Fluorescence lifetime imaging: an application to the detection ofskin tumors

A portable system based on fluorescence lifetime imaging has been developed and tested for the detection of skin tumors in humans. The Heme precursor δ-aminolevulinic acid, which promotes the preferential accumulation of the endogenous Protoporphyrin IX (PpIX) in proliferative tissues, is used as an exogenous marker to target the tumor, δ-aminolevulinic acid is topically administered to the patient 1 h before the measurement. Then, using a gated intensified camera, two or more images of the sample are acquired after different delays with respect to the excitation pulses. The images are processed in real time in order to calculate the spatial map of the fluorescence decay time of the sample. The localization of the tumor is based on the longer decay time detected in neoplastic tissues as a result of the stronger emission of PpIX, which has a long decay time, and the reduction in the short living natural tissue fluorescence     

Photodynamic therapy in ocular vascular disease

Photodynamic therapy (PDT) is a novel therapeutical approach which is noninvasive and potentially selective for neoplastic pathologies. Association of photosensitizers with low density lipoprotein (LDL) leads to direct targeting of the treated lesions with enhanced efficiency and selectivity. LDL-mediated PDT is particularly useful in the treatment of neo-vascular structures since LDL receptors are abundantly expressed on vascular endothelial cells. To evaluate the potential of selective photodynamic vasoocclusion in ocular neo-vascular disease a sequence of experiments was designed: Efficiency of the LDL-carrier was tested in vitro, the system was then transferred to an in vivo model demonstrating a vascularized neoplasm. Occlusion was successfully performed in experimentally induced neovascularization in the cornea, while selective photothrombosis of subretinal vasculature revealed lack of collateral damage. The experimental results were used to establish a first clinical trial for the use of PDT in age-related macular degeneration, one of the leading causes for blindness     

PDT effect of different photo sensitizers on a single nerve cell:electrophysiological and pharmacological study

The study of the single neuron response to photodynamic effect provides information on the dynamics of cytotoxic events leading to cell death and allows comparison of the phototoxicity of different photosensitizers. Isolated crayfish stretch receptor neurons were incubated 30 min with different concentrations of various photosensitizers and then irradiated with a He-Ne laser (632.8 nm; 0.3 W/cm²) until irreversible firing abolition. The dynamics of neuron impulse response was continuously recorded throughout the experiment. The following photosensitizers were studied: hematoporphyrin derivatives Photoheme and Photofrin II, 6 deuteroporphyrin IX derivatives and sulphonated aluminum and zinc phthalocyanines. Nerve cells were found to be insensitive to either He-Ne laser irradiation or photosensitization alone, but very vulnerable to photodynamic effect: neurons changed their firing rate and irreversibly ceased their impulse activity at nanomolar (phthalocyanines and hematoporphyrin derivatives) and even pikomolar (deuteroporphyrin derivatives) concentrations of photosensitizers. The dynamics of the neuron responses to photodynamic effects included stages of firing activation and/or inhibition followed by irreversible firing abolition. It depended on the photosensitizer type and concentration. Decrease of extracellular pH or action of pharmacological agents increasing intracellular Ca²⁺ concentration or inhibiting ATP synthesis exacerbated photodynamic neuron injury. In contrast, agents decreasing intracellular calcium concentration or bioenergetic substrates protected the neuron against photodynamic injury. The dependencies of neuron lifetimes on the photosensitizer concentration provide comparison of photodynamic efficiencies of different photosensitizers     

Photothermal imaging of nanoparticles and cells

This review summarizes the findings of recent applications of time-domain far-field photothermal (PT) technique to the detection and imaging of nanoscale absorbing particles. This two-beam (pump-probe) technique is based on time-resolved PT visualization of laser-induced thermal effects around nanoparticles. Imaging is accomplished, after an adjustable time delay after the pump laser pulse, with a second probe beam that senses the nanotarget. Using a tunable optical parametric oscillator laser (wavelength, 420 to 570 nm; energy, 0.1-300 /spl mu/J; pulse width, 8 ns) as the pump laser and a Raman shifter (639 nm, 10 nJ, 13 ns) as the probe laser, with a tunable delay of 0 to 5 000 ns of the probe pulse relative to the pump pulse, this approach has demonstrated the capability to visualize nanoscale gold particles (2 to 250 nm) alone and in cells, liposomes (30 to 90 nm), neutral red-stained particles (30 to 500 nm), and polystyrene beads. Different applications of the time-resolved PT technique are discussed, including imaging of absorbing cellular nanostructures and optimization of selective killing of cancer cells and bacteria.     

