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At least 73 records · Page 4Linked to original sources
PubMedDate not supplied

Spherical aberration correction in multiphoton fluorescence imaging using objective correction collar.

Multiphoton microscopy has evolved into a powerful bioimaging tool in three dimensions. However, the ability to image biological specimens in-depth can be hindered by sample spherical aberration and scattering. These two phenomena can result in the degradation of image resolution and the loss of detected multiphoton signal. In this work, we use the correction collar (for cover glass thickness) associated with a water immersion objective in an attempt to improve multiphoton imaging. In the two samples we examined (human skin and rat tail tendon), we found that while the improvement in image resolution was not visible qualitatively, the measured axial fluorescence or second harmonic generation signal profiles indicate that the use of the correction collar can help to improve the detected multiphoton signals. The maximum increases are 36% and 57% for the skin (sulforhodamine B fluorescence) and tendon (second harmonic generation) specimens, respectively. Our result shows that for in-depth multiphoton imaging, the correction collar may be used to correct for spherical aberration. However, each tissue type needs to be examined to determine the optimal correction collar setting to be used.

Animals↗
PubMed2006

Multiphoton fluorescence and second harmonic generation imaging of the structural alterations in keratoconus ex vivo.

PURPOSE: To demonstrate the application of multiphoton fluorescence (MF) and second harmonic generation (SHG) microscopy for ex vivo characterization of the structural alterations of human corneas due to keratoconus. METHODS: Buttons of keratoconic corneas derived from penetrating keratoplasty were sent for structural analysis with a custom-built multiphoton microscope. Fluorescence detected within the cytoplasm and the SHG signal obtained from collagen were used to demonstrate the morphologic changes in the corneal specimens. RESULTS: The fluorescent epithelial cells around the apical area were elongated and were aligned parallel to the adjacent collagen fibers. Parallel and centripetal distribution patterns of stromal collagen bundles were demonstrated at different depths within the keratoconic corneas. CONCLUSIONS: MF and SHG microscopy provides three-dimensional structural analysis of keratoconus ex vivo. It may provide important morphologic information for the investigation of the pathogenesis of keratoconus and may have potential in a clinical setting as an in vivo diagnostic and monitoring system for advancing keratoconus.

Cornea↗
PubMed2005

Optical microscopy studies of dynamics within individual polymer-dispersed liquid crystal droplets.

Optical devices based on polymer-dispersed liquid crystal (PDLC) thin films derive their functional properties from the electric-field-induced reorientation of (sub)micrometer-sized polymer-dispersed liquid crystal (LC) droplets. In these materials, the LC reorientation dynamics are strongly dependent on droplet size and shape, as well as polymer/LC interfacial interactions. The dynamics also vary spatially within individual droplets. This Account describes studies of individual PDLC droplets and their field-induced dynamics by high-resolution near-field scanning optical microscopy (NSOM) and multiphoton-excited fluorescence microscopy (MPEFM). Included are studies of native ("pure") PDLCs and those doped with ionic compounds and dyes; the latter are used in sophisticated photorefractive materials.

Coloring Agents↗
PubMed2006

Multiphoton microscopy for the investigation of dermal penetration of nanoparticle-borne drugs.

Multiphoton microscopy (MPM) of a dually fluorescence-labeled model system in excised human skin is employed for high-resolution three-dimensional (3D) visualization in order to study the release, accumulation, and penetration properties of drugs released from nanoscale carrier particles in dermal administration. Polymer particles were covalently labeled with fluorescein, whereas Texas Red as a drug-model was dissolved in the particles to be released to the formulation matrix. Single nanoparticles on skin could easily be localized and imaged with diffraction-limited resolution. The temporal evolution of the fluorescent drug-model concentration in various skin compartments over more than 5 hours was investigated by multiphoton spectral imaging of the same area of the specimen. The 3D penetration profile of the drug model in correlation with skin morphology and particle localization information is obtained by multiple laser line excitation experiments. MPM combined with spectral imaging was found to allow noninvasive long-term studies of particle-borne drug-model penetration into skin with subcellular resolution. By dual color labeling, a clear discrimination between particle-bound and released drug model was possible. The introduced technique was shown to be a powerful tool in revealing the dermal penetration properties and pathways of drugs and nanoscale drug vehicles on microscopic level.

Fluorescence↗
PubMed2002

Phase sensitive demodulation in multiphoton microscopy.

