Development of a narrow-band tunable picosecond dye laser and its application to excited-state lifetime measurement of a chlorinated aromatic hydrocarbon.

The development of a distributed-feedback dye laser, with a pulse width and a line width of 25 ps and 8.78 pm, respectively, is described. Using this nearly Fourier-transform-limited pulse, we measured the first singlet-excited-state lifetime of 1,2,4-trichlorobenzene. The tunable picosecond dye laser developed herein has a potential for the lifetime measurement and the efficient multiphoton ionization of aromatic hydrocarbons with a larger number of chlorine atoms and shorter excited-state lifetimes.     

Development of a compact alpha and beta camera for dismantlement applications

Journal of Radioanalytical and Nuclear Chemistry - A new method has been developed to image surface contamination on-site by short range radiation emitters (such as alpha or beta particle...     

Monitoring the migration behavior of living microorganisms in capillary electrophoresis using laser-induced fluorescence detection with a charge-coupled device imaging system

Remarkably high apparent peak efficiencies (10(6)-10(9) theoretical plates per meter) in capillary electrophoresis (CE) could be achieved in the separation of two different kinds of bacteria and Baker's yeast using poly(ethylene oxide) as a necessary buffer additive. In these applications no deliberate stacking procedure was implemented. Seemingly, the investigated organisms in this study behave differently than molecules under an applied electric field. For molecules, these extremely high efficiencies are very unusual. Using a 488 nm argon-ion laser coupled to a charge-coupled device (CCD) camera it was possible to monitor the migration behavior of stained microorganisms over a length of 10 cm. This part simulates the very beginning of the CE run. In specific cases 60-70% of the monitored detection window could be filled with analyte without significant loss in peak efficiency. For a mixture of two different microorganisms the occurring separation process could be followed in detail. The effect of buffer concentration, polymer type, polymer molecular weight, polymer concentration, pH, and the effect of injection time was investigated. The expansion of fast and reproducible CE separations to other unicellular organisms may become a powerful tool in microbiological science and technology.     

Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.     

Application of charge-coupled device technology for measurement of laser light and fluorescence distribution in tumors for photodynamic therapy

Laser and fluorescence light distributions with applications for photodynamic therapy were measured in mouse tumors using a non-invasive electronic optical imaging system. The system consists of a liquid-nitrogen-cooled, charge-coupled-device (CCD) array camera under computer control with 576 x 384 detection elements having dimensions of 23 microns x 23 microns. The available dynamic range of the array is approx. 10(3), and the effective wavelength range is 400-1000 nm. An interstitially placed cylindrical diffusing optical fiber was used to provide tumor illumination. The light distribution pattern from the fiber was determined by immersing the cylindrical diffusing tip in a fluorescing solution and recording the emission image. Fluorescence imaging facilitates an accurate measurement of light intensity distribution while avoiding problems associated with the directional nature of other detection methods used with diffusing fibers. Radiation-induced fibrosarcoma tumors on C3H mice were grown to about 1 cm diameter for in vivo recording of light distribution from the tumor volume and for determination of effective light penetration distance at 18 wavelengths in the range 458-995 nm. Endogenous tumor fluorescence and Photofrin II fluorescence intensity were measured over the wavelength range 585-725 nm to investigate the possible application of CCD imaging technology for drug distribution measurements. Model experiments were begun to evaluate the relative importance of potential distortions of light distribution measurements using this approach.     

Fluorescence imaging of potassium ions in living cells using a fluorescent probe based on a thrombin binding aptamer-peptide conjugate

When a biotinylated FRET probe based on a peptide-thrombin binding aptamer conjugate was introduced together with streptavidin and biotinylated nuclear export signal peptide into HeLa cells, the resulting ternary complex enabled visualization of K(+) concentration changes in the cell.     

Altered mitochondrial structure and motion dynamics in living cells with energy metabolism defects revealed by real time microscope imaging.

Using the real time microscope (RTM), a system applying new developments in light microscopy, we documented the spatial and temporal dynamics of mitochondrial behavior in human cultured skin fibroblasts. Without the use of stains or probes, we resolved fibroblast mitochondria as dark slender filaments of approximately 0.2 m wide and up to 10 m long, as well as a few smaller ovoid forms. In the living cell, the three most common mitochondrial movements were: (1) small oscillatory movements; (2) larger movements including filament extension, retraction, and branching as well as combinations of these actions; and (3) whole transit movements of single mitochondrial filaments. Skin fibroblasts from patients with mitochondrial complex I deficiency and normal fibroblasts during incubation with rotenone, or antimycin A, contained higher proportions of mitochondria in the swollen filamentous forms, nodal filaments, and ovoid forms rather than the slender filamentous forms in normal cells. Interestingly, decreased motility was observed with the more ovoid mitochondrial forms compared to the filamentous forms. We conclude that mitochondrial morphology and dynamic motion are strongly associated with changes in mitochondrial energy metabolism. Images documenting our observations are presented both at single time points and as QuickTime videos.     