Magnetic resonance imaging of coronary arteries and coronary stenosis

Coronary artery imaging is an important investigation for the management of coronary artery disease. Alternative noninvasive imaging would be useful, but the small caliber and tortuosity of the coronary vessels and cardiac and respiratory motion create formidable imaging problems. We first studied 21 normal subjects and 5 with coronary artery disease established by X-ray contrast angiography, of whom 2 had undergone bypass grafting. Of these, 22 were imaged successfully. Identification of the artery was possible for the left main stem, left anterior descending, right coronary, and left circumflex arteries respectively in 95%, 91%, 95%, and 76%. The arterial diameter at the origin could be measured in 77%, 77%, 81%, and 63%. The mean ±SD arterial diameter in each case (4.8±0.8, 3.7±0.5, 3.9±0.9, and 2.9±0.6 mm) was not significantly different from reference values (allp=ns). The mean length of artery visualized was 10.4±5.2,46.7±22.8,53.7±27.9, and 26.3±17.5 mm. In 12 normal males, the total coronary area was 30.9±9.2 mm² and the ratio compared with body surface area was 16.4±4.4 mm² m^–2 (bothp=ns compared with reference values). In seven patients, with X-ray contrast coronary angiography, the proximal arterial diameter measured by magnetic resonance was 3.9±1.1 mm, and by X-ray contrast angiography 3.7±1.0 mm (p=ns). We then studied 17 patients with angina. Imaging of just the relevant artery was performed and analysis was blinded to the X-ray angiography results. Stenosis was identified on the magnetic resonance (MR) images by localized reduction in vessel signal intensity. Stenosis location by MR was assessed by measurement of its distance from a reference vessel, with correlation to the X-ray findings. X-ray coronary angiography showed 23 stenoses of which 15 (65%) were correctly located by blind assessment of the MR images. Of the eight remaining stenoses, a further 5 (63%) were correctly located on the MR images after retrospective comparison (overall sensitivity 87%). There were three lesions thought to represent stenosis by MR, which on review of the X-ray angiogram proved to be a minor stenosis <50% (two cases) or a tortuous vessel (one case). Greater signal loss was seen in the more severe stenoses. The stenosis length by MRI was greater than by X-ray (8.4 versus 5.1 mm,p<0.001). The overestimation of stenosis length may be due to turbulence.     

Combining two-photon excitation with fluorescence lifetime imaging

Two-photon excitation (TPE) imaging with a microscope objective that is properly matched to the sample results in a two- to four-times larger penetration depth than with confocal laser scanning microscopy. The pulsed light source present in a TPE microscope makes time-gated fluorescence lifetime imaging an attractive alternative for quantitative imaging of ion concentrations. Using TPE fluorescence lifetime imaging, in-depth imaging of pH in biofilm can be accomplished     

Deuterium NMR study of the MP-2269: albumin interaction — a step forward to the dynamics of non-covalent binding

MP-2269, the Gd(III) complex of 4-pcntylbicyclo[2.2.2] octane-l-carboxyl-di-L-aspartyl-lysinc-derived-DTPA, is a small Gd-agent that binds non-covalently to scrum albumin in vivo to assume the enhanced relaxivities associated with macromolecular agents, (due in part to increased rotational correlation time, τ_R). To further explore the fundamental parameters that govern the dynamics of water proton relaxation enhancement by this prototypical albumin-binding agent, the rotational correlation time (τ_R) for the deuterated La(III) analog of MP-2269 has been independently measured in the presence and absence of 4% albumin using²H-NMR approaches. The diamagnetic La(III) analog of MP-2269 was deuterated at the α-position of the carbonyl groups.²H-NMR studies were conducted at 7.05T (46 MHz) and 310°K on a Bruker NMR spectrometer. Spectral deconvolution permitted calculation of transverse relaxation rates, 1/T₂, from the NMR linewidths and subsequently, τ_R. The results yielded a τ_R of the albumin bound complex of ∼ 8 ns. This value is intermediate between those earlier estimated by¹⁷O-NMR (∼ 1 ns) and¹H-NMRD (∼ 20–50 ns) and significantly shorter than that of albumin. The²H-NMR study results also indicate that the exchange between free and albumin-bound forms of the La(III) analog is slow (exchange lifetimes > 1 ms). This slow exchange does not affect the water residence lifetimes (τ_M 140-280 ns).     

