Using multiphoton excitation to explore the murky depths of developing embryos.
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Animals↗
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Results for “Microscopy, Fluorescence, Multiphoton”
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Improving the penetration depth in multiphoton excitation laser scanning microscopy.
More than a threefold increase in multiphoton laser scanning microscopy depth penetration using a passive predispersion compensation system is reported. Using dispersion-controlled pulses to counteract the effects of positive group delay dispersion in the imaging platform, optical sectioning of fluorescent samples to depths in excess of 800 microm was observed, compared with only 240 microm using a noncompensated setup. Experimental results obtained from both the predispersion compensated and noncompensated systems are compared with theoretical values of pulse broadening in a laser scanning microscope. The observed improvement in depth profiling potentially widens the applications and user base of nonlinear microscopy techniques.
Anatomy, Cross-Sectional↗
Intravital fluorescence microscopy in pulmonary research.
Over the last several years, microscopy as a scientific tool has reinvented itself evolving from a group of principally descriptive methodologies to encompass a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technology, the development of fluorescent proteins as well as the advent of inexpensive powerful computers, has led to the feasibility of simultaneous sub micron resolution and quantitation of multiple concurrent molecular markers for both protein and DNA. Confocal microscopy has allowed optical sectioning and reconstruction of tissues in three dimensions. Finally, the development of multiphoton methodologies as an extension of optical sectioning microscopy has further improved the potential utility of this technology when examining living or light scattering tissues such as the lung. In order to illustrate the utility of two-photon methods in pulmonary biology, we present the application of this approach to the study of cellular trafficking in situ and to the study of pulmonary vasoregulation in an ex vivo rodent model.
Animals↗
In vivo multiphoton imaging of a transgenic mouse model of Alzheimer disease reveals marked thioflavine-S-associated alterations in neurite trajectories.
Postmortem analyses of senile plaques reveal numerous dystrophic processes in their vicinity. We used in vivo multiphoton microscopy of a transgenic model of Alzheimer disease (AD) to simultaneously image senile plaques and nearby neuronal processes. Plaques were labeled by immunofluorescent staining or thioflavine-S and neuronal processes were labeled with a fluorescent dextran conjugate. Imaging of 3-dimensional volumes in the vicinity of plaques revealed subtle changes in neurite geometry in or near diffuse plaques. By contrast, disruptions in neurite morphology, including dystrophic neurites immediately surrounding plaques as well as major alterations in neurite trajectories, were seen in association with thioflavine-S-positive plaques. Nearly half of all labeled processes that came within 50 microm of a thioflavine-S-positive plaque were altered, suggesting a fairly large "halo" of neuropil alterations that extend beyond the discrete border of a thioflavine-S plaque. These results support the hypothesis that compact thioflavine-S-positive plaques disrupt the neuropil in AD.
Aging↗
Single-photon counting multicolor multiphoton fluorescence microscope.
We present a multicolor multiphoton fluorescence microscope with single-photon counting sensitivity. The system integrates a standard multiphoton fluorescence microscope, an optical grating spectrograph operating in the UV-Vis wavelength region, and a 16-anode photomultiplier tube (PMT). The major technical innovation is in the development of a multichannel photon counting card (mC-PhCC) for direct signal collection from multi-anode PMTs. The electronic design of the mC-PhCC employs a high-throughput, fully-parallel, single-photon counting scheme along with a high-speed electrical or fiber-optical link interface to the data acquisition computer. There is no electronic crosstalk among the detection channels of the mC-PhCC. The collected signal remains linear up to an incident photon rate of 10(8) counts per second. The high-speed data interface offers ample bandwidth for real-time readout: 2 MByte lambda-stacks composed of 16 spectral channels, 256 x 256 pixel image with 12-bit dynamic range can be transferred at 30 frames per second. The modular design of the mC-PhCC can be readily extended to accommodate PMTs of more anodes. Data acquisition from a 64-anode PMT has been verified. As a demonstration of system performance, spectrally resolved images of fluorescent latex spheres and ex-vivo human skin are reported. The multicolor multiphoton microscope is suitable for highly sensitive, real-time, spectrally-resolved three-dimensional imaging in biomedical applications.
Data Display↗
Effects of objective numerical apertures on achievable imaging depths in multiphoton microscopy.
Multiphoton microscopy is a powerful technique for achieving three-dimensional submicron imaging in biological specimens. However, specimen optical parameters such as refractive indices and scattering coefficients can result in the loss of image resolution and decreased signal in depth. These factors are coupled to the focusing objective's numerical aperture (NA) in limiting the achievable imaging depths. In this work, we performed multiphoton imaging on aqueous fluorescent solution, human skin, and rat tail tendon to show that, under the same immersion condition, lower NA objectives can examine more deeply into biological specimens and should be used when optimal imaging depths is desired.
