Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI).

Super-resolution optical microscopy is a rapidly evolving area of fluorescence microscopy with a tremendous potential for impacting many fields of science. Several super-resolution methods have been developed over the last decade, all capable of overcoming the fundamental diffraction limit of light. We present here an approach for obtaining subdiffraction limit optical resolution in all three dimensions. This method relies on higher-order statistical analysis of temporal fluctuations (caused by fluorescence blinking/intermittency) recorded in a sequence of images (movie). We demonstrate a 5-fold improvement in spatial resolution by using a conventional wide-field microscope. This resolution enhancement is achieved in iterative discrete steps, which in turn allows the evaluation of images at different resolution levels. Even at the lowest level of resolution enhancement, our method features significant background reduction and thus contrast enhancement and is demonstrated on quantum dot-labeled microtubules of fibroblast cells.

TIRF microscopy evanescent field calibration using tilted fluorescent microtubules.

Total internal reflection fluorescence microscopy has become a powerful tool to study the dynamics of sub-cellular structures and single molecules near substrate surfaces. However, the penetration depth of the evanescent field, that is, the distance at which the excitation intensity has exponentially decayed to 1/e, is often left undetermined. This presents a limit on the spatial information about the imaged structures. Here, we present a novel method to quantitatively characterize the illumination in total internal reflection fluorescence microscopy using tilted, fluorescently labelled, microtubules. We find that the evanescent field is well described by a single exponential function, with a penetration depth close to theoretically predicted values. The use of in vitro reconstituted microtubules as nanoscale probes results in a minimal perturbation of the evanescent field; excitation light scattering is eliminated and the refractive index of the sample environment is unchanged. The presented method has the potential to provide a generic tool for in situ calibration of the evanescent field.

Forbidden light detection from single molecules

We present a new concept for ultrasensitive detection of surface-generated fluorescence which is made possible by a new optical module. The detection method leads to an enhancement in fluorescence collection efficiency to more than 65% of the total of emitted light, whereas high-aperture microscope objectives are able to collect 44% at best. Moreover, by employing this new optical module, the detection volume can be restricted to approximately 10(-17) L. This allows for an exceptional discrimination of bulk-generated against surface-generated fluorescence, which may be of great value when surface-binding processes are monitored. We demonstrate the performance of the new detection system by detecting single fluorescent molecules and by determining antigen concentrations down to 5 fmol.

A theoretical investigation of single-molecule fluorescence detection on thin metallic layers.

In the present paper, the excitation and detection of single-molecule fluorescence over thin metallic films is studied theoretically within the framework of classical electrodynamics. The model takes into account the specific conditions of surface plasmon-assisted optical excitation, fluorescence quenching by the metal film, and detection geometry. Extensive numerical results are presented for gold, silver, and aluminum films, showing the detectable fluorescence intensities and their dependence on film thickness and the fluorescent molecule's position under optimal excitation conditions.

Theoretical study of detection of a dipole emitter through an objective with high numerical aperture.

The problem of detecting electric dipole emitters (such as a fluorescing molecule) through a microscope objective with a high numerical aperture is considered. Exact vector-wave-optics results of the field distribution of the electromagnetic field in image space are derived. On the basis of these results, the collection-efficiency function (CEF) is calculated. This calculation is compared with a semigeometric approximation of the CEF, for which a new analytical formula is presented. The derived results are important for applications such as fluorescence correlation spectroscopy and single-molecule fluorescence detection with a confocal microscope.

Theoretical investigation of aspects of single-molecule fluorescence detection in microcapillaries.

In the present report, the results of a theoretical investigation of two aspects of single-molecule detection by laser-induced fluorescence in microcapillaries are presented. The two issues studied are the scattering of the exciting laser beam on the microcapillary and the change of the fluorescence lifetime of the molecule due to the electrodynamic interaction between its fluorescence emission and the confining capillary. Numerical results for experimentally relevant conditions are provided.

Direct sequence spread spectrum differential phase shift keying SAW correlator on GaAs.

This paper presents the design, fabrication, and experimental results for a differential phase shift keying (DPSK) single SAW-based correlator on GaAs for direct sequence spread spectrum applications. The DPSK modulation format allows for noncoherent data demodulation; the SAW device correlator acts as the despreader. Unlike the conventional technique of using two parallel correlators and a one data bit delay element, this new system uses two inline correlators. When implemented on SAW devices, this in-line structure has the advantage of an inherent one data bit delay, lower insertion loss, and less signal distortion than the parallel structure. The DPSK correlator is fabricated on a {100} cut GaAs substrate with SAW propagation in the 110 direction, Using this cut, which is widely used in electronics, Rayleigh waves are generated with a piezoelectric coupling coefficient of the same order as ST-cut quartz. The piezoelectric semiconductor GaAs is of great interest because it is the only substrate that can be used to integrate SAW devices directly with electronics on the same chip, resulting in smaller packaging, reduction of packaging parasitics, lower cost, and greater system integration. This paper presents experimental results for SAW in-line correlator structures on GaAs along with their despreading system performances. Experimental measurements in both the time and frequency domains were performed and were found to be in good agreement with theoretical predictions.