Single-Pair FÖrster Resonance Energy Transfer With Multiparametric Excitation and Detection

Forster resonance energy transfer (FRET) between a donor and an acceptor dye molecule is a common method to study the distances between single molecules in living cells in the nanometer range. Quantitative distance measurements are difficult to obtain in spite of the strong distance dependency of the energy transfer efficiency. One problem is the incomplete fluorescent labeling of the molecules, which leads to the so-called zero-efficiency peak caused by FRET pairs with missing or nonfluorescing acceptor dyes. Other problems occur due to spectral bleed through, direct acceptor excitation, and the difficulty to obtain the quantum efficiencies of the dyes and the detection efficiencies of the corresponding detectors. In order to correct these defects, the donor as well as the acceptor are excited alternatingly using pulsed interleaved excitation. Time-correlated single-photon counting enables the measurement of fluorescence lifetime; hence, the FRET efficiency can also be derived from the decrease of the donor fluorescence lifetime. The universal time-tagged time-resolved data format allows to retrieve all the necessary information from the fluorescence photons detected during the measurement.     

Bioelectric changes in single neuron under photodynamic effect:comparison of different photosensitizers

An isolated crayfish mechanoreceptor neuron was used as a new test object for a cytophysiological study of various photosensitizers (methylene blue, janus green B, protoporphyrin IX, chlorins e6 and p6). The study of single neuron response provides evaluation of the dynamics of cytotoxic events leading from initial threshold changes to cell death. The typical response of a photosensitized neuron to He-Ne laser irradiation was firing acceleration and then irreversible block of spike generation. Crayfish neurons were very sensitive to the photodynamic effect; they changed firing rate and died at nanomolar concentrations of photosensitizers. Dependencies of spike acceleration rate and neuron lifetime on photosensitizer concentration allowed one to compare efficiencies of different photosensitizers. Chlorin p6, chlorin e6, and methylene blue were the most potent photosensitizers     

Effects of submicrosecond, high intensity pulsed electric fields on living cells - intracellular electromanipulation

Development of technology to produce nanosecond duration pulsed electric fields has allowed examination of the effects of ultrashort duration, high intensity electric fields on living cells. Theoretically, high intensity (MV/m) electric field applications with durations of less than one microsecond, when shortened toward nanoseconds, should increasingly affect intracellular rather than surface membranes of living cells. Experimentally, square-wave, 60 ns duration, high energy (36-53 kV/cm) pulses applied in trains of 1-10 pulses result in progressive increases in the numbers of permeabilized intracellular granules in a human eosinophil cell model-without large surface membrane effects. Electron micrographic examination of cells treated in this way demonstrates alteration of intracellular granule morphology consistent with permeabilization of granule membrane, i.e., intracellular electromanipulation. Continuous microscopic examination of individual living cells exposed to long or short duration pulsed electric field applications shows that permeabilization of surface membrane (median 5 minutes) with anodic preference (electroporation) and prompt cellular swelling follow a single, long duration (100 microsecond) pulse. In contrast, after a single short duration (60 ns) pulse, onset of surface membrane permeability is delayed (median 17 minutes), the increased permeability shows no anodic preference, and cellular swelling is absent suggesting that these effects are due to intracellular electromanipulation rather than direct effects on the surface membrane. Submicrosecond, intense pulsed electric fields applied to living cells achieve preferential effects on intracellular rather than surface membranes, potentially providing new approaches for selective/generalized cell or tissue ablation, growth stimulation and tissue remodeling.     