Animals↗
[New approach of fibrosis by multiphoton microscopy with second harmonic generation].
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Animals↗
High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution.
High-resolution four-dimensional (4-D) optical tomography of human skin based on multiphoton autofluorescence imaging and second harmonic generation (SHG) was performed with the compact femtosecond laser imaging system DermaInspect as well as a modified multiphoton microscope. Femtosecond laser pulses of 80 MHz in the spectral range of 750 to 850 nm, fast galvoscan mirrors, and a time-correlated single-photon counting module have been used to image human skin in vitro and in vivo with subcellular spatial and 250-ps temporal resolution. The nonlinear induced autofluorescence originates from naturally endogenous fluorophores and protein structures such as reduced nicotinamide adenine dinucleotide phosphate, flavins, collagen, elastin, porphyrins, and melanin. Second harmonic generation was used to detect collagen structures. Tissues of patients with dermatological disorders such as psoriasis, fungal infections, nevi, and melanomas have been investigated. Individual intratissue cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by fluorescence excitation spectra, by determination of the fluorescence decay per pixel, and by fluorescence lifetime imaging. The novel noninvasive multiphoton autofluorescence-SHG imaging technique provides 4-D (x,y,z,tau) optical biopsies with subcellular resolution and offers the possibility of introducing a high-resolution optical diagnostic method in dermatology.
Adult↗
Two-photon induced uncaging of a reactive intermediate. Multiphoton in situ detection of a potentially valuable label for biological applications.
[reaction: see text] Two-photon induced Wolff rearrangement of a terphenyl diazoketone 1 was achieved by using focused laser pulses of 532 nm from a Q-switched Nd:YAG laser. The nonfluorescent terphenyl diazoketone 1 was transformed into a fluorescent ester derivative 4, which can be detected in situ using the focused laser pulses at 532 nm. Laser power dependence studies show that the Wolff rearrangement is induced by two-photon absorption of the terphenyl diazoketone 1, but suggests that more than two photons of 532 nm are involved (a multiphoton process) in excitation of the ester derivative 4.
Diazomethane↗
Advances in laser sources for confocal and multiphoton microscopy.
The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.
Animals↗
Imaging vascular pathology.
Examining the mechanisms of renal microvascular alterations in disease and the ramifications they have for overall organ function has remained a challenge due to the complexity of the renal microvascular system and the inability to examine it with sufficient resolution in vivo. However, advances in intravital microscopy have provided opportunities to meet these challenges. In this review we will examine specific areas where intravital imaging has advanced our knowledge of renal disease processes and present areas where these techniques, especially intravital multiphoton microscopy, have even further potential to integrate our knowledge of renal vascular pathology.
Animals↗
Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy.
Neurotrophins such as nerve growth factor (NGF) may be useful for treating diseases in the central nervous system; our ability to harness the potential therapeutic benefit of NGF is directly related to our understanding of the fate of exogenously supplied factors in brain tissue. We utilized multiphoton microscopy to quantify the dynamic behavior of NGF in coronal, 400- micro m thick, fresh rat brain tissue slices. We administered a solution containing bioactive rhodamine nerve growth factor conjugate via pressure injection and monitored the dispersion in the striatal region of the coronal slices. Multiphoton microscopy facilitated repeated imaging deep ( approximately 200 micro m) into tissue slices with minimal photodamage of tissue and photobleaching of label. The pressure injection paradigm approximated diffusion from a point source, and we therefore used the corresponding solution to the diffusion equation to estimate an apparent diffusion coefficient in brain tissue (D(b)(34 degrees C)) of 2.75 +/- 0.24 x 10(-7) cm(2)/s (average +/- SE). In contrast, we determined a corresponding free diffusion coefficient in buffered solution (D(f)(34 degrees C)) of 12.6 +/- 0.9 x 10(-7) cm(2)/s using multiphoton fluorescence photobleaching recovery. The tortuosity, defined as the square root of the ratio of D(f) to D(b), was 2.14 and moderate in magnitude.
Algorithms↗
Multiphoton fluorescence lifetime imaging of human hair.
In vivo and in vitro multiphoton imaging was used to perform high resolution optical sectioning of human hair by nonlinear excitation of endogenous as well as exogenous fluorophores. Multiphoton fluorescence lifetime imaging (FLIM) based on time-resolved single photon counting and near-infrared femtosecond laser pulse excitation was employed to analyze the various fluorescent hair components. Time-resolved multiphoton imaging of intratissue pigments has the potential (i) to identify endogenous keratin and melanin, (ii) to obtain information on intrahair dye accumulation, (iii) to study bleaching effects, and (iv) to monitor the intratissue diffusion of pharmaceutical and cosmetical components along hair shafts.
