SEARCH · PubMed Health

Results for “Microscopy, Fluorescence, Multiphoton”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 163 records · Page 9Linked to original sources

PubMed2005

Intravital multiphoton microscopy of dynamic renal processes.

Recent advances in microscopy and optics, computer sciences, and the available fluorophores used to label molecules of interest have empowered investigators to utilize intravital two-photon microscopy to study the dynamic events within the functioning kidney. This emerging technique enables investigators to follow functional and structural alterations with subcellular resolution within the same field of view over seconds to weeks. This approach invigorates the validity of data and facilitates analysis and interpretation as trends are more readily determined when one is more closely monitoring indicative physiological parameters. Therefore, in this review we emphasize how specific approaches will enable studies into glomerular permeability, proximal tubule endocytosis, and microvascular function within the kidney. We attempt to show how visual data can be quantified, thus allowing enhanced understanding of the process under study. Finally, emphasis is given to the possible future opportunities of this technology and its present limitations.

Animals↗

PubMed2006

Imaging of cortical astrocytes using 2-photon laser scanning microscopy in the intact mouse brain.

A number of studies over the past decade have shown that astrocytes, the supportive cells of the brain, play important roles in synaptic transmission including regulating the strength of both excitatory and inhibitory synapses. A major challenge for the future is to define the role of astrocytes in complex tasks, such as functional hyperemia and sensory processing, as well as their contribution to acute and degenerative diseases of the nervous system. Multiphoton imaging approaches are ideally suited to study electrically non-excitable astrocytes. We here discuss novel in vivo studies aimed at defining the role of astrocytes in normal and pathological brain function. With a better understanding of the role astrocytes play in information processing and regulation of the brain microenvironment in vivo, and the understanding that astrocytes are heavily implicated in the pathology of many diseases such as epilepsy, Alzheimer's and Parkinson's diseases, astrocytes provide a promising target for future drug therapy approaches.

Animals↗

PubMed2005

Multiphoton-excited microfabrication in live cells via Rose Bengal cross-linking of cytoplasmic proteins.

We demonstrate the use of multiphoton-excited photochemistry to cross-link three-dimensional matrices directly from cytoplasmic proteins in a live cell (starfish oocyte). Fluorescence recovery after photobleaching measurements were used to determine diffusion coefficients inside intracellular cross-linked structures, and it was found that the diffusion was approximately 3 to 4 orders of magnitude slower than in free solution and 2-3 orders of magnitude slower than in cytoplasm and that the value can be tuned by controlling the laser exposure. Complex structures can be fabricated to construct channels and compartments that could be used to isolate cellular processes, and the method should thus be applicable to a broad range of problems in cell biology.

Animals↗

PubMed2006

Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye.

PURPOSE: The purpose of this work was to demonstrate the use of the combined imaging modality of multiphoton autofluorescence and second-harmonic generation (SHG) microscopy in obtaining spectrally resolved morphologic features of the cornea, limbus, conjunctiva, and sclera in whole, ex vivo porcine eyes. METHODS: The 780-nm output of a femtosecond, titanium-sapphire laser was used to induce broadband autofluorescence (435-700 nm) and SHG (390 nm) from various regions of the surface of ex vivo porcine eyes. A water-immersion objective was used for convenient imaging of the curved surface of the eye. RESULTS: Multiphoton autofluorescence was useful in identifying cellular structures of the different domains of the ocular surface, and the SHG signal can be used to resolve collagen organization within the cornea stroma and sclera of ex vivo porcine eyes. CONCLUSIONS: Multiphoton autofluorescence and SHG microscopy have been demonstrated to be an effective technique for resolving, respectively, the cellular and collagen structures within the ocular surface of ex vivo porcine eyes. SHG imaging resolved the difference in structural orientations between corneal and sclera collagen fibers. Specifically, the corneal collagen is organized in a depth-dependent fashion, whereas the scleral collagen is randomly packed. Because this technique does not require histologic preparation procedures, it has the potential to be applied for in vivo studies with minimal disturbance to the eye.

Animals↗

PubMed2003

Spatial localization of cardiac optical mapping with multiphoton excitation.

Depth and radius of regions interrogated by cardiac optical mapping with a laser beam depend on photon travel inside the heart. It would be useful to limit the range of depth and radius interrogated. We modeled the effects of a condensing lens to concentrate laser light at a target depth inside the heart, and near infrared excitation to increase penetration and produce two-photon absorption. A Monte Carlo simulation that incorporated a 0.55-NA lens, and absorption and scattering of 1064- or 488-nm laser light in 3-D cardiac tissue indicated the distribution of excitation fluence inside the tissue. A subsequent simulation incorporating absorption and scattering of transmembrane voltage-sensitive fluorescence (wavelength 669 nm) indicated locations from which fluorescence photons exiting the tissue surface originated. The results indicate that mapping at depths up to 300 microm in hearts can provide significant improvement in localization over existing cardiac optical mapping. The estimated interrogation region is sufficiently small to examine cardiac events at a cellular or subcellular scale and may allow mapping at various depths in the heart.