Investigating up-conversion fluorescence of Phloxine B

The steady-state and time-resolved emission of Phloxine B was examined when excited with 90 fs pulses from a mode-locked Ti:sapphire laser. Above the excitation wavelength (775 nm), Phloxine B was found to display two-photon excitation fluorescence with estimated cross-section for excitation of 0.87×10^-49 cm⁴ s/photon, and fluorescence spectrum consistent with one-photon excitation. Over the excitation wavelength range from 590 to 650 nm, Phloxine B was found to display one-photon excitation up-conversion fluorescence. The up-conversion fluorescence of Phloxine B was confirmed by frequency-domain measurements. The intensity decays revealed different double-exponential intensity decays for one-photon excitation at 390 nm and 633 nm. The longer fluorescence lifetimes were observed with 633 nm excitation. Hence, anti-Stokes excitation of Phloxine B causes delay emission at shorter wavelengths. Time-resolved anisotropy decay measurements revealed similar correlation times, but different amplitudes, as has been observed previously for two- versus one-photon excitation     

Quantification of metabolites from single-voxelin vivo 1H NMR data of normal human brain by means of time-domain data analysis

We present here a combination of time-domain signal analysis procedures for quantification of human brainin vivo ¹H NMR spectroscopy (MRS) data. The method is based on a separate removal of a residual water resonance followed by a frequency-selective time-domain line-shape fitting analysis of metabolite signals. Calculation of absolute metabolite concentrations was based on the internal water concentration as a reference. The estimated average metabolite concentrations acquired from six regions of normal human brain with a single-voxel spin-echo technique for theN-acetylaspartate, creatine, and choline-containing compounds were 11.4±1.0,6.5±0.5, and 1.7±0.2 mmol kg^–1 wet weight, respectively. The time-domain analyses ofin vivo ¹H MRS data from different brain regions with their specific characteristics demonstrate a case in which the use of frequency-domain methods pose serious difficulties.     

Perfecting and extending the near-infrared imaging window

In vivo fluorescence imaging in the second near-infrared window (NIR-II) has been considered as a promising technique for visualizing mammals. However, the defi...     

Photodynamic injury of isolated neuron and satellite glial cells: morphological study

Potential application of photodynamic therapy (PDT) for treatment of brain tumors including gliomas is currently studied. However, PDT effect on normal glial cells is unknown. We have studied PDT effect using a simple model system - isolated crayfish mechanoreceptor consisting of receptor neuron and surrounding glial cells. Sulphonated alumophthalocyanine photosens (AlPcS/sub n/) localizes predominately in the glial envelope around the neuron. PDT treatment with 10/sup -7/ M photosens inhibits and then irreversibly abolishes neuron activity for approximately 20 min. Then, in 1.7 h after PDT, the plasma membrane loses integrity and extracellular propidium iodide may enter into the cytosol and stain the nuclear chromatin. Neuron nucleus progressively shrinks but apoptotic nucleus fragmentation is not occurred. Such neuron death has been defined as delayed necrosis. Nuclei of the satellite glial cells also shrink. In 8 h after PDT treatment, some of them become fragmented that is characteristic for apoptosis, whereas others lose the plasma membrane integrity and die through necrosis. However, under PDT treatment glial cells not only die but also proliferate and their number is increased. This gliosis is probably aimed to neuron saving.     

In vitro imaging of single living human umbilical vein endothelial cells with a clinical 3.0-T MRI scanner

Iron oxide-labelled, single, living human umbilical vein endothelial cells (HUVECs) were imaged over time in vitro using a clinical 3.0-T magnetic resonance (MR) microscopy system. Labelling efficiency, toxicity, cell viability, proliferation and differentiation were assessed using flow cytometry, magnetic cell sorting and a phenanthroline assay. MR images were compared with normal light and fluorescence microscopy. Efficient uptake of iron oxide into HUVECs was shown, although with higher label uptake dose-dependent cytotoxic effects were observed, affecting cell viability. For MR imaging, a T2* weighted three-dimensional protocol was used with in-plane resolution of 39×48μm² and 100-μm slices with a scan time of 13 min. MRI could detect living cells in standard culture dishes at single-cell resolution, although label loss was observed that corresponded with the intracellular iron measurements. MR microscopy using iron oxide labels is a promising tool for studying HUVEC migration and cell biology in vitro and in vivo, but possible toxic effects of label uptake and loss of label over time should be taken into account.     

