1,721,069 research outputs found
Focusing astigmatic Gaussian beams through optical systems with a high numerical aperture
No full textWe theoretically derive the electric field distribution of an astigmatic Gaussian laser beam after it is focused through a high-aperture objective. We show that astigmatism values that are hard to detect in the collimated laser beam can have a large effect after diffraction-limited focusing. Such astigmatic beams may be frequently encountered in fluorescence correlation measurements and in laser-scanning confocal microscopy. We present experimental measurements of the excitation intensity distribution measured by 3D scanning of single fluorescent molecules immobilized on a glass surface. (c) 2005 Optical Society of America
Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy
No full textWe present a thorough introduction into the recently developed fluorescence lifetime correlation spectroscopy (FLCS). The theoretical basis of FLCS is explained, and the method is applied to the study of a dynamic transition between two fluorescence lifetime states in a dye-protein complex
Using fluorescence lifetime for discriminating detector afterpulsing in fluorescence-correlation spectroscopy
Full text linkFluorescence correlation spectroscopy (FCS) has become an important and widely used technique for many applications in physics, chemistry, and biology. Usually, FCS is measured with sensitive light detectors working in the photon-counting Geiger mode. A common property of such detectors is afterpulsing: the generation of spurious photon detection events after a genuine photon detection. Such afterpulsing causes a significant deviation of the measured autocorrelation function from its true value on a short time scale and can seriously influence derived parameters for fast processes such as triplet-state photophysics. Here, we discuss the impact of afterpulsing on FCS in detail. A new method is developed to eliminate afterpulsing effects by using time-correlated single-photon counting for separating the true fluorescence signal from afterpulsing events. (C) 2005 American Institute of Physics
Nanocavity-Based Determination of Absolute Values of Photoluminescence Quantum Yields
No full textWe present a new method for determining absolute values of quantum yield of luminescent emitters, which is based on the modification of the radiative transition of emitters within a tunable metallic nanocavity. The method presented is easy to set up and works without any calibration. It will thus be useful for all applications where absolute and calibration‐free measurements of luminescence quantum yields are needed. Moreover, it requires only a minute amount of low‐concentration fluorophore solution. We give a detailed description of the theory and data evaluation of the nanocavity measurements, and report experimental results for several common dyes in aqueous solution
Measuring rotational diffusion of macromolecules by fluorescence correlation spectroscopy
No full textWe describe a novel method to measure rotational diffusion of large biomolecules in solution based on fluorescence correlation on the nanosecond time scale. In contrast to conventional fluorescence anisotropy measurements, a correlation-based method will also work if the rotational diffusion time is much longer than the fluorescence decay time. Thus, the method is suited to study the rotational diffusion of macromolecules having rotational diffusion times of dozens to hundreds of nanoseconds, which is considerably larger than the fluorescence lifetime of most commercially available dyes or auto-fluorescent proteins. A pulsed interleaved excitation scheme with crossed excitation polarization maximizes the time-dependent amplitude of the measured correlation curve as caused by rotational diffusion. Using the determined rotational diffusion coefficient, precise values of the hydrodynamic radius can be obtained. The method is exemplified on sizing a set of common globular proteins
Instant three-color multiplane fluorescence microscopy
No full textOne of the most widely used microscopy techniques in biology and medicine is fluorescence microscopy, offering high specificity in labeling as well as maximal sensitivity. For live-cell imaging, the ideal fluorescence microscope should offer high spatial resolution, fast image acquisition, three-dimensional sectioning, and multicolor detection. However, most existing fluorescence microscopes have to compromise between these different requirements. Here, we present a multiplane, multicolor wide-field microscope that uses a dedicated beam splitter for recording volumetric data in eight focal planes and for three emission colors with frame rates of hundreds of volumes per second. We demonstrate the efficiency and performance of our system by three-dimensional imaging of multiply labeled fixed and living cells. The use of commercially available components makes our proposed microscope straightforward for implementation, thus promising for widely used applications
Feedback-controlled electro-kinetic traps for single-molecule spectroscopy
No full textA principal limitation of single-molecule spectroscopy in solution is the diffusion-limited residence time of a given molecule within the detection volume. A common solution to this problem is to immobilize molecules of interest on a passivated glass surface for extending the observation time to obtain reliable data statistics. However, surface tethering of molecules often introduces artifacts, particularly when studying the structural dynamics of biomolecules. To circumvent this limitation, we investigated alternative ways to extend single-molecule observation times in solution without surface immobilization. Among various possibilities, the so-called anti-Brownian electro-kinetic trap (or ABEL trap) seems best suited to achieve this goal. The essential part of that trap is a feedback-controlled electro-kinetic steering of a molecule’s position in reaction to its diffusive Brownian motion which is monitored by fluorescence, thus keeping the molecule within a sub-micron sized detection volume. Fluorescence trace recordings of over thousands of milliseconds duration on individual dye molecules within an ABEL trap have been reported. In this short review, we shall briefly discuss the principle and some results of ABEL trapping of individual molecules with possible extensions to future works
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