Action Potentials↗

PubMed2004

Three- and four-photon absorption of a multiphoton absorbing fluorescent probe.

High-order multiphoton excitation processes are becoming a reality for fluorescence imaging and phototherapy treatment because they afford minimization of scattered light losses and a reduction of unwanted linear absorption in the living organism transparency window, making them less susceptible to photodamage, while improving the irradiation penetration depth and spatial resolution. We report the four-photon-excited fluorescence emission of (7-benzothiazol-2-yl-9,-didecylfluoren-2-yl)diphenylamine in hexane and its four-photon absorption cross section sigma4' = 8.1 x 10(-109) cm8 s3 photon(-3) for the transition S0 --> S1 when excited at 1600 nm with a tunable optical parametric generator (OPG) pumped by picosecond laser pulses. When pumped at 1200 nm, three-photon absorption was observed, corresponding to the same transition.

Benzothiazoles↗

PubMed2006

In vivo assessment of human skin aging by multiphoton laser scanning tomography.

Changes of dermal collagen and elastin content are characteristic for skin aging as well as for pathological skin conditions. To evaluate these changes, we used in vivo multiphoton laser tomography to measure two-photon excited autofluorescence (AF) and second harmonic generation (SHG). We tested 18 patients of all ages and calculated the SHG-to-AF aging index of dermis (SAAID). We observed a negative relationship between the SAAID and age, which was accelerated for the female (n=7) subgroup. The current findings are the first in vivo demonstration of this relationship, and they show that specific characteristics of aged skin such as the ratio of extracellular matrix components collagen and elastin can be evaluated by in vivo AF and SHG measurements using near-IR femtosecond laser pulses.

Adult↗

PubMedDate not supplied

Multiphoton excitation of fluorescent DNA base analogs.

Multiphoton excitation was used to investigate properties of the fluorescent DNA base analogs, 2-aminopurine (2AP) and 6-methylisoxanthopterin (6MI). 2-aminopurine, a fluorescent analog of adenine, was excited by three-photon absorption. Fluorescence correlation measurements were attempted to evaluate the feasibility of using three-photon excitation of 2AP for DNA-protein interaction studies. However, high excitation power and long integration times needed to acquire high signal-to-noise fluorescence correlation curves render three-photon excitation FCS of 2AP not very useful for studying DNA base dynamics. The fluorescence properties of 6-methylisoxanthopterin, a guanine analog, were investigated using two-photon excitation. The two-photon absorption cross-section of 6MI was estimated to be about 2.5 x 10(-50) cm(4)s (2.5 GM units) at 700 nm. The two-photon excitation spectrum was measured in the spectral region from 700 to 780 nm; in this region the shape of the two-photon excitation spectrum is very similar to the shape of single-photon excitation spectrum in the near-UV spectral region. Two-photon excitation of 6MI is suitable for fluorescence correlation measurements. Such measurements can be used to study DNA base dynamics and DNA-protein interactions over a broad range of time scales.

2-Aminopurine↗

PubMedDate not supplied

Modeling aberrant wound healing using tissue-engineered skin constructs and multiphoton microscopy.

BACKGROUND: Keloids and hypertrophic scars result from aberrant wound healing and remain a potential complication of any surgical procedure or trauma. Investigation of aberrant wound healing has been limited to the study of growth factors, collagen precursors, and DNA synthesis in simple in vitro systems, which necessitate removal or destruction of cells or factors in the growth environment of cell cultures. Multiphoton microscopy (MPM) can use endogenous chromophores such as collagen and nicotinamide adenine dinucleotide hydrogenase to produce thin optical sections of thick living tissues without the use of dyes or stains. Endogenous second-harmonic-generation (SHG) signals in collagen can be collected to form an MPM image. OBJECTIVE: To present a novel wound-healing model used to investigate keloid-derived fibroblast activity and collagen production in the same intact tissue-engineered construct over time. METHODS: Artificial tissue constructs called RAFTs (produced by suspension of keloid or normal dermal fibroblasts in type I collagen gel with an overlying keratinocyte layer) were cultured at air-fluid interface. Multiphoton microscopy SHG images of collagen in the intact tissue constructs consisting of normal or keloid-derived fibroblasts were obtained. The constructs were then incised with a scalpel. Serial MPM and phase-contrast microscopy images were obtained to monitor changes in the extracellular matrix in response to wounding of the artificial skin construct over 8 days. RESULTS: The tissue-engineered constructs formed a bilayer resembling the dermis and epidermis of human skin. Phase-contrast microscopy revealed migration of keratinocytes into the defect created by scalpel wounding. The constructs were found to contract with time after wounding. The MPM SHG images showed collagen deposition in the tissue constructs after wounding. Tissue constructs with keloid-derived fibroblasts were found to deposit collagen at a higher rate than those with normal fibroblasts. CONCLUSIONS: The MPM model described herein permits serial observation of the same intact specimens without the need for fixation or cytotoxic stains. Furthermore, it demonstrates the biologic activity of RAFT artificial tissue constructs.

