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Results for “Microscopy, Fluorescence, Multiphoton”

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PubMed2005

Sequential compound exocytosis of large dense-core vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis.

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.

Anatomy, Cross-Sectional↗

PubMed2005

Exocytosis and endocytosis of small vesicles in PC12 cells studied with TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis.

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) in the lateral membranes not facing the glass-cover slip. Upon photolysis of a caged Ca2+ compound, TEP imaging with FM1-43 (a polar membrane tracer) detected massive exocytosis of vesicles with a time constant of about 1 s. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that the diameter of vesicles was small (55 nm). Extensive exocytosis of small vesicles (SVs) was shown to be mediated by the transient opening of a fusion pore with a diameter less than about 1.6 nm, and to be followed by direct ('kiss-and-run') endocytosis and translocation of the endocytic vesicles (EVs) deep into the cytoplasm. These processes were unaffected by GTP-gamma-S. In contrast, constitutive endocytic vesicles exhibited a diameter of 90 nm, took up molecules with a diameter of > 12 nm, and their formation was blocked by GTP-gamma-S. Electron-microscopic investigation with photoconversion of diaminobenzidine using FM1-43 confirmed an abundance of EVs with a diameter of 54 nm in stimulated cells. They rapidly translocated into the cytosol, and fused with endosomal organelles. The number of SV exocytosis events vastly exceeded the number of SVs morphologically docked at the plasma membrane. Simultaneous capacitance and FM1-43 measurements indicated that TEP imaging detected most SV exocytosis, and the fusion pore was closed within 2 s. Thus, we have, for the first time, directly visualized massive exocytosis of small vesicles in a non-synaptic preparation, and have revealed their fusion-pore mediated exocytosis and endocytosis.

Anatomy, Cross-Sectional↗

PubMed2003

Application of an ex ovo chicken chorioallantoic membrane model for two-photon excitation photodynamic therapy of age-related macular degeneration.

Two-photon excitation photodynamic therapy (TPE-PDT) is being investigated as a clinical treatment for age-related macular degeneration (AMD). TPE-PDT has the potential to provide a more specific and therefore advantageous therapy regime than traditional one-photon excitation PDT. The highly vascularized 8 to 9-day-old chicken chorioallantoic membrane (CAM) is used to model the rapid growth of blood vessels in the wet form of AMD. Using an ex ovo model system for the CAM, ablation studies were successful in mimicking the leaky vessels found in AMD. In addition, the distribution and localization of liposomal Verteporfin were investigated in order to characterize the photosensitizing drug in vivo. Localization of the photosensitizer appears to be greatest on the upper vessel wall, which indicates a potentially strong treatment locale for TPE-PDT.

Animals↗

PubMed2003

Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium.

In recent years, fluorescence microscopy based on two-photon excitation has become a popular tool for biological and biomedical imaging. Among its advantages is the enhanced depth penetration permitted by fluorescence excitation with the near-infrared photons, which is particularly attractive for deep-tissue imaging. To fully utilize two-photon fluorescence microscopy as a three-dimensional research technique in biology and medicine, it is important to characterize the two-photon imaging parameters in a turbid medium. We investigated the two-photon point spread functions (PSFs) in a number of scattering samples. Gel samples containing 0.1-microm fluorescent microspheres and Liposyn III were used as phantoms mimicking the turbid environment often found in tissue. A full characterization of the two-photon PSFs of a water and oil immersion objective was completed in samples composed of 0, 0.25, 0.5, 1, and 2% Liposyn III. Our results show that up to depths of about 100 (oil) and 200 microm (water), the presence of scatterers (up to 2% Liposyn III) does not appreciably degrade the PSF widths of the objectives.

Computer Simulation↗

PubMed2003

Mechanisms of membrane potential sensing with second-harmonic generation microscopy.