Analytical modeling and optimization of terahertz time-domainspectroscopy experiments, using photoswitches as antennas

From classical charge transport equations and dipole radiation equations, we derive a simple analytical model for the signal detected in terahertz time-domain spectroscopy using photoconducting antennas. The validity of our model is checked by comparison to published calculated results and to experimental data we have measured. This expression depends mostly on the carrier lifetime in the antennas' semiconductor material and on the laser pulse duration. The analytical form of the detected terahertz (THz) signal allows us to separate the contribution of the different experimental parameters. Together with a detailed noise analysis in THz time-domain spectroscopy, we optimize the parameter values in order to achieve the largest dynamics of measurement     

Enhanced Near-Field Imaging Contrasts of Silver Nanoparticles by Localized Surface Plasmon

Near-field images of Ag nanoparticles are studied using a near-field scanning optical microscopy (NSOM) operating at illumination mode with blue, green, and red probing lights. The obtained far-field intensity contrast between the nanoparticle and background strongly depends on the sizes of nanoparticles and the wavelength of probing light. Experimental NSOM images supported by theoretical 3-D finite-difference time-domain simulation demonstrate that the intensity contrast is enhanced at wavelength close to the localized surface plasmon resonance (LSPR) peak of the nanoparticle. The abilities to distinguish nanoparticles with different LSPR properties on the same substrate can lead to a material-specific NSOM imaging technique.      

Surface switching characteristics of variable permittivity dielectrics

Flashover voltage, lifetimes, and switch performance of insulators utilizing square thin and thick film electrodes were examined to determine the viability of using thin electrodes for reliable surface discharge switching. Gold, silver, and platinum were sputtered (0.25 /spl mu/m) and screen printed (15 /spl mu/m) onto Al/sub 2/O/sub 3/, TiO/sub 2/, and modified BaTiO/sub 3/ (MBT), then tested in air at 10/sup 5/ Pa, under vacuum (10/sup -3/ torr), and while immersed in an insulating fluid, SF-2 (manufactured by 3M). For the measured range of 0.5 to 3 mm in air, the flashover voltage for all three insulators was found to have a linear dependence on the electrode separation distance with 15 /spl mu/m thick screen printed electrodes and a square root dependence with 0.25 /spl mu/m thick sputtered electrodes. Delay times of approximately 20 ns with a corresponding jitter of 6 ns were observed across all three insulators under triggered flashover. Insulators in air with sputtered electrodes had lifetimes of approximately 5 flashovers for dc flashover and 40 for triggered flashover. Screen printed TiO/sub 2/ and MBT had dc lifetimes of approximately 10 flashovers in air, and 3 flashovers in vacuum and SF-2. Screen printed TiO/sub 2/ and MBT had triggered lifetimes of greater than 200 flashovers in air, and <3 flashovers in vacuum and SF-2. Screen printed Al/sub 2/O/sub 3/ had dc and triggered lifetimes of greater than 200 flashovers in air, vacuum and SF-2. Insulator failure during dc flashover was determined to be due to the formation of a conductive channel between the anode and cathode. Formation of the channel was attributed to insulator thermal and dielectric properties and the presence of vaporized electrode species in the gap region during flashover.     

Efficient calculation of lifetime based direct tunneling through stacked dielectrics

We present the efficient simulation of lifetime based tunneling in CMOS devices through layers of high-κ dielectrics which relies on the precise determination of quasi-bound states (QBS). The QBS are calculated using the perfectly matched layer (PML) method. Introducing a complex coordinate stretching allows artifical absorbing layers to be applied at the boundaries. The QBS appear as the eigenvalues of a linear, non-Hermitian Hamiltonian where the QBS lifetimes are directly related to the imaginary part of the eigenvalues. The PML method turns out to be an elegant, numerically stable, and efficient method to calculate QBS lifetimes for the investigation of direct tunneling through stacked gate dielectrics.     

Ultrafast method for the analysis of fluorescence lifetime imaging microscopy data based on the Laguerre expansion technique

We report a new deconvolution method for fluorescence lifetime imaging microscopy (FLIM) based on the Laguerre expansion technique. The performance of this method was tested on synthetic and real FLIM images. The following interesting properties of this technique were demonstrated. 1) The fluorescence intensity decay can be estimated simultaneously for all pixels, without a priori assumption of the decay functional form. 2) The computation speed is extremely fast, performing at least two orders of magnitude faster than current algorithms. 3) The estimated maps of Laguerre expansion coefficients provide a new domain for representing FLIM information. 4) The number of images required for the analysis is relatively small, allowing reduction of the acquisition time. These findings indicate that the developed Laguerre expansion technique for FLIM analysis represents a robust and extremely fast deconvolution method that enables practical applications of FLIM in medicine, biology, biochemistry, and chemistry.