Cells, Cultured↗

PubMed2005

Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer.

Protein labeling with green fluorescent protein derivatives has become an invaluable tool in cell biology. Protein quantification, however, is difficult when cells express constructs with overlapping fluorescent emissions. Under these conditions, signal separation using emission filters is inherently inefficient. Spectral imaging solves this problem by recording emission spectra directly. Unfortunately, linear unmixing, the algorithm used for quantifying individual fluorophores from emission spectra, fails when resonance energy transfer (RET) is present. We therefore sought to develop an unmixing algorithm that incorporates RET. An equation for spectral emission incorporating RET was derived and an assay based on this formalism, spectral RET (sRET), was developed. Standards with defined RET efficiencies and with known Cerulean/Venus ratios were constructed and used to test sRET. We demonstrate that sRET analysis is a comprehensive, photon-efficient method for imaging RET efficiencies and accurately determines donor and acceptor concentrations in living cells.

Algorithms↗

PubMed2006

Plaque-derived oxidative stress mediates distorted neurite trajectories in the Alzheimer mouse model.

Alzheimer disease (AD) is characterized both by senile plaques and neurodegeneration, although the details of the relationship between the 2 are not well understood. We postulated that oxidative stress resulting from senile plaques may mediate plaques' effects on local neuronal processes. Using multiphoton microscopy, we directly demonstrate the generation of reactive oxygen species by senile plaques. After screening of several natural antioxidants ex vivo, we assessed in vivo the effect of 2 orally administered antioxidants in APPswe/PS1d9 transgenic mice. Both Ginkgo biloba extract and vitamin E reduced the oxidative stress resulting from senile plaques in vivo as monitored with intracranial imaging. Both treatments also lead to a progressive reversal of the structural changes in dystrophic neurites associated with senile plaques. These results suggest a causal relationship between plaque-associated oxidative stress and neuritic alterations and demonstrate for the first time that the focal neurotoxicity associated with the senile plaques of AD is partially reversible with antioxidant therapies. The quantitative ex vivo screen combined with in vivo monitoring of efficacy should lead to more effective clinical therapies for the prevention of oxidative stress and neurotoxicity in AD.

Alzheimer Disease↗

PubMed2004

Poly-N-acetyl glucosamine-mediated red blood cell interactions.

BACKGROUND: Investigations were performed to assess the effect of poly-N-acetyl glucosamine (p-GlcNAc) fiber slurry-mediated hemostasis by interactions with red blood cells. METHODS: Red blood cell aggregation studies were performed using test material-coated microscope slides and multiphoton microscopic measurements. Enzymatic removal of red blood cell surface proteins was achieved using trypsin and neuraminidase treatment. Zeta-potential measurements (surface charge) were performed. RESULTS: Red blood cells interact directly with poly-N-acetyl glucosamine polymers through ionic interactions and cell-surface proteins. The effective concentration of poly-N-acetyl glucosamine fiber material for 50% red blood cell aggregation was 0.28 mg/mL. The p-GlcNAc beta-configuration fibers and an alpha-configuration structural modification of the fibers both produced maximal responses because of their zeta-potentials, whereas other chemically modified p-GlcNAcs and chitosans were ineffective. CONCLUSION: Poly-N-acetyl glucosamine-induced red blood cell aggregation is mediated by interactions with red blood cell surface charges.

Acetylglucosamine↗

PubMed2005

Metabolic mapping of MCF10A human breast cells via multiphoton fluorescence lifetime imaging of the coenzyme NADH.