We characterize the transmembrane voltage response of a novel second-harmonic generation (SHG) marker using a screening protocol with giant unilamellar vesicles. Two mechanisms are found to contribute to the voltage response: (1) an electro-optic-induced alteration of the molecular hyperpolarizability and (2) an electric-field-induced alteration of the degree of molecular alignment. We quantify the relative weights and of these contributions and provide an upper limit to their response time, which is found to be submillisecond. The identification of two voltage response mechanisms leads to new strategies for the molecular design of membrane potential markers.

Cell Membrane↗

PubMed2003

Two-photon interactions at single fluorescent molecule level.

Single-molecule spectroscopy and single-molecule detection are emerging areas that have many applications when combined with scanning, imaging, and spectroscopy techniques. We have combined a commercial confocal scanning head, to a Ti:sapphire laser and to an inverted microscope, for the detection of single molecule fluorescence of varies dyes by two-photon excitation. We collected spot images of fluorescent molecules that have been deposited on a substrate considering both blinking and photobleaching behavior of fluorescent spots. Here, we report data related to two-photon interactions that occur with the following fluorescent molecules: Indo-1, Rhodamine 6G, Fluorescein, and Pyrene. The choice of these specific dyes is based upon their wide use in biological and medical applications together with the varying complexity of their chemical structure that increases from Pyrene to Indo-1. Moreover, we report some data about single molecule studies related to denaturation of an enhanced green fluorescent protein, GFPmut2, under one photon excitation regime, that show a very similar trend to that observed for the already mentioned fluorescent molecules.

Fluorescein↗

PubMed2003

Application of two-photon flash photolysis to reveal intercellular communication and intracellular Ca2+ movements.

Two-photon excitation makes it possible to excite molecules in volumes of much less than 1 fl. In two-photon flash photolysis (TPFP) this property is used to release effector molecules from caged precursors with high three-dimensional resolution. We describe and examine the benefits of using TPFP in model solutions and in a number of cell systems to study their spatial and temporal properties. Using TPFP of caged fluorescein, we determined the free diffusion coefficient of fluorescein (D=4 x 0(-6) cm(2)/s at 20 degrees C, which is in close agreement with published values). TPFP of caged fluorescein in lens tissue in situ revealed spatial nonuniformities in intercellular fiber cell coupling by gap junctions. At the lens periphery, intercellular transport was predominantly directed along rows of cells, but was nearly isotropic further from the periphery. To test an algorithm aiming to reconstruct the Ca(2+) release flux underlying physiological Ca(2+) signals in heart muscle cells, TPFP of DM-Nitrophen was utilized to generate artificial microscopic Ca(2+) signals with known underlying Ca(2+) release flux. In an experiment with mouse oocytes, the recently developed Ca(2+) cage dimethoxynitrophenyl-ethyleneglycol-bis-(beta-aminoethylether)-N,N,N('),N(') tetraacetic acid-4 (DMNPE-4) was released in the oocyte cytosol and inside a nucleolus. Analysis of the resulting fluorescence changes suggested that the effective diffusion coefficient within the nucleolus was half of that in the cytosol. These experiments demonstrate the utility of TPFP as a novel tool for the optical study of biomedical systems.

Action Potentials↗

PubMed2003

Spectroscopic approach for monitoring two-photon excited fluorescence resonance energy transfer from homodimers at the subcellular level.

We have employed a spectroscopic approach for monitoring fluorescence resonance energy transfer (FRET) in living cells. This method provides excellent spectral separation of green fluorescent protein (GFP) mutant signals within a subcellular imaging volume using two-photon excited fluorescence imaging and spectroscopy (TPIS-FRET). In contrast to current FRET-based methodologies, TPIS-FRET does not rely on the selection of optical filters, ratiometric image analysis, or bleedthrough correction algorithms. Utilizing the intrinsic optical sectioning capabilities of TPIS-FRET, we have identified protein-protein interactions within discrete subcellular domains. To illustrate the applicability of this technique to the detection of homodimer formation, we demonstrated the in vivo association of promyleocyte (PML) homodimers within their corresponding nuclear body.

Cell Line, Tumor↗

PubMed2003

Illuminating protein interactions in tissue using confocal and two-photon excitation fluorescent resonance energy transfer microscopy.

