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Results for “Microscopy, Fluorescence, Multiphoton”

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At least 127 records · Page 7Linked to original sources
PubMed2007

Three-dimensional investigation and scoring of extracellular matrix remodeling during lung fibrosis using multiphoton microscopy.

The organization of collagen during fibrotic processes is poorly characterized because of the lack of appropriate methodologies. Here we show that multimodal multiphoton microscopy provides novel insights into lung fibrosis. We characterize normal and fibrotic pulmonary tissue in the bleomycin model, and show that second-harmonic generation by fibrillar collagen reveals the micrometer-scale three-dimensional spatial distribution of the fibrosis. We find that combined two-photon excited fluorescence and second-harmonic imaging of unstained lung tissue allows separating the inflammatory and fibrotic steps in this pathology, underlining characteristic features of fibroblastic foci in human Idiopathic Pulmonary Fibrosis samples. Finally, we propose phenomenological scores of lung fibrosis and we show that they unambiguously sort out control and treated mice, with a better sensitivity and reproducibility in the subpleural region. These results should be readily generalized to other organs, as an accurate method to assess extracellular matrix remodeling during fibrosis.

Animals↗
PubMed2006

Two-photon microscopes and in vivo multiphoton tomographs--powerful diagnostic tools for tissue engineering and drug delivery.

Near-infrared multiphoton microscopes and in vivo femtosecond laser tomographs are novel powerful diagnostic tools for intra-tissue drug screening and high-resolution structural imaging applicable to many areas of biomedical research. Deep tissue cells and extracellular matrix (ECM) compartments can be visualized in situ with submicron resolution without the need for tissue processing. In particular, the reduced fluorescent coenzyme NAD(P)H, flavoproteins, keratin, melanin, and elastin are detected by two-photon excited autofluorescence, whereas myosin, tubulin and the ECM protein collagen can be imaged additionally by second harmonic generation (SHG). Therefore, these innovative multiphoton technologies have been used to probe architecture and state of a variety of native tissues, as well as of tissue-engineered constructs, giving insights on the interaction between scaffolds and seeded cells in vitro prior implantation. Moreover, non-invasive 4-D multiphoton tomographs are employed in clinical studies to examine the diffusion behavior, the intra-tissue accumulation of topically applied cosmetic and pharmaceutical components, and their interaction with skin cells.

Animals↗
PubMed2006

Direct monitoring of vesicular release and uptake in brain slices by multiphoton excitation of the styryl FM 1-43.

Fluorescence imaging using FM 1-43 and related styryl dyes has provided invaluable insights into presynaptic function of synapses in culture preparations, but has been limited in use for studying central synapses in vivo or in brain slices, because of excessive fluorescence background due to nonspecific membrane binding of dye. We demonstrate here that focal excitation of FM dyes using two-photon laser-scanning microscopy (TPLSM) provides high resolution of FM 1-43-labeled nerve terminals in brain slices by suppressing out-of-focus background and that a readily releasable pool of vesicles can be selectively and stably labeled by hypertonic shock despite slice diffusion barriers. We find direct TPLSM of FM 1-43-labeled nerve terminals to be superior to treatment of slices with either the fluorescent quencher sulforhodamine 101 or dye scavenger ADVASEP-7 in resolving nerve terminal against background fluorescence, enabling continuous monitoring of vesicular uptake, and release of styryl dyes from individual nerve terminals in brain slices.

Animals↗
PubMed2002

Comparison of the axial resolution of practical Nipkow-disk confocal fluorescence microscopy with that of multifocal multiphoton microscopy: theory and experiment.

We compare the axial sectioning capability of multifocal confocal and multifocal multiphoton microscopy in theory and in experiment, with particular emphasis on the background arising from the cross-talk between adjacent imaging channels. We demonstrate that a time-multiplexed non-linear excitation microscope exhibits significantly less background and therefore a superior axial resolution as compared to a multifocal single-photon confocal system. The background becomes irrelevant for thin (< 15 microm) and sparse fluorescent samples, in which case the confocal parallelized system exhibits similar or slightly better sectioning behaviour due to its shorter excitation wavelength. Theoretical and experimental axial responses of practically implemented microscopes are given.

Fluorescence Polarization↗
PubMed2005

Dynamic multiphoton imaging: a live view from cells to systems.

