Selective Excitation Of Fluorescence Research Articles

Fast Fluorescence Microscopy with Light Sheets.

In light sheet microscopy, optical sectioning by selective fluorescence excitation with a sheet of light is combined with fast full-frame acquisition. This illumination scheme provides minimal photobleaching and phototoxicity. Complemented with remote focusing and multi-view acquisition, light sheet microscopy is the method of choice for acquisition of very fast biological processes, large samples, and high-throughput applications in areas such as neuroscience, plant biology, and developmental biology. This review explains why light sheet microscopes are much faster and gentler than other established fluorescence microscopy techniques. New volumetric imaging schemes and highlights of selected biological applications are also discussed.

Application of the method of selective fluorescence excitation to investigation of complexation and solvation processes of polar organic dye molecules in binary solvents

It is proposed to use the method of selective fluorescence excitation to find absorption spectra (fluorescence excitation spectra) of 1 : 1 primary solvated complexes between polar molecules of an organic dye and the active component of a binary solvent, whose neutral component is a nonpolar or low-polarity liquid. The technique was tested with diluted solutions of 4-dimethylaminochalcone (4-DMC) in mixtures of ethylbenzene with dimethyl formamide at extremely low contents of the latter. It is shown that the experimental absolute shift of the long-wavelength vibronic absorption band of three-component DMC solutions is in a good quantitative agreement with the analogous shift obtained independently based on the semi-empirical theory describing the joint effect of nonlinear (complexation) and linear (solvation) dipole-dipole interactions on the shift of spectral bands.

Adaptively controlled supercontinuum pulse from a microstructure fiber for two-photon excited fluorescence microscopy

Selective fluorescence excitation of specific molecular species is demonstrated by using coherent control of two-photon excitation with supercontinuum pulses generated with a microstructure fiber. Pulse shaping prior to pulse propagation through the fiber is controlled by a self-learning optimization loop so that the highest fluorescence signal contrast between two fluorescent proteins is obtainable. The self-learning optimization loop successfully controls both the optical nonlinarity of the microstructure fiber and the two-photon excitation of the fluorescent proteins.

Standing wave total internal reflection fluorescence microscopy to measure the size of nanostructures in living cells

We present the first application of standing wave fluorescence microscopy (SWFM) to determine the size of biological nanostructures in living cells. The improved lateral resolution of less than 100 nm enables superior quantification of the size of subcellular structures. We demonstrate the ability of SWFM by measuring the diameter of biological nanotubes (membrane tethers formed between cells). The combination of SWFM with total internal reflection (TIR), referred to as SW-TIRFM, allows additional improvement of axial resolution by selective excitation of fluorescence in a layer of about 100 nm.

Use of coherent control for selective two-photon fluorescence microscopy in live organisms

We demonstrate selective fluorescence We demonstrate selective fluorescence excitation of specific molecular species in live organisms by using coherent control of two-photon excitation. We have acquired quasi-simultaneous images in live fluorescently-labeled Drosophila embryos by rapid switching between appropriate pulse shapes. Linear combinations of these images demonstrate that a high degree of fluorophore selectivity is attainable through phase-shaping. Broadband phase-shaped excitation opens up new possibilities for single-laser, multiplex, in-vivo fluorescence microscopy.

Single cell imaging of PI3K activity and glucose transporter insertion into the plasma membrane by dual color evanescent wave microscopy.

Many signaling events involve the translocation of signaling molecules to or from the plasma membrane; however, suitable techniques to quantify the temporal relationships between such signaling events are lacking. Here, we describe an evanescent wave microscopy technique that allows parallel measurement of the recruitment and dissociation of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) labeled proteins to and from the plasma membrane in individual living cells. The selective excitation of fluorescence in a zone less than 100 nm above a cover glass enables selective imaging within the plasma membrane of adherent cells, with markedly improved resolution, lower background, and minimal phototoxicity compared to confocal microscopy and other microscopy-based assays. In the microscope design we have developed, the beams from helium-cadmium (442 nm) and argon (514 nm) lasers are merged and focused through a dove prism at an angle that yields total internal reflection. In this configuration, evanescent wave-excited fluorescence at the glass-water interface can be detected with either high or low magnification, to allow for high-resolution imaging or the study of many cells in parallel. We applied this technique to make parallel measurements of the time-course of insulin-triggered activation of phosphatidylinositol 3-kinase (PI3K) and GLUT4 glucose transporter insertion into the plasma membrane of individual differentiated 3T3L1 adipocytes using a phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P(3)]-binding pleckstrin homology domain fused to CFP, and GLUT4 conjugated to YFP. The technique should have wide applicability to various cell types and diverse signaling processes.