Traumatic brain injury (TBI) remains the most common cause of death in persons under age 45 in the Western world. One of the principal determinants of morbidity and mortality following TBI is traumatic axonal injury (TAI). Current hypotheses on the pathogenesis of TAI involve activation of apoptotic cascades secondary to TBI. While a number of studies have demonstrated direct evidence for the activation of apoptotic cascades in TAI, the precise pathway by which these cascades are initiated remains a subject of intense investigation. As axolemmal disruption with the subsequent intra-axonal influx of large molecular weight species has been demonstrated to occur in relation to local axonal breakdown, attention has focused on cascades that may occur as a result of loss of ionic homeostasis. One proposed pathway by which this has been hypothesized to occur is the Ca(2+)-mediated activation of calmodulin and subsequent activation of the phosphatase calcineurin with dephosphorylation of a protein known as BAD, leading to a proapoptotic interaction between BAD and the mitochondrial protein Bcl-xL. While this pathway is an intriguing route for traumatic axonal pathogenesis, neither conventional immunocytochemical/histochemical nor ultrastructural approaches have had the capacity to shed insight on whether BAD and Bcl-xL interact in TAI in vivo. We describe the implementation of confocal and two-photon excitation fluorescence resonance energy transfer (FRET) microscopy techniques through which we demonstrate interaction between the proapoptotic protein BAD and the prosurvival protein Bcl-xL within TAI following TBI. Further, we report on a method to reliably detect protein interactions within aldehyde fixed tissue sections through conventional immunohistochemical approaches.

Animals↗

PubMed2003

One- and two-photon fluorescence resonance energy transfer microscopy to establish a clustered distribution of receptor-ligand complexes in endocytic membranes.

One- and two-photon fluorescence resonance energy transfer (FRET) microscopy, using different bandwidth emission filters and a novel spectral spillover correction algorithm (PFRET algorithm), provides the basis for a quantitative approach to measure receptor clustering in endocytic membranes. Emission filters with wider bandwidth allow for an increased FRET signal and corresponding spillover. Treatment with the PFRET correction algorithm results in increasing correction levels and comparable energy transfer efficiency (E%) values, thus validating our algorithm-based approach. The relationship between E% and acceptor and donor levels and donor:acceptor (D:A) ratio is used to characterize the distribution of receptor-ligand complexes in endocytic membranes. In addition to the standard test for clustering (E%'s independence from acceptor levels), we describe a second parameter: the negative dependence of E% on increasing donor levels and D:A ratio. A donor geometric exclusion hypothesis is proposed to explain this phenomenon. One- and two-photon FRET microscopy assays show that polymeric IgA-receptor-ligand complexes are organized in clusters within apical endocytic membranes of polarized Madin-Darby canine kidney cells.

Algorithms↗

PubMed2003

Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods.

Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.

Adenylate Kinase↗

PubMedDate not supplied

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging.

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

Animals↗

PubMedDate not supplied

Two-photon laser scanning fluorescence microscopy using photonic crystal fiber.

We report the application of a simple yet powerful modular pulse compression system based on photonic crystal fibers that improves on incumbent two-photon laser scanning fluorescence microscopy techniques. This system provides more than a sevenfold increase in fluorescence yield when compared with a commercial two-photon microscopy system. From this, we infer pulses of IR radiation of less than 35 fs duration reaching the sample.

Animals↗

PubMedDate not supplied

Detection of dual-gene expression in arteries using an optical imaging method.

We evaluate the in vivo use of an optical imaging method to detect the vascular expression of green fluorescent protein (GFP) or red fluorescent protein (RFP), and to detect the simultaneous expression of GFP and RFP after transduction into arteries by a dual-promoter lentiviral vector driving their concurrent expression. This method involves using a charge-coupled device camera to detect fluorescence, a fiber optic probe to transmit light, and optical filters to distinguish each marker. In animal models, these vectors are locally delivered to target arteries, whereas the gene for a nonfluorescent cell-surface protein is transduced into contralateral arteries as the sham control. The images show distinct areas of bright fluorescence from GFP and RFP along the target arteries on excitation; no exogenous fluorescence is observed in the controls. Measured signal intensities from arteries transduced with the single- and dual-promoter vectors exceed the autofluorescence signal from the controls. Transgene expression of GFP and RFP in vivo is confirmed with confocal microscopy. We demonstrate the use of an optical imaging method to concurrently detect two distinct fluorescent proteins, potentially permitting the expression of multiple transgenes and their localization in the vasculature to be monitored.