Leaps in scientific technology often occur at the interface of seemingly disparate disciplines. This holds true with the recent application of multiphoton microscopy to the biological sciences, leading to a new generation of imaging-based studies extending from the tracking of individual molecules within living cells to the observation of whole organisms.

Animals↗
PubMed2004

Improved "optical highlighter" probes derived from discosoma red fluorescent protein.

The tetrameric red fluorescent protein, DsRed, undergoes a rapid red to green color change evoked by short wavelength (lambda < 760 nm) femtosecond irradiation--a phenomenon that underpins the application of DsRed as an "optical highlighter" probe for tracking live cells, organelles, and fusion proteins. This color change results from selective bleaching of the "mature" red-emitting species of DsRed and an enhancement of emission from the "immature" green species, likely caused by dequenching of fluorescence resonance energy transfer occurring within the protein tetramer. Here, we have examined the role of residues known to influence the rate and completeness of chromophore maturation on the cellular and biophysical properties of DsRed mutants. Surprisingly, a single amino acid mutation (N42Q) with increased basal green emission yet rapid chromophore maturation displayed a multiphoton-evoked color change that was brighter, more consistent, more vivid, and easier to evoke than DsRed, despite the larger proportion of green chromophores. Rapidly maturing mutants with more complete chromophore maturation, exhibited little color change and increased resistance to multiphoton bleaching. We describe improved optical and cell biological properties for two DsRed-derived variants which we showcase in photolabeling studies, and discuss these data in terms of implications for fluorescence resonance energy transfer-based probes.

Cell Line↗
PubMed2006

Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism.

The dispersive nature of the acousto-optical deflector (AOD) presents a challenge to applications of two sequential orthogonal AODs (a two-dimensional AOD) as XY scanners in multiphoton microscopy. Introducing a prism before the two-dimensional (2D) AOD allows both temporal and spatial dispersion to be compensated for simultaneously. A 90 fs laser pulse was broadened to 572 fs without compensation, and 143 fs with compensation. The ratio of long axis to short axis of the exiting laser beam spot was 3.50 without compensation and 1.14 with compensation. The insertion loss was 37%. Two-photon fluorescence microscopy used the compensated 2D AOD scanner to image a fluorescent microsphere, which improves signal intensity -15-fold compared with the uncompensated scanner.

Artifacts↗
PubMed2006

Probing the microenvironment of mammary tumors using multiphoton microscopy.

Advances in optical imaging technologies that allow the subcellular resolution of undissected tissue have begun to offer new clues into the biology of development and disease. For cancer, such advances mean that the primary tumor is no longer a black box and that the disease can be studied throughout the metastatic cascade and not just as an endpoint. In this review we examine the advances in multiphoton imaging technology that have been used to define the microenvironment and its role in delineating the invasion and intravasation steps of metastasis inside living mammary tumors. Results show that the tumor microenvironment is a dynamic place where interactions between tumor cells, macrophages, blood vessels, and extracellular matrix fibers define the metastatic phenotype.

Animals↗
PubMed2004

Multiphoton switching dynamics of single green fluorescent proteins.

Multi-photon driven photo-switching between dark and bright (fluorescent) states of a green fluorescent protein (GFP) mutant is demonstrated. A single-protein investigation shows the existence of two distinct bright states that display sharp two-photon cross-section bands peaked at 780 nm and at 870 nm. Fluorescence of these two species can be independently switched on and off. These results highlight a new photoconversion pathway for photochromic GFPs and can have significant applications in multi-photon confocal microscopy and in optical data-storage architectures.

Green Fluorescent Proteins↗
PubMed2005

Multiphoton excitation-evoked chromophore-assisted laser inactivation using green fluorescent protein.

Noninvasive, straightforward methods to inactivate selected proteins in living cells with high spatiotemporal resolution are needed. Chromophore-assisted laser inactivation (CALI) can be used to photochemically inactivate proteins, but it has several drawbacks, such as procedural complexity and nonspecific photodamage. Here we show that by application of multiphoton excitation to CALI, enhanced green fluorescent protein (EGFP) is an effective chromophore for inactivation of a protein's function without nonspecific photodamage in living mammalian cells.

Connexin 43↗
PubMedDate not supplied

Multiphoton excited fabrication of collagen matrixes cross-linked by a modified benzophenone dimer: bioactivity and enzymatic degradation.