Multiphoton laser scanning microscopy offers advantages in depth of penetration into intact samples over other optical sectioning techniques. To achieve these advantages it is necessary to detect the emitted light without spatial filtering. In this nondescanned (nonconfocal) approach, ambient room light can easily contaminate the signal, forcing experiments to be performed in absolute darkness. For multiphoton microscope systems employing mode-locked lasers, signal processing can be used to reduce such problems by taking advantage of the pulsed characteristics of such lasers. Specifically, by recovering fluorescence generated at the mode-locked frequency, interference from stray light and other ambient noise sources can be significantly reduced. This technology can be adapted to existing microscopes by inserting demodulation circuitry between the detector and data collection system. The improvement in signal-to-noise ratio afforded by this approach yields a more robust microscope system and opens the possibility of moving multiphoton microscopy from the research lab to more demanding settings, such as the clinic.

Amplifiers, Electronic↗
PubMedDate not supplied

Effect of pulse duration on two-photon excited fluorescence and second harmonic generation in nonlinear optical microscopy.

We have developed a multiphoton microscopy (MPM) system using a 12-fs Ti:sapphire laser with adjustable dispersion precompensation in order to examine the impact of pulse duration on nonlinear optical signals. The efficiencies of two-photon-excited fluorescence (TPEF) and second harmonic generation (SHG) were studied for various pulse durations, measured at the sample, ranging from approximately 400 fs to sub-20 fs. Both TPEF and SHG increased proportionally to the inverse of the pulse duration for the entire tested range. Because of improved signal-to-noise ratio, sub-20-fs pulses were used to enhance MPM imaging depth by approximately 160%, compared to 120-fs pulses, in human skin.

Algorithms↗
PubMedDate not supplied

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging.

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.

Animals↗
PubMed2006

Preclinical characterization of amyloid imaging probes with multiphoton microscopy.

Multiphoton microscopy is an optical imaging technique that allows high resolution detection of fluorescence in thick, scattering tissues. The technique has been used for trans-cranial imaging of the brains of living transgenic mouse models of Alzheimer's disease. Direct detection of senile plaques in these mice has allowed the characterization of the natural history of individual senile plaques, the evaluation of plaque clearance during immunotherapy, and the characterization of the kinetics and biodistribution of the PET ligand, PIB. With the expanding repertoire of structural and functional fluorescent probes, and the preclinical characterization of new contrast agents for complementary imaging modalities like MRI, PET, SPECT, and NIRS, multiphoton microscopy will continue to be a powerful tool in understanding and combating Alzheimer's disease.

Alzheimer Disease↗
PubMed2006

4Pi microscopy of quantum dot-labeled cellular structures.

The most prominent restrictions of fluorescence microscopy are the limited resolution and the finite signal. Established conventional, confocal, and multiphoton microscopes resolve at best approximately 200nm in the focal plane and only 500nm in depth. Additionally, organic fluorophores and fluorescent proteins are bleached after 10(4)-10(5) excitation cycles. To overcome these restrictions, we synergistically combine the 3- to 7-fold improved axial resolution of 4Pi microscopy with the greatly enhanced photostability of semiconductor quantum dots. Co-localization studies of immunolabeled microtubules and mitochondria demonstrate the feasibility of this approach for routine biological measurements. In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail.

Animals↗
PubMed2005

Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo.

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot-labeled bone marrow-derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

Animals↗
PubMed2004

Imaging coronary artery microstructure using second-harmonic and two-photon fluorescence microscopy.

The microstructural basis for the mechanical properties of blood vessels has not been directly determined because of the lack of a nondestructive method that yields a three-dimensional view of these vascular wall constituents. Here, we demonstrate that multiphoton microscopy can be used to visualize the microstructural basis of blood vessel mechanical properties, by combining mechanical testing (distension) of excised porcine coronary arteries with simultaneous two-photon excited fluorescence and second-harmonic generation microscopy. Our results show that second-harmonic generation signals derived from collagen can be spectrally isolated from elastin and smooth muscle cell two-photon fluorescence. Two-photon fluorescence signals can be further characterized by emission maxima at 495 nm and 520 nm, corresponding to elastin and cellular contributions, respectively. Two-dimensional reconstructions of spectrally fused images permit high-resolution visualization of collagen and elastin fibrils and smooth muscle cells from intima to adventitia. These structural features are confirmed by coregistration of multiphoton microscopy images with conventional histology. Significant changes in mean fibril thickness and overall wall dimension were observed when comparing no load (zero transmural pressure) and zero-stress conditions to 30 and 180 mmHg distension pressures. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric properties of the microstructure of the blood vessel wall.

