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PubMed2006

Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization.

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 microm, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 microm3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

Animals↗
PubMed2006

Resonance-enhanced multiphoton ionization time-of-flight mass spectrometry for detection of nitrogen containing aliphatic and aromatic compounds: resonance-enhanced multiphoton ionization spectroscopic investigation and on-line analytical application.

Resonance-enhanced multiphoton ionization (REMPI) combined with time-of-flight mass spectrometry (TOFMS) is an analytical method capable of on-line monitoring of trace compounds in complex matrices. A necessary prerequisite for substance selective detection is spectroscopic investigation of the target molecules. Several organic nitrogen compounds comprising aliphatic and aromatic amines, nitrogen heterocyclic compounds, and aromatic nitriles are spectroscopically investigated with a tunable narrow bandwidth optical parametric oscillator (OPO) laser system providing a scannable wavelength range between 220 and 340 nm. These species are known as possible precursors in fuel-NO formation from combustion of solid fuels such as biomass and waste. A newly conceived double inlet system was used in this study, which allows rapid change between effusive and supersonic molecular beams. The resulting REMPI spectra of the compounds are discussed with respect to electronic transitions that could be utilized for a selective ionization of these compounds in complex mixtures such as combustion and process gases. The practicability of this approach is demonstrated by wavelength selected on-line REMPI-TOFMS detection of aniline and cyanonaphthalene in the burning chamber of a waste incineration plant. REMPI mass spectra recorded at different excitation wavelengths as well as variations in time show the utilization of species-selective REMPI-TOFMS detection for on-line monitoring of crucial substances in pollutant formation.

Air Pollutants↗
PubMed2006

One- and two-photon induced fluorescence of Pacific Blue-labeled human serum albumin deposited on different core size silver colloids.

We studied one- and two-photon induced fluorescence of Pacific Blue (PB)-labeled human serum albumin (HSA) in the presence of different size silver colloids. The PB fluorescence emission intensity was observed with small (30-40 nm) and large (about 120 nm) colloids and compared with PB emission in absence of colloids. For the system with a small core size colloids we did not detect any fluorescence enhancement with one-photon excitation and the enhancement observed with two-photon excitation was about 2.5-fold. In contrast, for large silver colloids we observed about a 2-fold increase in PB fluorescence brightness for one-photon excitation, and the enhancement with two-photon excitation excided 13-folds. Much stronger increases in brightness observed with two-photon excitation, compared to one-photon excitation, indicate a dominant role of enhanced local field in fluorescence enhancement on silver colloids in solutions.

Colloids↗
PubMed2007

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy.

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.

Biofilms↗
PubMed2005

Molecular photobleaching kinetics of Rhodamine 6G by one- and two-photon induced confocal fluorescence microscopy.

Under high-excitation irradiance conditions in one- and two-photon induced fluorescence microscopy, the photostability of fluorescent dyes is of crucial importance for the detection sensitivity of single molecules and for the contrast in fluorescence imaging. Herein, we report on the dependence of photobleaching on the excitation conditions, using the dye Rhodamine 6G as a typical example. The different excitation modes investigated include 1) one-photon excitation into the first-excited singlet state in the range of 500 to 528 nm by continuous wave and picosecond-pulsed lasers and 2) two- and one-photon excitation to higher-excited singlet states at 800 and 350 nm, respectively, by femtosecond pulses. Experimental strategies are presented, which allow resolving the photophysics. From single-molecule trajectories and fluorescence correlation spectroscopy, as well as with a simple theoretical model based on steady-state solutions of molecular rate equation analysis, we determined the underlying photobleaching mechanisms and quantified the photokinetic parameters describing the dependence of the fluorescence signal on the excitation irradiance. The comparison with experimental data and an exact theoretical model show that only minor deviations between the different theoretical approaches can be observed for high-pulsed excitation irradiances. It is shown that fluorescence excitation is in all cases limited by photolysis from higher-excited electronic states. In contrast to picosecond-pulsed excitation, this is extremely severe for both one- and two-photon excitation with femtosecond pulses. Furthermore, the photostability of the higher-excited electronic states is strongly influenced by environmental conditions, such as polarity and temperature.

