downregulation, suggesting C17orf37 acts as a signaling molecule that increases invasive potential of prostate cancer cells by NF-kappaB-mediated downstream target genes. Our data strongly suggest C17orf37 overexpression in prostate cancer functionally enhances migration and invasion of tumor cells, and is an important target for cancer therapy.

Fluorescence lifetime of actin in the familial hypertrophic cardiomyopathy transgenic heart.

Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin cross-bridge due to alterations in the rates of cross-bridge cycling. In this report, we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (tau) of Alexa488-phalloidin-labeled actin. The fluorescence lifetime is the average rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 ms from the center half of the I-band during 60 s of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin.

Fluorescence correlation spectroscopy in a reverse Kretchmann surface plasmon assisted microscope.

Fluorescence Correlation Spectroscopy (FCS) demands a high rate of photon detection per molecule, low background, and large fluctuations of fluorescence associated with translational motion. The new approach presented here, Surface Plasmon Assisted Microscope (SPAM), meets these requirements by drastically limiting the observation volume. In this method, the observational layer is made so thin that fluctuations are mostly due to the axial motion of molecules. This is conveniently realized by placing a sample on a thin metal film and illuminating it with a laser beam through an aqueous medium. The excited fluorophores close to the surface couple (via near-field interactions) to surface plasmons in the metal. Propagated surface plasmons decouple on opposite side of the metal film as a far-field radiation and emit in directional manner. Fluorescence is collected with a high Numerical Aperture objective. A confocal aperture inserted in its conjugate image plane reduces lateral dimensions of the detection volume to a diffraction limit. The thickness of the detection layer is reduced further by metal quenching of excited fluorophores at a close proximity (about 30 nm) to the surface. We used a suspension of fluorescent microspheres to show that FCS-SPAM is an efficient method to measure molecular diffusion.

Monolayers of silver nanoparticles decrease photobleaching: application to muscle myofibrils.

Studying single molecules in a cell has the essential advantage that kinetic information is not averaged out. However, since fluorescence is faint, such studies require that the sample be illuminated with the intense light beam. This causes photodamage of labeled proteins and rapid photobleaching of the fluorophores. Here, we show that a substantial reduction of these types of photodamage can be achieved by imaging samples on coverslips coated with monolayers of silver nanoparticles. The mechanism responsible for this effect is the interaction of localized surface plasmon polaritons excited in the metallic nanoparticles with the transition dipoles of fluorophores of a sample. This leads to a significant enhancement of fluorescence and a decrease of fluorescence lifetime of a fluorophore. Enhancement of fluorescence leads to the reduction of photodamage, because the sample can be illuminated with a dim light, and decrease of fluorescence lifetime leads to reduction of photobleaching because the fluorophore spends less time in the excited state, where it is susceptible to oxygen attack. Fluorescence enhancement and reduction of photobleaching on rough metallic surfaces are usually accompanied by a loss of optical resolution due to refraction of light by particles. In the case of monolayers of silver nanoparticles, however, the surface is smooth and glossy. The fluorescence enhancement and the reduction of photobleaching are achieved without sacrificing the optical resolution of a microscope. Skeletal muscle myofibrils were used as an example, because they contain submicron structures conveniently used to define optical resolution. Small nanoparticles (diameter approximately 60 nm) did not cause loss of optical resolution, and they enhanced fluorescence approximately 500-fold and caused the appearance of a major picosecond component of lifetime decay. As a result, the sample photobleached approximately 20-fold more slowly than the sample on glass coverslips.

Cross-bridge duty cycle in isometric contraction of skeletal myofibrils.

During interaction of actin with myosin, cross-bridges impart mechanical impulses to thin filaments resulting in rotations of actin monomers. Impulses are delivered on the average every tc seconds. A cross-bridge spends a fraction of this time (ts) strongly attached to actin, during which it generates force. The "duty cycle" (DC), defined as the fraction of the total cross-bridge cycle that myosin spends attached to actin in a force generating state (ts/ tc), is small for cross-bridges acting against zero load, like freely shortening muscle, and increases as the load rises. Here we report, for the first time, an attempt to measure DC of a single cross-bridge in muscle. A single actin molecule in a half-sarcomere was labeled with fluorescent phalloidin. Its orientation was measured by monitoring intensity of the polarized TIRF images. Actin changed orientation when a cross-bridge bound to it. During isometric contraction, but not during rigor, actin orientation oscillated between two values, corresponding to the actin-bound and actin-free state of the cross-bridge. The average ts and tc were 3.4 and 6 s, respectively. These results suggest that, in isometrically working muscle, cross-bridges spend about half of the cycle time attached to actin. The fact that 1/ tc was much smaller than the ATPase rate suggests that the bulk of the energy of ATP hydrolysis is used for purposes other than performance of mechanical work.

