Heparin acts as a structural component of ß-endorphin amyloid fibrils rather than a simple aggregation promoter.

The aggregation promoter heparin is commonly used to study the aggregation kinetics and biophysical properties of protein amyloids. However, the underlying mechanism for amyloid promotion by heparin remains poorly understood. In the case of the neuropeptide ß-endorphin that can reversibly adopt a functional amyloid form in nature, aggregation in the presence of heparin leads to a loss of function. Applying correlative optical super-resolution microscopy methods, we show that heparin incorporates into emerging ß-endorphin fibrils forming an integral component and is essential for amyloid templating. This will have direct implications on ß-endorphin's normal physiological function and raises concerns on the biological relevance of heparin-promoted amyloid models.

Development of broadband cavity ring-down spectroscopy for biomedical diagnostics of liquid analytes.

We present a spectrometer for sensitive absorption measurements in liquids across broad spectral bandwidths. The spectrometer combines the unique spectral properties of incoherent supercontinuum light sources with the advantages of cavity ring-down spectroscopy, which is a self-calibrating technique. A custom-built avalanche photodiode array is used for detection, permitting the simultaneous measurement of ring-down times for up to 64 different spectral components at nanosecond temporal resolution. The minimum detectable absorption coefficient was measured to be 3.2 × 10(-6) cm(-1) Hz(-1/2) at 527 nm. We show that the spectrometer is capable of recording spectral differences in trace levels of blood before and after hemolysis.

Solitary pulse generation by backward Raman scattering in H2-filled photonic crystal fibers.

Using a hydrogen-filled hollow-core photonic crystal fiber as a nonlinear optical gas cell, we study amplification of ns-laser pulses by backward rotational Raman scattering. We find that the amplification process has two characteristic stages. Initially, the pulse energy grows and its duration shortens due to gain saturation at the trailing edge of the pulse. This phase is followed by formation of a symmetric pulse with a duration significantly shorter than the phase relaxation time of the Raman transition. Stabilization of the Stokes pulse profile to a solitonlike hyperbolic secant shape occurs as a result of nonlinear amplification at its front edge and nonlinear absorption at its trailing edge (caused by energy conversion back to the pump field), leading to a reshaped pulse envelope that travels at superluminal velocity.

mhFLIM: resolution of heterogeneous fluorescence decays in widefield lifetime microscopy.

Frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate way of measuring fluorescence lifetimes in widefield microscopy. However, the resolution of multiple exponential fluorescence decays has remained beyond the reach of most practical FD-FLIM systems. In this paper we describe the implementation of FD-FLIM using a 40 MHz pulse train derived from a supercontinuum source for excitation. The technique, which we term multi-harmonic FLIM (mhFLIM), makes it possible to accurately resolve biexponential decays of fluorophores without any a priori information. The system's performance is demonstrated using a mixture of spectrally similar dyes of known composition and also on a multiply-labeled biological sample. The results are compared to those obtained from time correlated single photon counting (TCSPC) microscopy and a good level of agreement is achieved. We also demonstrate the first practical application of an algorithm derived by G. Weber [1] for analysing mhFLIM data. Because it does not require nonlinear minimisation, it offers potential for realtime analysis during acquisition.

A method to unmix multiple fluorophores in microscopy images with minimal a priori information.

The ability to quantify the fluorescence signals from multiply labeled biological samples is highly desirable in the life sciences but often difficult, because of spectral overlap between fluorescent species and the presence of autofluorescence. Several so called unmixing algorithms have been developed to address this problem. Here, we present a novel algorithm that combines measurements of lifetime and spectrum to achieve unmixing without a priori information on the spectral properties of the fluorophore labels. The only assumption made is that the lifetimes of the fluorophores differ. Our method combines global analysis for a measurement of lifetime distributions with singular value decomposition to recover individual fluorescence spectra. We demonstrate the technique on simulated datasets and subsequently by an experiment on a biological sample. The method is computationally efficient and straightforward to implement. Applications range from histopathology of complex and multiply labelled samples to functional imaging in live cells.

Dynamic control of higher-order modes in hollow-core photonic crystal fibers.

We present a versatile method for selective mode coupling into higher-order modes of photonic crystal fibers, using holograms electronically generated by a spatial light modulator. The method enables non-mechanical and completely repeatable changes in the coupling conditions. We have excited higher order modes up to LP(31) in hollow-core photonic crystal fibers. The reproducibility of the coupling allows direct comparison of the losses of different guided modes in both hollow-core bandgap and kagome-lattice photonic crystal fibers. Our results are also relevant to applications in which the intensity distribution of the light inside the fiber is important, such as particle- or atom-guidance.

Cavity enhanced absorption spectroscopy of multiple trace gas species using a supercontinuum radiation source.