Adult↗
Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.
Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.
Angiotensin II↗
Two-dimensional imaging without scanning by multifocal multiphoton microscopy.
We describe multifocal multiphoton microscopy giving images without laser scanning. A multitude of 8 x 8 laser beams is focused into a sample yielding two-photon excitation in a plane. The focal spots are arranged in a rectangular array with close spacing between individual points (approximately 0.5 microm). The fluorescence emission from the sample is recorded with a CCD camera, but, owing to the close distance between the beams, they can no longer be regarded as individual points but rather as an illumination of the plane that is covered by the array of focal points. The axial sectioning capability is comparable with an ordinary single-beam two-photon microscope. Interference between the beams that could compromise the axial sectioning capability does not occur in our setup owing to small temporal delays between the individual beams. The axial sectioning capability of the setup is discussed in detail by means of the step response in which the foci are scanned axially into a uniformly fluorescent medium.
Equipment Design↗
In vivo imaging of reactive oxygen species specifically associated with thioflavine S-positive amyloid plaques by multiphoton microscopy.
Amyloid-beta, the primary constituent of senile plaques in Alzheimer's disease, is hypothesized to cause neuronal damage and cognitive failure, but the mechanisms are unknown. Using multiphoton imaging, we show a direct association between amyloid-beta deposits and free radical production in vivo in live, transgenic mouse models of Alzheimer's disease and in analogous ex vivo experiments in human Alzheimer tissue. We applied two fluorogenic compounds, which become fluorescent only after oxidation, before imaging with a near infrared laser. We observed fluorescence associated with dense core plaques, but not diffuse plaques, as determined by subsequent addition of thioflavine S and immunohistochemistry for amyloid-beta. Systemic administration of N-tert-butyl-alpha-phenylnitrone, a free radical spin trap, greatly reduced oxidation of the probes. These data show directly that a subset of amyloid plaques produces free radicals in living, Alzheimer's models and in human Alzheimer tissue. Antioxidant therapy neutralizes these highly reactive molecules and may therefore be of therapeutic value in Alzheimer's disease.
Alzheimer Disease↗
Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy.
We describe novel approaches for compensating dispersion effects that arise when acousto-optic (AO) beam deflection of ultrafast laser pluses is used for multiphoton laser-scanning microscopy (MPLSM). AO deflection supports quick positioning of a laser beam to random locations, allowing high frame-rate imaging of user-selected sites of interest, in addition to conventional raster scanning. Compared to standard line-scan approaches, this results in improved signal strength (and thus increased signal-to-noise) as well as reduced photobleaching and photodamage. However, 2-D AO scanning has not yet been applied for multiphoton microscopy, largely because ultrafast laser pulses experience significant spatial and temporal dispersion while propagating through AO materials. We describe and quantify spatial dispersion, demonstrating it to be a significant barrier to achieving maximal spatial resolution. We also address temporal dispersion, which is a well-documented effect that limits multiphoton excitation efficacy, and is particularly severe for AO devices. To address both problems, we have developed a single diffraction grating scheme that reduces spatial dispersion more than three-fold throughout the field of view, and a novel four-pass stacked-prism prechirper that fully compensates for temporal dispersion while reducing by two-fold the required physical length relative to commonly employed designs. These developments enable the construction of a 2-D acousto-optic multiphoton laser-scanning microscope system.
Acoustics↗
Combined multiphoton microscopy and optical coherence tomography using a 12-fs broadband source.
A 12-fs broadband (100-nm) source is used to combine multiphoton microscopy (MPM) and optical coherence tomography (OCT) in a single platform. An ultrafast Ti:sapphire laser simultaneously provides short pulses necessary for efficient MPM excitation and the broad bandwidth required for high-resolution OCT. Using 0.3-microm microspheres and a 63x, 0.95 numerical aperture objective, we demonstrate that MPM and OCT channels are coregistered with lateral resolution of approximately 0.5 microm and axial resolution of approximately 1.5 microm. Preliminary studies of a 3-D organotypic epithelial tissue model show that multiphoton images of fluorescence and second harmonic signals are derived from cellular and extracellular matrix structures, respectively, while OCT images are generated from scattering interfaces due to tissue variations in refractive index. The combined MPM/OCT microscope is capable of providing simultaneous functional and structural information from cells and extracellular matrix and is potentially a powerful tool for studying biological processes in thick tissues.
Equipment Design↗