Home About us Contact
|
Home About us Contact | ||
|
|
||
Multiphoton Microscopy (multiphoton + microscopy)
Selected AbstractsThe Primary Cilium of Connective Tissue Cells: Imaging by Multiphoton MicroscopyTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 9 2008Eve Donnelly Three-dimensional reconstruction of a multiphoton image stack showing primary cilia (green) tenocyte nuclei (blue), and collagen (red) in extensor tendon. From "The Primary Cilium of Connective Tissue Cells: Imaging by Multiphoton Microscopy," by Eve Donnelly, et al., on page 1062, in this issue. Anatomical Record 291:1062,1073. [source] Multiphoton microscopy in life sciencesJOURNAL OF MICROSCOPY, Issue 2 2000K. König Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm,2 to GW cm,2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm,2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible. [source] In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopyTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 1 2006Cornelia E. Farnum Abstract Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. © 2005 Wiley-Liss, Inc. [source] CNS-irrelevant T-cells enter the brain, cause blood,brain barrier disruption but no glial pathologyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Alina Smorodchenko Abstract Invasion of autoreactive T-cells and alterations of the blood,brain barrier (BBB) represent early pathological manifestations of multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). Non-CNS-specific T-cells are also capable of entering the CNS. However, studies investigating the spatial pattern of BBB alterations as well as the exact localization and neuropathological consequences of transferred non-CNS-specific cells have been thus far lacking. Here, we used magnetic resonance imaging and multiphoton microscopy, as well as histochemical and high-precision unbiased stereological analyses to compare T-cell transmigration, localization, persistence, relation to BBB disruption and subsequent effects on CNS tissue in a model of T-cell transfer of ovalbumin (OVA)- and proteolipid protein (PLP)-specific T-cells. BBB alterations were present in both EAE-mice and mice transferred with OVA-specific T-cells. In the latter case, BBB alterations were less pronounced, but the pattern of initial cell migration into the CNS was similar for both PLP- and OVA-specific cells [mean (SEM), 95 × 103 (7.6 × 103) and 88 × 103 (18 × 103), respectively]. Increased microglial cell density, astrogliosis and demyelination were, however, observed exclusively in the brain of EAE-mice. While mice transferred with non-neural-specific cells showed similar levels of rhodamine-dextran extravasation in susceptible brain regions, EAE-mice presented huge BBB disruption in brainstem and moderate leakage in cerebellum. This suggests that antigen specificity and not the absolute number of infiltrating cells determine the magnitude of BBB disruption and glial pathology. [source] Quantified characterization of human cutaneous normal scar using multiphoton microscopyJOURNAL OF BIOPHOTONICS, Issue 1-2 2010Xiaoqin Zhu Abstract The morphological alterations of human cutaneous normal scar were quantitatively analyzed using multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation. High-contrast, high-resolution images of normal scar and uninjured skin were obtained for comparison. In addition, some quantitative parameters have been extracted to quantitatively discriminate between normal scar and uninjured skin. The MPM combined with quantitative method enable a better understanding of microstructual alterations of the epidermis, elastic fiber, and collagen in normal scar. It may lead the way to making know the mechanism of normal scar formation and identifying feasible therapeutic options. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Imaging engineered tissues using structural and functional optical coherence tomographyJOURNAL OF BIOPHOTONICS, Issue 11 2009Xing Liang Abstract As the field of tissue engineering evolves, there will be an increasingly important need to visualize and track the complex dynamic changes that occur within three-dimensional constructs. Optical coherence tomography (OCT), as an emerging imaging technology applied to biological materials, offers a number of significant advantages to visualize these changes. Structural OCT has been used to investigate the longitudinal development of engineered tissues and cell dynamics such as migration, proliferation, detachment, and cell-material interactions. Optical techniques that image functional parameters or integrate multiple imaging modalities to provide complementary contrast mechanisms have been developed, such as the integration of optical coherence microscopy with multiphoton microscopy to image structural and functional information from cells in engineered tissue, optical coherence elastography to generate images or maps of strain to reflect the spatially-dependent biomechanical properties, and spectroscopic OCT to differentiate different cell types. From these results, OCT demonstrates great promise for imaging and visualizing engineered tissues, and the complex cellular dynamics that directly affect their practical and clinical use. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Multiphoton microscopy in life sciencesJOURNAL OF MICROSCOPY, Issue 2 2000K. König Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three-dimensional fluorescence imaging based on non-resonant two-photon or three-photon fluorophor excitation requires light intensities in the range of MW cm,2 to GW cm,2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi-gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non-invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth-resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non-invasive fluorophore loading into single living plant cells. Non-destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis-like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two-photon excitation process rather than a one-photon or three-photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two-photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid-induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm,2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non-invasive NIR laser surgery within a living cell or within an organelle is therefore possible. [source] Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cellsJOURNAL OF NEUROCHEMISTRY, Issue 5 2005J. Balaji Abstract Synthesis and subsequent sequestration into vesicles are essential steps that precede neurotransmitter exocytosis, but neither the total neurotransmitter content nor the fraction sequestered into vesicles have been measured in individual live neurons. We use multiphoton microscopy to directly observe intracellular and intravesicular serotonin in the serotonergic neuronal cell line RN46A. We focus on how the relationship between synthesis and sequestration changes as synthesis is up-regulated by differentiation or down-regulated by chemical inhibition. Temperature-induced differentiation causes an increase of about 60% in the total serotonin content of individual cells, which goes up to about 10 fmol. However, the number of vesicles per cell increases by a factor of four and the proportion of serotonin sequestered inside the vesicles increases by a factor of five. When serotonin synthesis is inhibited in differentiated cells and the serotonin content goes down to the level present in undifferentiated cells, the sequestered proportion still remains at this high level. The total neurotransmitter content of a cell is, thus, an unreliable indicator of the sequestered amount. [source] Intact corneal stroma visualization of GFP mouse revealed by multiphoton imagingMICROSCOPY RESEARCH AND TECHNIQUE, Issue 12 2006Wen Lo Abstract The aim of this work is to demonstrate that multiphoton microscopy is a preferred technique to investigate intact cornea structure without slicing and staining. At the micron resolution, multiphoton imaging can provide both large morphological features and detailed structure of epithelium, corneal collagen fibril bundles and keratocytes. A large area multiphoton cross-section across an intact eye excised from a GFP mouse was obtained by a homebuilt multiphoton microscope. The broadband multiphoton fluorescence (435,700 nm) and second harmonic generation (SHG, 360,400 nm) signals were generated by the 760 nm output of a femtosecond titanium-sapphire laser. A water immersion objective (Fluor Ō, 40X, NA 0.8; Nikon) was used to facilitate imaging the curve ocular surface. The multiphoton image over entire cornea provides morphological information of epithelial cells, keratocytes, and global collagen orientation. Specifically, our planar, large area multiphoton image reveals a concentric pattern of the stroma collagen, indicative of the laminar collagen organization throughout the stroma. In addition, the green fluorescence protein (GFP) labeling contributed to fluorescence contrast of cellular area and facilitated visualizing of inactive keratocytes. Our results show that multiphoton imaging of GFP labeled mouse cornea manifests both morphological significance and structural details. The second harmonic generation imaging reveals the collagen orientation, while the multiphoton fluorescence imaging indicates morphology and distribution of cells in cornea. Our results support that multiphoton microscopy is an appropriate technology for further in vivo investigation and diagnosis of cornea. Microsc. Res. Tech., 2006. © 2006 Wiley-Liss, Inc. [source] Use of confocal and multiphoton microscopy for the evaluation of micro-optical components and emittersMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2004J.M. Girkin Abstract We report on the application of confocal and multiphoton microscopic techniques for the evaluation of the latest generation of micro optical components. The optical emitting characteristics of arrays of matrix addressable GaN micrometer-sized light emitting diodes (micro-LEDs) have been measured using a commercial confocal microscope utilising the LEDs' own emission along with reflection confocal microscopy to determine the surface structure. Multiphoton induced luminescence from the 10,20-micron diameter emitters has also been used to examine the structure of the device and we compare this with electrically induced emission. In related work, the optical properties of micro lens arrays (10,100-micron diameter) fabricated in SiC, Sapphire, and Diamond have been determined using transmission confocal microscopy. Such optical microscopy techniques offer a simple, non-destructive method to determine the structure and performance of such novel devices. Microsc. Res. Tech. 64:293,296, 2004. © 2004 Wiley-Liss, Inc. [source] Three-dimensional microscopy migrates to the web with "PowerUp Your Microscope"MICROSCOPY RESEARCH AND TECHNIQUE, Issue 2 2004Paola Bonetto Abstract "PowerUp Your Microscope" is a software package designed and realized for the optimization of 3D optical microscopy image quality using the Internet and inverse problems computational approaches. The package is mainly devoted to 3D microscopy users, being operative for wide-field, confocal, and multiphoton microscopy. It provides the microscopy community with an extremely easy and comparatively powerful access to advanced image restoration methods. The core of the computational section is the optical system modeling and inverse deconvolution implementation, which is strongly linked to Web-based software and technology. This project constitutes a real and effective migration to the Web, extending computational approaches to image restoration to the whole microscopy user community, regardless of their background. Microsc. Res. Tech. 64:196,203, 2004. © 2004 Wiley-Liss, Inc. [source] In vivo delivery of fluoresceinated dextrans to the murine growth plate: Imaging of three vascular routes by multiphoton microscopyTHE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 1 2006Cornelia E. Farnum Abstract Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes. © 2005 Wiley-Liss, Inc. [source] | |||||||||||||