Supercontinuum radiation sources are attractive for spectroscopic applications owing to their broad wavelength coverage, which enables spectral signatures of multiple species to be detected simultaneously. Here we report the first use of a supercontinuum radiation source for broadband trace gas detection using a cavity enhanced absorption technique. Spectra were recorded at bandwidths of up to 100 nm, encompassing multiple absorption bands of H(2)O, O(2) and O(2)-O(2). The same instrument was also used to make quantitative measurements of NO(2) and NO(3). For NO(3) a detection limit of 3 parts-per-trillion in 2 s was achieved, which corresponds to an effective 3sigma sensitivity of 2.4 x 10(-9) cm(-1)Hz(-1/2). Our results demonstrate that a conceptually simple and robust instrument is capable of highly sensitive broadband absorption measurements.

Transoral access for endoscopic thyroid resection.

BACKGROUND: Endoscopic neck surgery is requested by an increasing number of patients. The access trauma of the axillary, breast, and chest approaches is greater than with open or video-assisted surgery. The authors tested the feasibility of the sublingual transoral access, which they believe is the most promising minimally invasive endoscopic access to the thyroid gland from outside the neck region. METHODS: The sublingual transoral access was first evaluated in two fresh human cadavers. An experimental investigation then was performed using a porcine model. A total of 10 endoscopic transoral thyroidectomies were performed in 10 pigs using a modified axilloscope with an obturator, ultrasonic scissors, and a neuromonitoring system to identify the recurrent laryngeal nerve. RESULTS: A complete transoral thyroid resection was achieved with both the human cadavers and all the living pigs. Despite the complexity of the anatomic region, the transoral procedure was astonishingly easy to perform. In the animal study, the time from the introduction of the obturator just above the larynx to its removal was 59 s. The average overall operation time was 50 min. The neuromonitoring system permitted the regular function of the recurrent laryngeal nerves on both sides to be proved after removal of the thyroid gland. The pigs were observed for another 2 h after the operation. No complications occurred during the operation or afterward. CONCLUSIONS: Endoscopic transoral thyroid resection is possible. It proved to be a safe procedure in living pigs and astonishingly easy to perform. The results may be helpful for thyroid resections in humans using a similar access, as suggested by the thyroidectomies in human cadavers preceding this study.

Effects of sodium nitroprusside on splanchnic microcirculation in a resuscitated porcine model of septic shock.

BACKGROUND: We tested the hypothesis that sodium nitroprusside (SNP) might improve the impairment of hepatosplanchnic microcirculatory blood flow (MBF) in septic shock. METHODS: Fourteen pigs were anaesthetized and their lungs mechanically ventilated. Sepsis was induced with i.v. infusion of live Pseudomonas aeruginosa [1x10(8) colony forming units (CFU) ml(-1) kg(-1)] for 1 h. Sixty minutes later, the animals received in a random succession either SNP or normal saline for 30 min. Mean arterial pressure (MAP), cardiac index (CI), mean pulmonary artery pressure (MPAP), carbon dioxide tension of the ileal mucosa (PCO2; by gas tonometry), ileal mucosal and hepatic MBF by laser Doppler flowmetry, blood gases, and lactates were assessed before, during administration, and 30 min after discontinuing the test drug. RESULTS: Bacterial infusion promoted hypodynamic shock (MAP -18%, CI -33%, ileal MBF -19%, and hepatic MBF -27%), which was converted to normodynamic shock by resuscitation. During SNP infusion, ileal mucosal MBF significantly increased (+19%) compared with control (P = 0.033). Although hepatic MBF increased (+42% from baseline), this did not differ from control. In order to maintain a constant central venous pressure and MAP, fluid loading and norepinephrine (P < 0.01) were increased. Acid-base status was not altered by SNP. CONCLUSIONS: In a resuscitated porcine model of the early phase of septic shock, SNP improved ileal mucosal MBF but required a concomitant increase in fluid and norepinephrine supplements to maintain constant systemic haemodynamic parameters.

[Taking venous blood samples in the daily routine of German hospitals]. / Die venöse Blutentnahme im klinischen Alltag.

BACKGROUND AND OBJECTIVE: In Germany, taking blood samples is generally considered to be the task of a physician. Doctors can legally delegate the taking of blood samples to nurses only after having made sure that they are qualified for this task. In daily routine at many hospitals blood samples are almost always taken by doctors and nearly exclusively by qualified assistants in the medical practices of resident doctors. METHODS: Answers to standardized questionnaires by 538 nurses were analysed for the way in which five German hospitals of a corporate group of hospitals dealt with this task, and what circumstances and reasons played a role. RESULTS: It was found that in the East German states blood samples were significantly more often taken by nurses than in the rest of the Federal Republic. 476 of all nurses questioned (89.14% of total) considered themselves capable of taking blood samples. However taking these samples has remained part of doctors' daily routine. CONCLUSION: In times of necessary cost cutting and a growing lack of physicians it would be reasonable to delegate this task to nurses who have been specifically trained for it.

A white light confocal microscope for spectrally resolved multidimensional imaging.