Animals↗
PubMed2006

Intact corneal stroma visualization of GFP mouse revealed by multiphoton imaging.

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, 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.

Animals↗
PubMed2003

Monte Carlo simulation of multiphoton fluorescence microscopic imaging through inhomogeneous tissuelike turbid media.

Image resolution and signal level in fluorescence microscopy through inhomogeneous turbid media consisting of scatterers of multiple sizes under single- (1p), two- (2p), and three-photon (3p) excitation have been investigated based on a modified Monte Carlo model. The effects of the size distribution and the concentration distribution of scattering particles are explored. Simulation results reveal that the size and the concentration distribution both have an impact on image formation in media consisting of small particles and that 3p excitation has the most significant impact. In media with scatterers of a large size, both size and concentration distributions lead to a slight effect. Image formation in a mixed medium containing small and large scattering particles is more affected by the large particles.

Anisotropy↗
PubMedDate not supplied

Excitation beyond the monochromatic laser limit: simultaneous 3-D confocal and multiphoton microscopy with a tapered fiber as white-light laser source.

Confocal and multiphoton microscopy are essential tools in modern life sciences. They allow fast and highly resolved imaging of a steadily growing number of fluorescent markers, ranging from fluorescent proteins to quantum dots and other fluorophores, used for the localization of molecules and the quantitative detection of molecular properties within living cells and organisms. Up to now, only one physical limitation seemed to be unavoidable. Both confocal and multiphoton microscopy rely on lasers as excitation sources, and their monochromatic radiation allows only a limited number of simultaneously usable dyes, which depends on the specific number of laser lines available in the used microscope. We have overcome this limitation by successfully replacing all excitation lasers in a standard confocal microscope with pulsed white light ranging from 430 to 1300 nm generated in a tapered silica fiber. With this easily reproducible method, simultaneous confocal and multiphoton microscopy was demonstrated. By developing a coherent and intense laser source with spectral width comparable to a mercury lamp, we provide the flexibility to excite any desired fluorophore combination.

Animals↗
PubMed2006

Setup and characterization of a multiphoton FLIM instrument for protein-protein interaction measurements in living cells.

BACKGROUND: Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in Förster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed. METHODS: We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching. RESULTS: We develop and validate here a protocol for rigorous characterization of time-domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET. CONCLUSIONS: We report a well-designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal-based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET.

Bacterial Proteins↗
PubMedDate not supplied

Second-harmonic imaging of cornea after intrastromal femtosecond laser ablation.

Nonlinear laser scanning microscopy is widely used for noninvasive imaging in cell biology and tissue physiology. However, multiphoton fluorescence imaging of dense, transparent connective tissue (e.g., cornea) is challenging since sophisticated labeling or slicing is necessary. High-resolution, high-contrast second harmonic generation (SHG) imaging of corneal tissue based on the intrinsic structure of collagen is discussed. The three-dimensional corneal ultrastructure in depths up to hundreds of microns can be probed noninvasively, without any staining or mechanical slicing. As an important application of second harmonic imaging in ophthalmology, the modification of corneal ultrastructure using femtosecond laser intrastromal ablation is systematically investigated to evaluate next-generation refractive surgical approaches.

Animals↗
PubMed2006

MPScope: a versatile software suite for multiphoton microscopy.

MPScope is a software suite to control and analyze data from custom-built multiphoton laser scanning fluorescence microscopes. The acquisition program MPScan acquires, displays and stores movies, linescans, image stacks or arbitrary regions from up to four imaging channels and up to two analog inputs, while plotting the intensity of regions of interest in real-time. Bidirectional linescans allow 256 x 256 pixel frames to be acquired at up to 10 fps with typical galvanometric scanners. A fast stack mode combines movie acquisition with continuous z-focus motion and adjustment of laser intensity for constant image brightness. Fast stacks can be automated by custom programs running in an integrated scripting environment, allowing a 1 mm(3) cortical volume to be sampled in 1 billion voxels in approximately 1 h. The analysis program MPView allows viewing of stored frames, projections, automatic detection of cells and plotting of their average intensity across frames, direct frame transfer to Matlab, AVI movie creation and file export to ImageJ. The combination of optimized code, multithreading and COM (Common Object Model) technologies enables MPScope to fully take advantage of custom-built two-photon microscopes and to simplify their realization.

Animals↗