Metal Enhanced Fluorescence on Silicon Wafer Substrates.

We report on the fluorescence enhancement induced by silver island film (SIF) deposited on a silicon wafer. The model immunoassay was studied on silvered and unsilvered wafers. The fluorescence brightness of Rhodamine Red X increased about 300% on the SIF, while the lifetime was reduced by several fold and the photostability increased substantially. We discuss potential uses of silicon wafer substrates in multiplex assays in which the fluorescence is enhanced due to the SIF, and the multiplexing is achieved by using micro transponders.

Decreasing photobleaching by silver island films: application to muscle.

Recently it has become possible to study interactions between proteins at the level of single molecules. This requires collecting data from an extremely small volume, small enough to contain one molecule-typically of the order of attoliters (10(-18) L). Collection of data from such a small volume with sufficiently high signal-to-noise ratio requires that the rate of photon detection per molecule be high. This calls for a large illuminating light flux, which in turn leads to rapid photobleaching of the fluorophores that are labeling the proteins. To decrease photobleaching, we measured fluorescence from a sample placed on coverslips coated with silver island films (SIF). SIF reduce photobleaching because they enhance fluorescence brightness and significantly decrease fluorescence lifetime. Increase in the brightness effectively decreases photobleaching because illumination can be attenuated to obtain the same fluorescence intensity. Decrease of lifetime decreases photobleaching because short lifetime minimizes the probability of oxygen attack while the fluorophore is in the excited state. The decrease of photobleaching was demonstrated in skeletal muscle. Myofibrils were labeled lightly with rhodamine-phalloidin, placed on coverslips coated with SIF, illuminated by total internal reflection, and observed through a confocal aperture. We show that SIF causes the intensity of phalloidin fluorescence to increase 4-5 fold and its fluorescence lifetime to decrease on average 23-fold. As a consequence, the rate of photobleaching of four or five molecules of actin of a myofibril on Olympus coverslips coated with SIF decreased at least 30-fold in comparison with photobleaching on an uncoated coverslip. Significant decrease of photobleaching makes the measurement of signal from a single cross-bridge of contracting muscle feasible.

Application of surface plasmon coupled emission to study of muscle.

Muscle contraction results from interactions between actin and myosin cross-bridges. Dynamics of this interaction may be quite different in contracting muscle than in vitro because of the molecular crowding. In addition, each cross-bridge of contracting muscle is in a different stage of its mechanochemical cycle, and so temporal measurements are time averages. To avoid complications related to crowding and averaging, it is necessary to follow time behavior of a single cross-bridge in muscle. To be able to do so, it is necessary to collect data from an extremely small volume (an attoliter, 10(-18) liter). We report here on a novel microscopic application of surface plasmon-coupled emission (SPCE), which provides such a volume in a live sample. Muscle is fluorescently labeled and placed on a coverslip coated with a thin layer of noble metal. The laser beam is incident at a surface plasmon resonance (SPR) angle, at which it penetrates the metal layer and illuminates muscle by evanescent wave. The volume from which fluorescence emanates is a product of two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The fluorescence is quenched at the metal interface (up to approximately 10 nm), which further limits the thickness of the fluorescent volume to approximately 50 nm. The fluorescence is detected through a confocal aperture, which limits the lateral dimensions of the detection volume to approximately 200 nm. The resulting volume is approximately 2 x 10(-18) liter. The method is particularly sensitive to rotational motions because of the strong dependence of the plasmon coupling on the orientation of excited transition dipole. We show that by using a high-numerical-aperture objective (1.65) and high-refractive-index coverslips coated with gold, it is possible to follow rotational motion of 12 actin molecules in muscle with millisecond time resolution.

Minimization of detection volume by surface-plasmon-coupled emission.

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.