Animals↗

PubMedDate not supplied

Mitigating thermal mechanical damage potential during two-photon dermal imaging.

Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin in vivo. We describe a technique to mitigate specimen thermal mechanical damage based on the use of a laser pulse picker that reduces the laser repetition rate by selecting a fraction of pulses from a laser pulse train. Since the laser pulse picker decreases laser average power while maintaining laser pulse peak power, thermal mechanical damage can be minimized while two-photon fluorescence excitation efficiency is maximized.

Burns↗

PubMedDate not supplied

Nonlinear anisotropic diffusion filtering of three-dimensional image data from two-photon microscopy.

Two-photon microscopy in combination with novel fluorescent labeling techniques enables imaging of three-dimensional neuronal morphologies in intact brain tissue. In principle it is now possible to automatically reconstruct the dendritic branching patterns of neurons from 3-D fluorescence image stacks. In practice however, the signal-to-noise ratio can be low, in particular in the case of thin dendrites or axons imaged relatively deep in the tissue. Here we present a nonlinear anisotropic diffusion filter that enhances the signal-to-noise ratio while preserving the original dimensions of the structural elements. The key idea is to use structural information in the raw data-the local moments of inertia-to locally control the strength and direction of diffusion filtering. A cylindrical dendrite, for example, is effectively smoothed only parallel to its longitudinal axis, not perpendicular to it. This is demonstrated for artificial data as well as for in vivo two-photon microscopic data from pyramidal neurons of rat neocortex. In both cases noise is averaged out along the dendrites, leading to bridging of apparent gaps, while dendritic diameters are not affected. The filter is a valuable general tool for smoothing cellular processes and is well suited for preparing data for subsequent image segmentation and neuron reconstruction.

Algorithms↗

PubMedDate not supplied

Imaging of Caenorhabditis elegans neurons by second-harmonic generation and two-photon excitation fluorescence.

Second-harmonic generation (SHG) and two-photon excitation fluorescence (TPEF) are relatively new and promising tools for the detailed imaging of biological samples and processes at the microscopic level. By exploiting these nonlinear phenomena phototoxicity and photobleaching effects on the specimens are reduced dramatically. The main target of this work was the development of a compact inexpensive and reliable experimental apparatus for nonlinear microscopy measurements. Femtosecond laser pulses were utilized for excitation. We achieved high-resolution imaging and mapping of Caenorhabditis elegans (C. elegans) neurons and muscular structures of the pharynx, at the microscopic level by performing SHG and TPEF measurements. By detecting nonlinear phenomena such as SHG and TPEF it is feasible to extract valuable information concerning the structure and the function of nematode neurons.

Animals↗

PubMedDate not supplied

Two-photon 3-D mapping of ex vivo human skin endogenous fluorescence species based on fluorescence emission spectra.

Spectral resolved tissue imaging has a broad range of biomedical applications such as the minimally invasive diagnosis of diseases and the study of wound healing and tissue engineering processes. Two-photon microscopy imaging of endogenous fluorescence has been shown to be a powerful method for the quantification of tissue structure and biochemistry. While two-photon excited autofluorescence is observed ubiquitously, the identities and distributions of endogenous fluorophores have not been completely characterized in most tissues. We develop an image-guided spectral analysis method to analyze the distribution of fluorophores in human skin from 3-D resolved two-photon images. We identify five factors that contribute to most of the luminescence signals from human skin. Luminescence species identified include tryptophan, NAD(P)H, melanin, and elastin, which are autofluorescent, and collagen that contributes to a second harmonic signal.

Equipment Design↗