Biochemistry↗
PubMed2006

Quantitative linear unmixing of CFP and YFP from spectral images acquired with two-photon excitation.

BACKGROUND: Spectrally distinct fluorescent proteins (FPs) have been developed permitting the visualization of several proteins simultaneously in living cells. The emission spectra of FPs, in most cases, overlap, making signal separation based on filter technology inefficient and in cases of bleed-through, inaccurate. Spectral imaging can overcome these obstacles through a process called linear unmixing. Given a complex spectra composed of multiple fluorophores, linear unmixing can reduce the complex signal to its individual, weighted, component spectra. Spectral imaging with two-photon excitation allows the collection of nontruncated emission spectra. The accuracy of linear unmixing under these conditions needs to be evaluated. METHODS: Capillaries containing defined mixtures of CFP and YFP were used to test the accuracy of linear unmixing using spectral images obtained with two-photon excitation. RESULTS: Linear unmixing can be accurate when wavelength and power-matched reference spectra are provided to the algorithm. Linear unmixing errors can occur due to (1) excitation laser contamination of emission signals, (2) the presence of FRET, (3) poor selection of excitation wavelength, and (4) failure to background subtract reference spectra. CONCLUSIONS: Linear unmixing, when judiciously performed, can accurately measure the abundance of CFP and YFP in mixed samples, even when their relative intensities range from 90:1.

Algorithms↗
PubMed2006

Macrophages promote collagen fibrillogenesis around terminal end buds of the developing mammary gland.

Development of the ductal network in the mammary gland is dependent in part on the presence of macrophages. Here we utilize multi-photon microscopy and second harmonic generation to describe terminal end bud 3-dimensional structure and the organization of the surrounding collagen matrix. We have applied this approach to analyze the effect of macrophage deficiency on terminal end bud structure and collagen organization, using mice homozygous for a null mutation in the colony stimulating factor-1 gene (Csf1op/Csf1op). Primary terminal end buds have an oblong shape, with long collagen I fibers close to the neck of the terminal end bud and radiating upwards in the direction of growth. Around the terminal end buds, the amount of total collagen I detected by antibody staining was not affected by macrophage deficiency. However the amount of collagen I organized into long fibers, detected by second harmonic generation signal, was reduced in Csf1op/Csf1op mice. Macrophage deficiency also caused terminal end buds to be rounder and shorter. These studies reveal a role for macrophages in collagen fibrillogenesis and in organization of the structure of terminal end buds.

Animals↗
PubMed2004

Two-photon imaging of brain pericytes in vivo using dextran-conjugated dyes.

Pericytes in the central nervous system (CNS) are hypothesized to be involved in important circulatory functions, including local blood flow regulation, angiogenesis, immune reaction, and regulation of blood-brain barrier. Despite these putative functions, functional correlates of pericytes in vivo are scarce. We have labeled CNS pericytes using the dextran-conjugated fluorescent calcium indicator Calcium Green I and imaged them in somatosensory cortex of the mouse in vivo. Intracellular calcium concentration in pericytes showed spontaneous surges lasting for several seconds. Furthermore, population bursts of neuronal activity were associated with increased Ca(2+) signal in a portion of the pericytes. Selective in vivo labeling of pericytes with functional markers may help reveal their physiological function in neuronal activity-associated regulation of local cerebral blood flow.

Animals↗
PubMed2004

Distance measurement by circular scanning of the excitation beam in the two-photon microscope.

We developed a method to measure relative distances with nanometer accuracy of fluorescent particles of different color in a two-photon scanning fluorescence microscope, with two-channel photon counting detection. The method can be used in the 10-500 nm range, for distances below the resolution limit of standard far field microscopy. The proposed technique is more efficient than the methods using raster scanning. To achieve maximum sensitivity in the radial direction, the excitation beam is moved periodically in a circular orbit with a radius of the size of the point spread function. The phase and the modulation of the periodic fluorescence signal, calculated by fast Fourier transform, gives the phase and the radial distance of the particle from the center of scanning. The coordinates of particles are recovered simultaneously in the two channels and the relative distance is calculated in real time. Particles can be tracked by moving the center of scanning to the recovered position, while measuring the distance from the second particle. Intensity data are saved and fitted later by a model accounting for light leakage between the channels. The total number of detected photons limited the accuracy of the position and distance measurement. Experiments demonstrating the advantages of the method were performed on fluorescent spheres and single dye molecules immobilized on quartz surface.