Multiphoton excited (MPE) photochemistry is used to fabricate model tissue engineering scaffolds directly from types I, II, and IV collagen. A modified benzophenone dimer (BPD) provides the photoactivation and becomes incorporated into the resulting collagen matrixes. Unlike xanthene photochemistries, the benzophenone dimer can be used in acidic environments, where most forms of collagen have the greatest solubility. The minimum feature sizes are investigated by using two- and three-photon excitation, where the latter provides for superior "resolution" and suggests that collagen structures can be fabricated on the size scales of focal contacts. The resulting structures display excellent retention of bioactivity as evidenced by highly specific cell adhesion as well as immunofluorescence labeling. Structural and chemical aspects of the collagen matrixes are probed through measuring the enzymatic degradation through specific and nonspecific proteases, as the resulting relative rates are consistent with the activity of these enzymes. The degradation rates can also be controlled through varying the cross-link density in the matrixes, which is achieved through tuning the exposure dose during the fabrication process. The degradation rates are also found to be consistent with swelling/shrinking measurements and thus the average mesh size of the matrixes. In all cases the enzymatic degradations are well-fit single exponentials, suggesting that the matrixes can be fabricated with a priori knowledge of their structural properties. These results coupled with the resulting bioactivity suggest that the multiphoton fabrication process may be a powerful tool for the creation of cell-sized tissue engineering scaffolds.

Benzophenones↗
PubMedDate not supplied

Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy.

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.

Animals↗
PubMed2004

Optical biopsy of liver fibrosis by use of multiphoton microscopy.

We demonstrate the application of multiphoton microscopy in diagnosing toxin- (CCl4-) induced liver fibrosis in mice. Although hepatocyte autofluorescence does not vary significantly, different degrees of necrosis and stellate cell proliferation at necrotic sites in livers with fibrosis (ex vivo) can be detected easily from multiphoton-induced autofluorescence images by use of 780-nm excitation. Our result suggests that multiphoton microscopy can be developed into an effective technique for the detection and diagnosis of liver fibrosis in vivo.

Animals↗
PubMed2003

Multiphoton excitation spectra in biological samples.

Multiphoton microscopy is becoming a popular mode of live and fixed cell imaging. This mode of imaging offers several advantages due to the fact that fluorochrome excitation is a nonlinear event resulting in excitation only at the plane of focus. Multiphoton excitation is enhanced by the use of ultrafast lasers emitting in the near IR, offering better depth penetration coupled with efficient excitation. Because these lasers, such as titanium:sapphire lasers, offer tunable output it is possible to use them to collect multiphoton excitation spectra. We use the software-tunable Coherent Chameleon laser coupled to the Zeiss LSM 510 META NLO to acquire x-y images of biological samples at multiple excitation wavelengths, creating excitation lambda stacks. The mean intensity of pixels within the image plotted versus excitation wavelength reveals the excitation spectra. Excitation lambda stacks can be separated into individual images corresponding to the signal from different dyes using linear unmixing algorithms in much the same way that emission fingerprinting can be used to generate crosstalk free channels from emission lambda stacks using the META detector. We show how this technique can be used to eliminate autofluorescence and to produce crosstalk-free images of dyes with very close overlap in their emission spectra that cannot be separated using emission fingerprinting. Moreover, excitation finger- printing can be performed using nondescanned detectors (NDDs), offering more flexibility for eliminating autofluorescence or crosstalk between fluorochromes when imaging deep within the sample. Thus, excitation fingerprinting complements and extends the functions offered by the META detector and emission fingerprinting. We correct biases in the laser and microscope transmission to acquire realistic multiphoton excitation spectra for fluorochromes within cells using the microscope, which enables the optimization of the excitation wavelength for single and multilabel experiments and provides a means for studying the influence of the biological environment on nonlinear excitation.

3T3 Cells↗
PubMed2004

Laser spectral characterization in multiphoton microscopy.

Spectral and temporal characterization is a fundamental task when a tunable Ti:sapphire ultrafast laser system is operated for multiphoton microscopy applications. In the present paper simple procedures are reported that perform laser-peak-emission wavelength and bandwidth measurements without the need of any further instrumentation but a simple and inexpensive diffraction grating, by taking advantage of the confocal microscope imaging capabilities.

Equipment Design↗
PubMed2006

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells.

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.

Algorithms↗