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Journal Article

[Two-photon laser scanning fluorescence microscopy].

01 Oct 2007-Tanpakushitsu kakusan koso. Protein nucleic acid enzyme (Tanpakushitsu Kakusan Koso)-Vol. 52, pp 1778-1779
About: This article is published in Tanpakushitsu kakusan koso. Protein nucleic acid enzyme.The article was published on 2007-10-01 and is currently open access. It has received 1480 citations till now. The article focuses on the topics: Scanning confocal electron microscopy & Microscopy.
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01 Jan 2006
TL;DR: In this paper, the authors proposed a method for propagating and focusing of optical fields in a nano-optics environment using near-field optical probes and probe-sample distance control.
Abstract: 1. Introduction 2. Theoretical foundations 3. Propagation and focusing of optical fields 4. Spatial resolution and position accuracy 5. Nanoscale optical microscopy 6. Near-field optical probes 7. Probe-sample distance control 8. Light emission and optical interaction in nanoscale environments 9. Quantum emitters 10. Dipole emission near planar interfaces 11. Photonic crystals and resonators 12. Surface plasmons 13. Forces in confined fields 14. Fluctuation-induced phenomena 15. Theoretical methods in nano-optics Appendices Index.

3,772 citations

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TL;DR: Functional photoacoustic microscopy (fPAM) is reported, which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100.
Abstract: Although optical absorption is strongly associated with the physiological status of biological tissue, existing high-resolution optical imaging modalities, including confocal microscopy, two-photon microscopy and optical coherence tomography, do not sense optical absorption directly. Furthermore, optical scattering prevents these methods from imaging deeper than ~1 mm below the tissue surface. Here we report functional photoacoustic microscopy (fPAM), which provides multiwavelength imaging of optical absorption and permits high spatial resolution beyond this depth limit with a ratio of maximum imaging depth to depth resolution greater than 100. Reflection mode, rather than orthogonal or transmission mode, is adopted because it is applicable to more anatomical sites than the others. fPAM is demonstrated with in vivo imaging of angiogenesis, melanoma, hemoglobin oxygen saturation (sO_2) of single vessels in animals and total hemoglobin concentration in humans.

1,766 citations

Journal ArticleDOI
TL;DR: This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales, according to the tissue depth at which they operate.
Abstract: Optical microscopy has been a fundamental tool of biological discovery for more than three centuries, but its in vivo tissue imaging ability has been restricted by light scattering to superficial investigations, even when confocal or multiphoton methods are used. Recent advances in optical and optoacoustic (photoacoustic) imaging now allow imaging at depths and resolutions unprecedented for optical methods. These abilities are increasingly important to understand the dynamic interactions of cellular processes at different systems levels, a major challenge of postgenome biology. This Review discusses promising photonic methods that have the ability to visualize cellular and subcellular components in tissues across different penetration scales. The methods are classified into microscopic, mesoscopic and macroscopic approaches, according to the tissue depth at which they operate. Key characteristics associated with different imaging implementations are described and the potential of these technologies in biological applications is discussed.

1,607 citations

Journal ArticleDOI
TL;DR: Three tumor-associated collagen signatures (TACS) are observed and defined that provide novel markers to locate and characterize tumors and should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.
Abstract: Stromal-epithelial interactions are of particular significance in breast tissue as misregulation of these interactions can promote tumorigenesis and invasion. Moreover, collagen-dense breast tissue increases the risk of breast carcinoma, although the relationship between collagen density and tumorigenesis is not well understood. As little is known about epithelial-stromal interactions in vivo, it is necessary to visualize the stroma surrounding normal epithelium and mammary tumors in intact tissues to better understand how matrix organization, density, and composition affect tumor formation and progression. Epithelial-stromal interactions in normal mammary glands, mammary tumors, and tumor explants in three-dimensional culture were studied with histology, electron microscopy, and nonlinear optical imaging methodologies. Imaging of the tumor-stromal interface in live tumor tissue ex vivo was performed with multiphoton laser-scanning microscopy (MPLSM) to generate multiphoton excitation (MPE) of endogenous fluorophores and second harmonic generation (SHG) to image stromal collagen. We used both laser-scanning multiphoton and second harmonic generation microscopy to determine the organization of specific collagen structures around ducts and tumors in intact, unfixed and unsectioned mammary glands. Local alterations in collagen density were clearly seen, allowing us to obtain three-dimensional information regarding the organization of the mammary stroma, such as radiating collagen fibers that could not have been obtained using classical histological techniques. Moreover, we observed and defined three tumor-associated collagen signatures (TACS) that provide novel markers to locate and characterize tumors. In particular, local cell invasion was found predominantly to be oriented along certain aligned collagen fibers, suggesting that radial alignment of collagen fibers relative to tumors facilitates invasion. Consistent with this observation, primary tumor explants cultured in a randomly organized collagen matrix realigned the collagen fibers, allowing individual tumor cells to migrate out along radially aligned fibers. The presentation of these tumor-associated collagen signatures allowed us to identify pre-palpable tumors and see cells at the tumor-stromal boundary invading into the stroma along radially aligned collagen fibers. As such, TACS should provide indications that a tumor is, or could become, invasive, and may serve as part of a strategy to help identify and characterize breast tumors in animal and human tissues.

1,524 citations


Cites background from "[Two-photon laser scanning fluoresc..."

  • ...Multiphoton excitation (MPE) with MPLSM allows imaging of endogenous fluorophores from deep inside live biological tissues with the fluorescence excitation primarily restricted to the plane of focus due to a quadratic dependence on the laser light intensity and a low probability of multiple low-energy photons being absorbed outside the focal plane [16,17,23]....

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  • ...Specifically, multiphoton microscopy results from the nonlinear excitation of molecular fluorescence and can produce images deep into thick tissues [16,17], while SHG signals depend on non-linear interactions of illumination with a non-centrosymmetric environment (e....

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Journal ArticleDOI
TL;DR: The structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging are focused on, with particular attention to recent techniques.
Abstract: Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.

1,214 citations


Cites methods from "[Two-photon laser scanning fluoresc..."

  • ...A number of techniques have been developed to improve the resolution of optical microscopy, including optical sectioning by confocal microscopy (62) and multiphoton excitation (90, 97), improvement of axial resolution by two objective lenses employed in 4Pi (159) and I(5)M (138)....

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