Algorithms↗
PubMed2004

Antecedents of two-photon excitation laser scanning microscopy.

In 1931, Maria Göppert-Mayer published her doctoral dissertation on the theory of two-photon quantum transitions (two-photon absorption and emission) in atoms. This report describes and analyzes the theoretical and experimental work on nonlinear optics, in particular two-photon excitation processes, that occurred between 1931 and the experimental implementation of two-photon excitation microscopy by the group of Webb in 1990. In addition to Maria Göppert-Mayer's theoretical work, the invention of the laser has a key role in the development of two-photon microscopy. Nonlinear effects were previously observed in different frequency domains (low-frequency electric and magnetic fields and magnetization), but the high electric field strength afforded by lasers was necessary to demonstrate many nonlinear effects in the optical frequency range. In 1978, the first high-resolution nonlinear microscope with depth resolution was described by the Oxford group. Sheppard and Kompfner published a study in Applied Optics describing microscopic imaging based on second-harmonic generation. In their report, they further proposed that other nonlinear optical effects, such as two-photon fluorescence, could also be applied. However, the developments in the field of nonlinear optical stalled due to a lack of a suitable laser source. This obstacle was removed with the advent of femtosecond lasers in the 1980s. In 1990, the seminal study of Denk, Strickler, and Webb on two-photon laser scanning fluorescence microscopy was published in Science. Their paper clearly demonstrated the capability of two-photon excitation microscopy for biology, and it served to convince a wide audience of scientists of the potential capability of the technique.

History, 20th Century↗
PubMed2004

Fluorescence lifetime imaging by time-correlated single-photon counting.

We present a time-correlated single photon counting (TCPSC) technique that allows time-resolved multi-wavelength imaging in conjunction with a laser scanning microscope and a pulsed excitation source. The technique is based on a four-dimensional histogramming process that records the photon density over the time of the fluorescence decay, the x-y coordinates of the scanning area, and the wavelength. The histogramming process avoids any time gating or wavelength scanning and, therefore, yields a near-perfect counting efficiency. The time resolution is limited only by the transit time spread of the detector. The technique can be used with almost any confocal or two-photon laser scanning microscope and works at any scanning rate. We demonstrate the application to samples stained with several dyes and to CFP-YFP FRET.

Animals↗
PubMed2004

Probing microscopic diffusion by 2-photon flash photolysis: measurement of isotropic and anisotropic diffusion in lens fiber cells.

Two-photon excited flash photolysis (TPEFP) was used to photorelease caged fluorescein in test solutions and inside fiber cells of the eye lens. Accurate alignment between the focus of the IR beam and the probe beam from the confocal microscope was achieved with an accessory focussing lens and computer models of diffusion were fit to experimental data to extract apparent diffusion coefficients. Inside a fiber cell, the diffusion coefficient for fluorescein was 4 x 10(-7) cm(2)/s at 21 degrees C, a value an order of magnitude lower than observed in free solution. Fluorescence also diffused between fiber cells via gap junctions. In the periphery, diffusion between cells occurred mainly in a radial direction while deep in the lens the diffusion between cells appeared more isotropic. Diffusion between cells was slower than inside cells and corresponded to less than approximately 1% of the area between cells being available for diffusion. This value is in good agreement with that expected from measurements of gap junction structure and packing density if a 1-1.5-nm aqueous gap junction pore is nearly always open.

Animals↗
PubMed2004

Practical limits of resolution in confocal and non-linear microscopy.

Calculated and measured resolution figures are presented for confocal microscopes with different pinhole sizes and for nonlinear (2-photon and second harmonic) microscopes. A modest degree of super-resolution is predicted for a confocal microscope but in practice this is not achievable and confocal fluorescence gives little resolution improvement over widefield. However, practical non-linear microscopes do approach their theoretical resolution and therefore show no resolution disadvantage relative to confocal microscopes in spite of the longer excitation wavelength.

Animals↗
PubMed2004

Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization.

Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.

Animals↗