Multiphoton microscopy: a potential "optical biopsy" tool for real-time evaluation of lung tumors without the need for exogenous contrast agents.

Context: Multiphoton microscopy (MPM) is an emerging, nonlinear, optical-biopsy technique, which can generate subcellular-resolution images from unprocessed and unstained tissue in real time.

Objective: To assess the potential of MPM for lung tumor diagnosis.

Design: Fresh sections from tumor and adjacent nonneoplastic lung were imaged with MPM and then compared with corresponding hematoxylin-eosin slides.

Miniature varifocal objective lens for endomicroscopy.

A miniature catadioptric lens for endoscopic imaging based on the principle of wavelength division multiplexing is presented. We demonstrate change of the magnification and the field of view (FOV) of the lens without any mechanical adjustment of the optical elements. The lens provides magnifications of ~-1.

Dual modality endomicroscope with optical zoom capability.

We present a miniature endomicroscope that combines large field-of-view (FOV) (1.15 mm) reflectance imaging with high-resolution (~0.5 μm) multiphoton intrinsic fluorescence imaging.

In vivo imaging of unstained tissues using long gradient index lens multiphoton endoscopic systems.

We characterize long (up to 285 mm) gradient index (GRIN) lens endoscope systems for multiphoton imaging. We fabricate a portable, rigid endoscope system suitable for imaging unstained tissues, potentially deep within the body, using a GRIN lens system of 1 mm diameter and 8 cm length. The portable device is capable of imaging a ~200 µm diameter field of view at 4 frames/s.

In vivo imaging of unstained tissues using a compact and flexible multiphoton microendoscope.

We use a compact and flexible multiphoton microendoscope (MPME) to acquire in vivo images of unstained liver, kidney, and colon from an anesthetized rat. The device delivers femtosecond pulsed 800 nm light from the core of a raster-scanned dual-clad fiber (DCF), which is focused by a miniaturized gradient-index lens assembly into tissue. Intrinsic fluorescence and second-harmonic generation signal from the tissue is epi-collected through the core and inner clad of the same DCF.

Multiphoton microscopy in the evaluation of human bladder biopsies.

Context: Multiphoton microscopy (MPM) is a nonlinear imaging approach, providing cellular and subcellular details from fresh (unprocessed) tissue by exciting intrinsic tissue emissions. With miniaturization and substantially decreased cost on the horizon, MPM is an emerging imaging technique with many potential clinical applications.

Objectives: To assess the imaging ability and diagnostic accuracy of MPM for human bladder biopsies.

Multifocal multiphoton endoscope.

We report a miniaturized resonant/non-resonant multi-fiber raster scanner that is paired with a gradient-index lens assembly to achieve a compact and flexible multifocal multiphoton endoscope capable of longitudinal parallel image acquisition. Multiphoton images are obtained simultaneously at three axial depths, separated by ≥4.8  μm, by incorporating three axially offset double clad optical fibers into the miniaturized scanner.

Multiphoton microscopy and microspectroscopy for diagnostics of inflammatory and neoplastic lung.

Limitations of current medical procedures for detecting early lung cancers inspire the need for new diagnostic imaging modalities for the direct microscopic visualization of lung nodules. Multiphoton microscopy (MPM) provides for subcellular resolution imaging of intrinsic fluorescence from unprocessed tissue with minimal optical attenuation and photodamage. We demonstrate that MPM detects morphological and spectral features of lung tissue and differentiates between normal, inflammatory and neoplastic lung.

A desolvation model for trifluoroethanol-induced aggregation of enhanced green fluorescent protein.

Studies of amyloid disease-associated proteins in aqueous solutions containing 2,2,2-trifluoroethanol (TFE) have shown that the formation of structural intermediates is often correlated with enhanced protein aggregation. Here, enhanced green fluorescent protein (EGFP) is used as a model protein system to investigate the causal relationship between TFE-induced structural transitions and aggregation. Using circular dichroism spectroscopy, light scattering measurements, and transmission electron microscopy imaging, we demonstrate that population of a partially α-helical, monomeric intermediate is roughly correlated with the growth of β-sheet-rich, flexible fibrils for acid-denatured EGFP.

Use of a lensed fiber for a large-field-of-view, high-resolution, fiber-scanning microendoscope.