Biochemical estimation of NADH concentration is a useful method for monitoring cellular metabolism, because the NADH/NAD+ reduction-oxidation pair is crucial for electron transfer in the mitochondrial electron chain. In this article, we present a novel method for deriving functional maps of intracellular reduction-oxidation ratio in vivo via measurement of the fluorescence lifetimes and the ratio of free and protein-bound NADH using two-photon fluorescence lifetime imaging (FLIM). Through systematic analysis of FLIM data from the control cells, it was observed that there is a statistically significant decrease in the fluorescence lifetime of both free and protein-bound NADH and the contribution of protein-bound NADH as cells progress from an early to logarithmic to confluent phase. Potassium cyanide (KCN) treatment and serum starvation of cells yielded similar changes. There was a statistically significant decrease in the fluorescence lifetime of protein-bound and free NADH at the early and logarithmic phase of the growth curve and a statistically significant decrease in the contribution of protein-bound NADH relative to that observed in the control cells at all three phases of the growth curve. The imposed perturbations (confluence, serum starvation, and KCN treatment) are all expected to result in an increase in the ratio of NADH/NAD+. Our studies suggest that the fluorescence lifetime of both the free and the protein-bound components of NADH and the ratio of free to protein-bound NADH is related to changes in the NADH/NAD+ ratio.

Cell Line↗

PubMed2006

Effects of different immersion media in multiphoton imaging of the epithelium and dermis of human skin.

In this work, we compared the performance of objectives with similar numerical aperture of 0.75 but different immersion media of air, water, glycerin, and oil in the imaging of human skin epithelium and dermis. In general, we found that the oil immersion objective recorded the strongest intensity at the same mechanical depth. We also characterized the focal shifts and found that with decreasing refractive index, the focal shift becomes increasingly more negative (for both the epithelium and dermis). In imaging the dermis, we estimated the image resolution at the depths of 18.8 and 30.2 microm, and found that the image resolution were comparable at these depths under the four types of immersion conditions. Our results demonstrate that by changing the immersion media, the main microscopic imaging effects are the recorded axial intensities and the focal shifts. The effects on the image resolution are negligible.

Dermis↗

PubMed2005

Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy.

We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.

Algorithms↗

PubMed2003

Multiphoton microscopy: an optical approach to understanding and resolving sulfur mustard lesions.

Sulfur mustard (SM; 2,2(')-dichloroethyl sulfide) is a percutaneous alkylating agent first used as a chemical weapon at Ypres, Belgium, in World War I. Despite its well-documented history, the primary lesions effecting dermal-epidermal separation and latent onset of incapacitating blisters remain poorly understood. By immunofluorescent imaging of human epidermal keratinocytes (HEK) and epidermal tissues exposed to SM (400 microM for 5 min), we have amassed unequivocal evidence that SM disrupts adhesion complex molecules, which are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Images of keratin 14 (K14) in control cells showed tentlike filament networks linking the HEK's basolateral anchoring sites to the dorsal surface of its nuclei. Images from 6-h postexposure profiles revealed early disruption (</=1 h) and progressive collapse of the K14 cytoskeleton. Collapse involved focal erosions, loss of functional asymmetry, and displacement of nuclei beneath a mat of jumbled filaments. In complementary studies, 1-h images showed statistically significant (p<0.01) decreases of 25 to 30% in emissions from labeled alpha(6)beta(4) integrin and laminin 5, plus disruption of their receptor-ligand organization. Results indicate that SM alkylation destabilizes dermal-epidermal attachments and potentiates vesication by disrupting adhesion complex molecules and associated signaling mechanisms required for their maintenance and repair.

Cell Adhesion Molecules↗

PubMed2003

Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy.

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Animals↗

PubMed2004

Mechanisms of dendritic elaboration of sensory neurons in Drosophila: insights from in vivo time lapse.

In vivo time-lapse multiphoton microscopy was used to analyze the remodeling of the dendritic arborizing (da) sensory neuron known as dorsal dendritic arborizing neuron E (ddaE) during metamorphosis. After its larval processes have been removed, the cell body of ddaE repositions itself on the body wall between 25 and 40 hr after puparium formation (APF) and begins its adult outgrowth at 40 hr APF. The scaffold of the arbor is laid down between 40 and 54 hr APF, when growth is characterized by high filopodial activity at both terminal and interstitial positions and by branch retraction along with branch establishment. Later in development, filopodial activity remains high but is confined to terminal branches, and branch retraction is no longer seen. Treatment with the insect hormone juvenile hormone (JH), a key regulator of metamorphosis, alters the shape and complexity of the adult dendritic tree in a time-dependent manner. Early treatments with juvenile hormone mimic (JHm) appear to repress extension programs and maintain retraction programs. With later JHm treatments, extension programs appear normal, but retraction programs are maintained beyond their normal time. The JH treatments show the importance of retraction programs in establishing the overall arbor shape.

Animals↗