Determination of pah in soil samples by high-performance thin-layer chromatography (HPTLC)

A thin-layer Chromatographic screening-method is presented for the determination of polycyclic aromatic hydrocarbons (PAH) in soil samples. This screening method is intended for a quick overview of the composition of a contaminant. The developed method of separation is well suited for the semi-quantitative determination of PAH in soil samples, and can be used to identify samples that require further analysis by means of GC or HPLC. The separation of 8 PAH groups with a maximum of 2 PAH (discernible by selective excitation of fluorescence) each is possible. The results of the analysis based on PAH group separation can be regarded as semi-quantitative. The extraction of PAH is effected by means of a solvent mixture consisting of n-hexane-acetone (1:1, v/v). The extraction is aided by ultrasonic treatment. The extract is purified by application to an activated silica gel column (solid-phase extraction). The qualitative analysis can be carried out either by visual observation or by fluorimetric scanning (TLC-Scanner). The characteristic fluorescent colours facilitate a reliable visual identification of PAH. The applicability of the method is shown and a comparison to other analytical methods is carried out. Some of the remarkable features of this method are its user-friendly handling, the low consumption of solvents and the applicability without the necessity for extra equipment.

Redistribution of O2 p-states in La 2CuO 4 by Sr-doping

The difference between the oxygen K α X-ray emission spectra of in-plane oxygen in La 2−xSr xCuO 4 (x=0.07; 0.15), measured with selective fluorescence excitation, is compared with the difference of the corresponding oxygen 1 s X-ray absorption spectra. It is concluded that metallic hole band formed under doping originates from σ-bonding and antibonding bands.

Photoionization of single rotational levels in excited B 2Π, C 2Π, and D 2Σ+ states of 14N16O

Rotational state selective fluorescence excitation and two-color resonantly enhanced ionization of nitric oxide has been investigated under collision free conditions. Single photon absorption from a tunable VUV laser source prepared the molecules in the intermediate B 2Π, C 2Π, and D 2Σ+ states energetically close to the dissociation limit of NO. The dissociation of C 2Π(v′=0) at J′=2.5(2Π3/2) and J′=3.5(2Π1/2) as well as B 2Π(v′=7) at J′=4.5(2Π3/2) and J′=7.5 (2Π1/2) was clearly established. Time delayed photoionization was used to measure the lifetimes of stable and weakly predissociative states. Photoionization cross sections of single rotational levels in the electronically excited states have been determined by saturating the ionization step.

Determination of polycyclic aromatic hydrocarbons in unfractionated solid solvent-refined coal by matrix isolation fluorescence spectrometry

An untreated solid solvent-refined coal sample yields matrix isolation fluorescence spectra suitable for identification and determination of polycyclic aromatic hydrocarbons. Selective excitation of fluorescence in Shpol'skii matrices and background suppression using the time resolution capabilities of a dye-laser spectrometer permit the unambiguous identification of PAHs including benzo(a)pyrene. Determination of four polycyclic aromatic hydrocarbons (benzo(a)pyrene, perylene, benz(a)anthracene, and benzo(b)fluorene) in Solvent Refined Coal I is reported, as is the determination, by identical spectrometric technique, of the benzo(a)pyrene content of a NBS shale oil standard reference material. 5 figures, 2 tables.

The transverse Zeeman effect in calcium fluoride crystals containing erbium

A detailed study has been made of the optical transverse Zeeman effect for trivalent erbium ions at sites of tetragonal C4v symmetry in crystals of calcium fluoride with magnetic fields of up to 6T. Interpretation of the optical absorption spectrum is complicated by the presence of magnetically non-equivalent C4v sites and Er3+ sites of other point symmetries, but selective excitation of fluorescence with a tunable dye laser has been used to isolate the spectrum of ions in the required transverse C4v sites. Selective laser excitation spectroscopy has also been used to locate several previously undetected Er3+ crystal-field energy levels for the C4v site. A crystal-field analysis has successfully accounted for the observed transverse Zeeman patterns, including the variation in these patterns as a function of orientation of the magnetic field in the (100) crystal plane and as a function of magnetic field strength.