Spectrofluorometric imaging microscopy is demonstrated in a confocal microscope using a supercontinuum laser as an excitation source and a custom-built prism spectrometer for detection. This microscope system provides confocal imaging with spectrally resolved fluorescence excitation and detection from 450 to 700 nm. The supercontinuum laser provides a broad spectrum light source and is coupled with an acousto-optic tunable filter to provide continuously tunable fluorescence excitation with a 1-nm bandwidth. Eight different excitation wavelengths can be simultaneously selected. The prism spectrometer provides spectrally resolved detection with sensitivity comparable to a standard confocal system. This new microscope system enables optimal access to a multitude of fluorophores and provides fluorescence excitation and emission spectra for each location in a 3D confocal image. The speed of the spectral scans is suitable for spectrofluorometric imaging of live cells. Effects of chromatic aberration are modest and do not significantly limit the spatial resolution of the confocal measurements.

Use of (130)Te(2) for frequency referencing and active stabilisation of a violet extended cavity diode laser.

This paper reports on the use of (130)Te(2) absorption lines in active laser-locking, and in frequency referencing, of the emission of a violet extended cavity diode laser with a wavelength of around 410 nm. We note the existence of closely spaced tellurium absorption lines, suitable for referencing purposes in gas sensing applications, at wavelengths below the lower limit (417 nm) of the spectral region covered by the tellurium atlas [J. Cariou, P. Luc, Atlas du spectre d'Absorption de la Molecule de Tellure, CNRS, Paris, 1980]. The absolute positions of the lines in the acquired spectra were estimated by comparison to a simultaneously acquired fluorescence spectrum of atomic indium, and were identified using calculations based on fundamental spectroscopic data. The laser frequency was stabilised within a range of 40 MHz, which is negligible compared to typical transition widths at atmospheric pressure.

Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources.

High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research.

Application of frequency-domain Fluorescence Lifetime Imaging Microscopy as a quantitative analytical tool for microfluidic devices.

We describe the application of wide-field frequency domain Fluorescence Lifetime Imaging Microscopy (FLIM) to imaging in microfluidic devices. FLIM is performed using low cost, intensity modulated Light Emitting Diodes (LEDs) for illumination. The use of lifetime imaging for quantitative analysis within such devices is demonstrated by mapping the molecular diffusion of iodide ions across a microchannel.

Flame front tracking by laser induced fluorescence spectroscopy and advanced image analysis.

This paper presents advanced image analysis methods for extracting information from high speed Planar Laser Induced Fluorescence (PLIF) data obtained from turbulent flames. The application of non-linear anisotropic diffusion filtering and of Active Contour Models (Snakes) is described to isolate flame boundaries. In a subsequent step, the detected flame boundaries are tracked in time using a frequency domain contour interpolation scheme. The implementations of the methods are described and possible applications of the techniques are discussed.

Nonlinear diffusion filtering of images obtained by planar laser-induced fluorescence spectroscopy.

The application of nonlinear anisotropic diffusion filtering to reduce noise and enhance contours in images obtained by two-dimensional planar laser-induced fluorescence (PLIF) spectroscopy is presented. In this process the diffusion coefficient is locally adapted, becoming negligible as object boundaries are approached. Noise is efficiently removed, and object contours are strongly enhanced. The technique is demonstrated with PLIF images obtained from the OH radical recorded in turbulent flames. We show that nonlinear diffusion is suitable as a preprocessing step, before image segmentation becomes possible, and we demonstrate how the technique is applied for the quantitative extraction of flame reaction boundaries from PLIF data.

Two-line atomic fluorescence as a temperature probe for highly sooting flames.

We investigate the applicability of two-line atomic fluorescence (TLAF) from seeded indium atoms for temperature measurements in highly sooting flames. The results show that TLAF holds promise for two-dimensional temperature measurements in sooting and fuel-rich flames under conditions in which other thermometry techniques fail, a result that is attributed to the superior characteristics of the indium atomization process. Furthermore, no native species was found to interfere spectrally with the detected TLAF wavelengths. Advantages of and problems with the technique are discussed.

Investigation of two-photon-induced polarization spectroscopy of the a-X (1,0) transition in molecular nitrogen at elevated pressures.

Two-photon-induced polarization spectroscopy of molecular nitrogen in the alpha 1IIg(nu' =) <-- X 1Sigma(g)+ (nu" =) system near 283 nm was performed, and its signal dependence investigated over the pressure range from 1.2 to 5 bars at 300 K. A significant increase of the signal intensity with pressure beyond the expected square law for a two-photon process was observed for pure nitrogen. Similar behavior was also found for a constant nitrogen partial pressure with increasing partial pressures of argon buffer gas. In both cases the spectral linewidth of the excited transitions increased dramatically with overall pressure. A possible explanation is given for the observed behavior in terms of contributions to the nonlinear susceptibility of the medium from the population of one-photon resonantly absorbing excited-state nitrogen and ground state N(2)(+) ions created in the multiphoton absorption process at the high laser intensities required.