Excited state properties of a new photoinitiating system for laser imaging applications

The design of an efficient photosenzitizer/photoinitiator combination is partly governed by a better understanding of the excited state processes involved. In the present paper, the photochemistry of a thiopyrylium salt (TP) as photosensitizer and of a tetraperester of benzophenone, tetra t-butyl peroxycarbonylbenzophenone (BTTB) as initiator, used in laser imaging applications has been investigated. The reactivity of the triplet states of both compounds BTTB and TP was studied by time-resolved laser absorption spectroscopy. The laser excitation of TP leads to a long-lived triplet state (lifetime 20–25 μsec) and a second species arising from the triplet state which cannot yet be characterized. Under laser excitation, BTTB gives a longlived transient arising from the cleavage of the peroxy bond. The short-lived triplet state cannot be observed on the nanosecond timescale. The triplet state lifetime has been evaluated from quenching experiments and found to be about 1 ns in acetonitrile. The deactivation of the TP triplet state by BTTP was considered, the deactivation constant was found to be equal to 6.6 × 10⁷ m^?1/sec in acetonitrile. The initiation mechanism is discussed.     

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.     

Transient absorption spectra of pinacyanol and cyanine photoisomers obtained with a sync-pumped picosecond dye laser and independently tunable probe light

We have measured the absorption difference spectra and quantum yields of photoisomers of pinacyanol iodide and 1,1′-diethyl-4,4′-cyanine iodide on a picosecond time scale using syne-pumped picosecond dye laser pulses for excitation and independently tunable analyzing light.     

A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence

In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores. This flexibility and excitation accuracy is key to multicolour applications and future adaptation of the instrument to address the application requirements and newly emerging dyes.     

A fluorescent probe directly detect peroxynitrite based on boronate oxidation and its applications for fluorescence imaging in living cells

We present the design, synthesis, spectroscopy, and biological applications of PyBor, a new type of fluorescent probe for peroxynitrite detection in aqueous solution and living cells. The probe employs pyrene as the fluorophore, and is equipped with a chemically responsive unit boronate. The fluorescent probe can selectively detect peroxynitrite with fluorimetric determination and high-performance liquid chromatography analyses in aqueous solution and RAW264.7 cells intracellular free extracts. We also study our probe to time dependent peroxynitrite release from 3-morpholinylsydnonimine hydrochloride. Confocal microscopy experiments using mouse macrophage cell line RAW264.7 show that PyBor is able to detect the different intracellular peroxynitrite levels. In addition, we have performed quantum chemical calculations with TD-DFT/M06/TZVP level with COSMO solvation model basis sets using a suite of Gaussian 09 programs to provide insights into the structure optical properties of PyBor and PyOH.     

Fluorescence behavior of 7-hydroxycoumarine excited by one-photon and two-photon absorption by means of a tunable dye laser

The fluorescence spectrum of 7-hydroxycoumarine in ethanol excited by a pulsed tunable dye laser reveals different features when excitation proceeds via one-photon and two-photon absorption. In the former case the spectrum shows two peaks delayed in time by approximately 2 ns and characterized by different lifetimes. The relative intensity of these peaks is unaffected by significant changes in the dye concentration and therefore the formation of an “exciplex” species seems to be here confirmed. In the spectrum obtained by two-photon excitation the second peak at longer wavelength is absent. Results are presented for both room and liquid nitrogen temperatures as well as for other solvents such as glycerin and EPA. These results are considered to be important for the evaluation of absolute two-photon cross sections where the quantum efficiencies of one- and two-photon processes are assumed to be the same.     

Development of FRET-based ratiometric fluorescent Cu2+ chemodosimeters and the applications for living cell imaging

Coumarin-rhodamine-based compounds 1a,b were rationally designed and synthesized as novel FRET ratiometric fluorescent chemodosimeters. Ratiometric chemodosimeters 1a,b exhibit several favorable features, including a large variation in the emission ratio, well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell membrane permeability. Importantly, these excellent attributes enable us to demonstrate ratiometric imaging of Cu(2+) in living cells by using these novel ratiometric fluorescent chemodosimeters.     

Radiative lifetime of the Te2 B0+u and A0+u states excited by a pulsed dye laser

A pulsed dye laser has been used to measure the radiative lifetimes and quenching rates of transitions of the B0⁺_u and A0⁺_u states of Te₂. The observed zero pressure lifetimes vary from 55 to 730 ns. The quenching rates vary from 0.9 × 10⁶ to 40 × 10⁶ s⁻¹ Torr⁻¹.     

Fluorescent chemodosimeter for Cys/Hcy with a large absorption shift and imaging in living cells

A novel molecule T1 with efficient intramolecular charge transfer was designed as a fluorescent chemodosimeter for cysteine (Cys) and homocysteine (Hcy). Upon addition of Cys/Hcy, T1 exhibited greatly enhanced fluorescence intensity as well as a large absorption peak shift (70 nm), and can be used for bioimaging of Cys/Hcy in living cells and detection in human plasma by visual color change. The detection mechanism was proved by (1)H NMR, mass spectrometry analysis and Gaussian calculations.