Highly efficient optical detection of surface-generated fluorescence.

We present a theoretical study of a new highly efficient system for optical light collection, designed for ultrasensitive fluorescence detection of surface-bound molecules. The main core of the system is a paraboloid glass segment acting as a mirror for collecting the fluorescence. A special feature of the system is its ability to sample not only fluorescence that is emitted below the angle of total internal reflection (the critical angle) but also particularly the light above the critical angle. As shown, this is especially advantageous for collecting the fluorescence of surface-bound molecules. A comparison is made with conventional high-aperture microscope objectives. Furthermore, it is shown that the system allows not only for highly efficient light collection but also for confocal imaging of the detection region, which is of great importance for rejecting scattered light in potential applications such as the detection of only a few molecules.

Optical collection efficiency function in single-molecule detection experiments.

The optical collection efficiency function for an optical system such as that used in single-molecule detection experiments is studied. Closed analytical expressions based on a geometrical optics approximation are presented. Comparison is made with exact wave optics calculations.

Maximum-likelihood criterion and single-molecule detection.

The maximum-likelihood criterion is shown to be a powerful method for analyzing fluorescence-detection data with small signal-to-noise ratios. A probability study of the maximum-likelihood criterion for a supposed single-molecule detection experiment is presented that takes into account the photokinetics of the molecule to be detected, its diffusion, and the laser-beam geometry. Furthermore, the efficiency of time-integrated and time-correlated single-photon counting methods are studied and compared.

Substance P in human essential hypertension.

Substance P (SP) plays an important role in central nervous and peripheral blood pressure regulation. Its effects include modulating influence on the adrenergic system and inhibition of stress-induced plasma noradrenaline increase in animal studies. In patients with essential hypertension (n = 45, WHO stages 1 and 2) the SP-like immunoreactivity (SPLIR) was found significantly (p less than 0.01) lower (1.36 +/- 0.23 pg/100 microliters) than in 24 normotensive subjects (4.54 +/- 0.72 pg/100 microliters). Furthermore, the influence of a mental stress test on SPLIR was investigated in patients with essential hypertension (n = 11, WHO stage 1) and compared with nine normotensive subjects. Whereas in normotensive subjects plasma SP increased under a standardized mental arithmetic test (4.03 +/- 0.48 to 4.74 +/- 0.56 pg/100 microliters), in hypertensive patients a decrease of SP from lower baseline levels (2.85 +/- 0.54 to 2.57 +/- 0.54 pg/100 microliters) was demonstrated. The significantly different changes of plasma SP in normotensive and hypertensive subjects under mental stress conditions had the opposite direction in comparison with the adrenergic reaction [higher and prolonged increase of plasma noradrenaline (NA) in the hypertensive group]. Under antihypertensive drug treatment with prazosin (4.5 mg/day, n = 10) or with captopril (450 mg/day, n = 10) an increase of plasma SP was registered. The results support the participation of SP in the pathogenesis of human hypertension and in therapeutic mechanisms. Lower plasma levels and decreased responsiveness of SP possibly represent the enhanced stress sensitivity in primary hypertension.

[The blood pressure-regulating role of the renin-angiotensin system in essential hypertension before and after emotional stress]. / Zur blutdruckregulativen Rolle des Renin-Angiotensin-Systems bei essentiellen Hypertonikern vor und nach psychoemotionalem Stress.

The aim of the study was to prove the correlations between systolic and diastolic blood pressure (BP), heart rate (HR) and plasma renin activity (PRA) under mental arithmetic stress without and with beta-blockade. 22 male hypertensive patients (aged 19-39 years) were examined, divided into a group treated with d, 1-propranolol (Obsidan) and a control group receiving placebo. Under arithmetic stress the kinetics of systolic BP showed no differences even after beta-blockade. In contrast, the increases of HR and PRA before, during and after mental arithmetic were either significantly reduced (HR) or completely blocked (PRA) under beta-blockade. However, these suppressions of HR and PRA did not prevent the stress-induced elevations of systolic and diastolic BP under beta-blockade.