We report the application of a lensed fiber to a miniaturized fiber raster scanner in order to reduce the fiber's output beam size, thereby allowing for a compact and flexible endoscope capable of a large field of view (FOV) and high spatial resolution. For a proof of principle, the fabricated lensed fiber scanner is paired with a miniaturized gradient-index assembly to achieve a one-photon lateral resolution of 1.1 μm with a FOV that has a diameter of 440 μm.

Ionic strength-dependent persistence lengths of single-stranded RNA and DNA.

Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions.

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates.

Background: Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).

Compact and flexible raster scanning multiphoton endoscope capable of imaging unstained tissue.

We present a compact and flexible endoscope (3-mm outer diameter, 4-cm rigid length) that utilizes a miniaturized resonant/nonresonant fiber raster scanner and a multielement gradient-index lens assembly for two-photon excited intrinsic fluorescence and second-harmonic generation imaging of biological tissues. The miniaturized raster scanner is fabricated by mounting a commercial double-clad optical fiber (DCF) onto two piezo bimorphs that are aligned such that their bending axes are perpendicular to each other. Fast lateral scanning of the laser illumination at 4.

Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores.

Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network.

Multiphoton microscopy for structure identification in human prostate and periprostatic tissue: implications in prostate cancer surgery.

Objective: • To test whether multiphoton microscopy (MPM) might allow identification of prostatic and periprostatic structures with magnification and resolution similar to gold standard histopathology.

Material And Methods: • The present study included 95 robotic radical prostatectomy patients who consented to participate in an Institutional Review Board-approved study starting in 2007. • The types of specimens used for imaging were excised surgical margins and biopsies, and sections obtained from the excised prostate.

Multiphoton polarization imaging of steady-state molecular order in ternary lipid vesicles for the purpose of lipid phase assignment.

We have investigated lipid acyl chain order parameters of giant unilamellar vesicles (GUVs) using multiphoton fluorescence microscopy. We compare two widely used models of lipid acyl chain order parameters: the "wobble-on-a-cone" model and the Gaussian distribution model. For the first time, we systematically address a ternary system for which the phase diagram encompassing both composition and temperature space has been mapped in order to determine tie-line directions and thus phase assignment.

Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation.

Because oligomers and aggregates of the protein α-synuclein (αS) are implicated in the initiation and progression of Parkinson's disease, investigation of various αS aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, β-sheet-rich fibrillar species by incubation of αS in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of αS, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly α-helical conformation at high [TFE].

Precise and millidegree stable temperature control for fluorescence imaging: application to phase transitions in lipid membranes.

We present the design of a custom temperature-controlled chamber suitable for water or oil immersion fluorescence microscopy and its application to phase behavior in lipid bilayer vesicles. The apparatus is self-contained and portable, suitable for multiuser microscopy facilities. It offers a higher temperature resolution and stability than any comparable commercial apparatus, on the order of millidegrees.

Spatiotemporal dynamics of rhythmic spinal interneurons measured with two-photon calcium imaging and coherence analysis.

In rhythmic neural circuits, a neuron often fires action potentials with a constant phase to the rhythm, a timing relationship that can be functionally significant. To characterize these phase preferences in a large-scale, cell type-specific manner, we adapted multitaper coherence analysis for two-photon calcium imaging. Analysis of simulated data showed that coherence is a simple and robust measure of rhythmicity for calcium imaging data.

Endoscope lens with dual fields of view and resolutions for multiphoton imaging.

We report the development of a miniaturized dual-optical-zone endoscope objective lens. The lens has two foci, with 0.18 and 0.

Mechanisms of quenching of Alexa fluorophores by natural amino acids.

Quenching of fluorophores by the same proteins that they covalently label is a phenomenon that is neither well-known nor well-characterized. It is often assumed that fluorophores are unperturbed by their target proteins. However, it has been observed that attached fluorophores can be quenched by contact with amino acids within the same protein, and this property has been exploited to report on changing conformational states or intramolecular dynamics of proteins.

Multiphoton microscopy as a diagnostic imaging modality for lung cancer.

Lung cancer is the leading killer among all cancers for both men and women in the US, and is associated with one of the lowest 5-year survival rates. Current diagnostic techniques, such as histopathological assessment of tissue obtained by computed tomography guided biopsies, have limited accuracy, especially for small lesions. Early diagnosis of lung cancer can be improved by introducing a real-time, optical guidance method based on the in vivo application of multiphoton microscopy (MPM).

GUV preparation and imaging: minimizing artifacts.

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases.