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SignificanceThree-photon (3P) microscopy significantly increases the depth and resolution of in vivo imaging due to decreased scattering and nonlinear optical sectioning. Simultaneous excitation of multiple fluorescent proteins is essential to study multicellular interactions and dynamics in the intact brain.AimWe characterized the excitation laser pulses at a range of wavelengths for 3P microscopy, and then explored the application of tdTomato or mScarlet and EGFP for dual-color single-excitation structural 3P imaging deep in the living mouse brain.ApproachWe used frequency-resolved optical gating to measure the spectral intensity, phase, and retrieved pulse widths at a range of wavelengths. Then, we performed in vivo single wavelength-excitation 3P imaging in the 1225 - 1360 nm range deep in the mouse cerebral cortex to evaluate the performance of tdTomato or mScarlet in combination with EGFP.ResultsWe find that tdTomato and mScarlet, expressed in oligodendrocytes and neurons respectively, have high signal-to-background in the 1300–1360 nm range, consistent with enhanced 3P cross sections.ConclusionsThese results suggest that a single excitation wavelength source is advantageous for multiple applications of dual-color brain imaging and highlight the importance of empirical characterization of individual fluorophores for 3P microscopy.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

We demonstrate a two photon (2P) fiber STED microscope in which the excitation and STED light are delivered to the sample in polarization maintaining (PM) fiber.

The DyMIN method reduces photobleaching, a problem in STED microscopy. Labs implementing custom-built STED microscopes would greatly benefit from DyMIN capabilities. We present an inexpensive, open-source version utilizing an FPGA and multiplexer.

Targeting specifics subsets of peripheral pathways of the autonomic nervous system will enable new avenues to study organ control and develop new disease therapies. Vagus nerve stimulation (VNS) has shown many therapeutic benefits but current approaches involve imprecise electrical stimulation that gives rise to adverse effects, and the functionally relevant pathways are poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate highly specific neural communication with light-sensitive actuators (opsins). Opsins can be targeted to cell populations of interest based on the location of viral delivery and genetic control of expression. Here, we tested whether holographic photostimulation of subsets of axons of the cervical vagus nerve that innervate the heart can be used to modulate cardiac function. Viral injection of retrograde adeno-associated virus (rAAV2-retro) in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Selective holographic photostimulation of axons resulted in changes in heart rate, surface cardiac electrogram, and respiratory responses that were different from responses elicited by whole nerve photostimulation.

A diode-based time lens laser with nonlinear pulse compression at 976 nm produces 700 fs pulses with 17 kW peak power. Two-photon imaging of a mouse brain slice is demonstrated with the laser.

We present widefield structured illumination in a miniature, light-weight fiber-coupled microscope with electrowetting axial scanning. We demonstrate imaging of YFP-labeled neurons in mouse brain tissue showing potential for fast volumetric imaging in freely moving animals.

We demonstrate a near-infrared, femtosecond, diode laser-based source with kW peak power for two-photon microscopy. At a wavelength of 976 nm, the system produces sub-ps pulses operating at a repetition rate of 10 MHz with kilowatt class peak powers suitable for deep tissue two-photon microscopy. The system, integrated with a laser-scanning microscope, images to a depth of 900 µm in a fixed sample of PLP-eGFP labeled mouse brain tissue. This represents a significant development that will lead to more efficient, compact, and accessible laser sources for biomedical imaging.

Detection of circulating tumor cells (CTCs) in a blood sample is limited by the sensitivity and specificity of the biomarker panel used to identify CTCs over other blood cells. In this work, we present Bayesian theory that shows how test sensitivity and specificity set the rarity of cell that a test can detect. We perform our calculation of sensitivity and specificity on our image cytometry biomarker panel by testing on pure disease positive (D+ ) populations (MCF7 cells) and pure disease negative populations (D- ) (leukocytes). In this system, we performed multi-channel confocal fluorescence microscopy to image biomarkers of DNA, lipids, CD45, and Cytokeratin. Using custom software, we segmented our confocal images into regions of interest consisting of individual cells and computed the image metrics of total signal, second spatial moment, spatial frequency second moment, and the product of the spatial-spatial frequency moments. We present our analysis of these 16 features. The best performing of the 16 features produced an average separation of three standard deviations between D+ and D- and an average detectable rarity of ∼1 in 200. We performed multivariable regression and feature selection to combine multiple features for increased performance and showed an average separation of seven standard deviations between the D+ and D- populations making our average detectable rarity of ∼1 in 480. Histograms and receiver operating characteristics (ROC) curves for these features and regressions are presented. We conclude that simple regression analysis holds promise to further improve the separation of rare cells in cytometry applications. © 2017 International Society for Advancement of Cytometry.

We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and in vivo neural imaging in awake and freely-behaving animals.

Electrowetting devices are compact, low power, transmissive, and adaptable. We have demonstrated nonmechanical beam steering in a LIDAR system and imaging in a multiphoton microscope, with an electrowetting prism.

The term “acquired brain injury” refers to any type of brain damage that occurs after birth. Two main types of acquired brain injury are stroke and traumatic brain injury (TBI). A stroke occurs when there is a blockage or bleed in the vascular system of the brain, while a TBI results from an external force to the head. Older adults are at a higher risk of both stroke and TBI; thus, overall incidence is increasing as the proportion of older adults in the population is growing. Stroke and TBI result in immediate and long-term cognitive changes. Impairments in the domains of language, attention, memory, executive functions, perception, and social cognition have been documented following stroke and TBI. However, strokes tend to cause focal or selective cognitive disorders, while cognitive deficits following TBI are widespread and can be generalized. Individuals who have suffered a stroke or TBI may also experience psychosocial changes; for example, symptoms of depression and anxiety are common. Functional outcomes, including independence in activities, are varied and are associated with a range of factors including age, injury severity, cognitive disorders, and psychosocial factors. To achieve optimal outcomes for individuals following stroke and TBI, and to reduce the impact of the injury on everyday functioning, a multidisciplinary rehabilitation process is recommended.

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. Full three-dimensional two-photon imaging of GCaMP6s showing individual neuron activity in multiple focal planes was achieved in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling cells in multiple focal planes to be imaged simultaneously. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ∼2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

We report a miniature head mounted two-photon fiber-coupled microscope (TP-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. Full three-dimensional two-photon imaging of GCaMP6s showing individual neuron activity in multiple focal planes was achieved in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is demonstrated. Additionally, the axial scanning of the electrowetting lens allows dynamic control of tilt to the focal plane allowing rapid scanning of different regions of interest in three dimensions. Two-photon imaging allows increased penetration depth in tissue with a field-of-view of 240 μm diameter and 200 μm variable axial focus. The TP-FCM has a light-weight design (~4 g) and excellent image stability. TP-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening up unique opportunities in neuroscience research.

Two-photon microscopy is a powerful tool of current scientific research, allowing optical visualization of structures below the surface of tissues. This is of particular value in neuroscience, where optically accessing regions within the brain is critical for the continued advancement in understanding of neural circuits. However, two-photon imaging at significant depths have typically used Ti:Sapphire based amplifiers that are prohibitively expensive and bulky. In this study, we demonstrate deep tissue two-photon imaging using a compact, inexpensive, turnkey operated Ytterbium fiber laser (Y-Fi, KM Labs). The laser is based on all-normal dispersion (ANDi) that provides short pulse durations and high pulse energies. Depth measurements obtained in ex vivo mouse cortex exceed those obtainable with standard two-photon microscopes using Ti:Sapphire lasers. In addition to demonstrating the capability of deep-tissue imaging in the brain, we investigated imaging depth in highly-scattering white matter with measurements in sciatic nerve showing limited optical penetration of heavily myelinated nerve tissue relative to grey matter.

We have demonstrated an electrowetting prism as a lateral scanning element for a 2-photon excitation microscope. We show imaging of mouse hippocampal neurons, with a field of view of 130 ×130 μm2

Sub-diffraction limited fluorescent images using a fiber-based stimulated emission depletion (STED) microscope are reported. Both excitation and depletion beams are transported through polarization-maintaining fiber and a lateral resolution of 100nm has been achieved.

We present numerical simulations of multielectrode electrowetting devices used in a novel optical design to correct wavefront aberration. Our optical system consists of two multielectrode devices, preceded by a single fixed lens. The multielectrode elements function as adaptive optical devices that can be used to correct aberrations inherent in many imaging setups, biological samples, and the atmosphere. We are able to accurately simulate the liquid-liquid interface shape using computational fluid dynamics. Ray tracing analysis of these surfaces shows clear evidence of aberration correction. To demonstrate the strength of our design, we studied three different input aberrations mixtures that include astigmatism, coma, trefoil, and additional higher order aberration terms, with amplitudes as large as one wave at 633 nm.

We present a miniature head mounted two-photon fiber-coupled microscope (2P-FCM) for neuronal imaging with active axial focusing enabled using a miniature electrowetting lens. We show three-dimensional two-photon imaging of neuronal structure and record neuronal activity from GCaMP6s fluorescence from multiple focal planes in a freely-moving mouse. Two-color simultaneous imaging of GFP and tdTomato fluorescence is also demonstrated. Additionally, dynamic control of the axial scanning of the electrowetting lens allows tilting of the focal plane enabling neurons in multiple depths to be imaged in a single plane. Two-photon imaging allows increased penetration depth in tissue yielding a working distance of 450 μm with an additional 180 μm of active axial focusing. The objective NA is 0.45 with a lateral resolution of 1.8 μm, an axial resolution of 10 μm, and a field-of-view of 240 μm diameter. The 2P-FCM has a weight of only ~2.5 g and is capable of repeatable and stable head-attachment. The 2P-FCM with dynamic axial scanning provides a new capability to record from functionally distinct neuronal layers, opening new opportunities in neuroscience research.

Laser scanners are an integral part of high resolution biomedical imaging systems such as confocal or 2-photon excitation (2PE) microscopes. In this work, we demonstrate the utility of electrowetting on dielectric (EWOD) prisms as a lateral laser-scanning element integrated in a conventional 2PE microscope. To the best of our knowledge, this is the first such demonstration for EWOD prisms. EWOD devices provide a transmissive, low power consuming, and compact alternative to conventional adaptive optics, and hence this technology has tremendous potential. We demonstrate 2PE microscope imaging of cultured mouse hippocampal neurons with a FOV of 130 × 130 μm2 using EWOD prism scanning. In addition, we show simulations of the optical system with the EWOD prism, to evaluate the effect of propagating a Gaussian beam through the EWOD prism on the imaging quality. Based on the simulation results a beam size of 0.91 mm full width half max was chosen to conduct the imaging experiments, resulting in a numerical aperture of 0.17 of the imaging system.

The seismic risk potential for Malawi is high because traditional adobe and earthen structures are seismically vulnerable and large earthquakes of Mw7.0 or greater can be originated from the Malawi Rift. To assess seismic risk of the Malawian communities quantitatively, data and models for exposure, hazard, and vulnerability modules that are suitable for Malawi are integrated. The developed risk model is applied to a retrospective appraisal of the past damaging 2009 Karonga earthquake sequence and to the future earthquake scenarios for long-term risk management purposes. The earthquake impact assessment results highlight that the collapse risk predictions of the Malawian settlements are particularly dependent on the inclusion of large-magnitude earthquakes from the active faults around Lake Malawi and the selection and combination of seismic vulnerability models.

We describe a fiber-coupled miniature microscope with tunable focus using no mechanical parts for two-photon scanning microscopy in thick mouse brain tissue. A frequency-resolved optical grating (FROG) is used to measure the dispersive effects in the fiber-bundle.

Triclosan is an antimicrobial found in personal care and sanitizing products, such as soaps, toothpaste, and hair products. There have been recent concerns for the possible effects on human health, as triclosan has been detected in human breast milk, blood, and urine samples. In a previous study, we found that triclosan alters serum thyroid hormone and testosterone concentrations in male rats. In the current study, we evaluated the effects of triclosan in the female Wistar rat following exposure for 21 days in the Endocrine Disruptor Screening Program pubertal protocol and the weanling uterotrophic assay (3-day exposure). In the pubertal study, triclosan advanced the age of onset of vaginal opening and increased uterine weight at 150 mg/kg, indicative of an estrogenic effect. In the uterotrophic assay, rats received oral doses of triclosan (1.18, 2.35, 4.69, 9.37, 18.75, 37.5, 75, 150, and 300 mg/kg) alone, 3 microg/kg ethinyl estradiol (EE), or triclosan (same doses as above) plus 3 microg/kg EE. Uterine weight was increased in the EE group (positive control) as compared with the control but was not affected by triclosan alone. However, there was a significant dose-dependent increase in the group cotreated with EE and triclosan (>or= 4.69 mg/kg) as compared with EE alone, indicating a potentiation of the estrogen response on uterine weight. This result was well correlated with potentiated estrogen-induced changes in uterine histology. Serum thyroid hormone concentrations were also suppressed by triclosan in this study, similar to other studies in the male and female rat. In conclusion, triclosan affected estrogen-mediated responses in the pubertal and weanling female rat and also suppressed thyroid hormone in both studies. The lowest effective concentrations in the rodent model are approximately 10 (for estrogen) and 40 (for thyroid hormone) times higher than the highest concentrations reported in human plasma.

Summary Two-dimensional Fourier transform spectral interferometry is used to characterize the spatio-temporal aberrations of a UV microscope objective. The spatial and temporal profiles of a 420 nm, 38 fs pulse at the focus of a 0.32 NA UV objective are then deduced using a wave propagation code incorporating the measured aberrations.

We performed stimulated emission depletion (STED) imaging of isolated olfactory sensory neurons (OSNs) using a custom-built microscope. The STED microscope uses a single pulsed laser to excite two separate fluorophores, Atto 590 and Atto 647N. A gated timing circuit combined with temporal interleaving of the different color excitation/STED laser pulses filters the two channel detection and greatly minimizes crosstalk. We quantified the instrument resolution to be ∼81 and ∼44 nm, for the Atto 590 and Atto 647N channels. The spatial separation between the two channels was measured to be under 10 nm, well below the resolution limit. The custom-STED microscope is incorporated onto a commercial research microscope allowing brightfield, differential interference contrast, and epifluorescence imaging on the same field of view. We performed immunolabeling of OSNs in mice to image localization of ciliary membrane proteins involved in olfactory transduction. We imaged Ca2+-permeable cyclic nucleotide gated (CNG) channel (Atto 594) and adenylyl cyclase type III (ACIII) (Atto 647N) in distinct cilia. STED imaging resolved well-separated subdiffraction limited clusters for each protein. We quantified the size of each cluster to have a mean value of 88±48 nm and 124±43 nm, for CNG and ACIII, respectively. STED imaging showed separated clusters that were not resolvable in confocal images.

Coherent beams at soft X-ray (SXR) wavelengths can be generated using extreme nonlinear optics by focusing an intense laser into a gas. In this paper, we discuss phase-matching and quasi-phase-matching techniques that use gas-filled modulated waveguides to enhance the frequency conversion process. This leads to the generation of SXR beams that are both spatially and temporally coherent.

We discuss coherent up-conversion of near-infrared femtosecond laser light into the extreme ultraviolet and soft-X-ray regions of the spectrum using the process of high-harmonic generation. We show that by using concepts from visible wavelength photonics, it is now possible to extend nonlinear optics well into the X-ray region of the spectrum. By manipulating the dynamics of the high harmonic process using optimized waveguide structures and pulse shapes, and on attosecond timescales, we can control the energy of the emitted photons, as well as increase the efficiency of the up-conversion process. Use of these optimized waveguide structures and pulse shapes can also improve the spatial and temporal coherence of the light. This work demonstrates that sophisticated concepts from nonlinear-optical photonics and coherent control can be applied to "extreme" nonlinear optics.

We demonstrate enhanced generation of coherent light in the “water window” region of the soft x-ray spectrum at 4.4 nanometers, using quasi–phase-matched frequency conversion of ultrafast laser pulses. By periodically modulating the diameter of a gas-filled hollow waveguide, the phase mismatch normally present between the laser light and the generated soft x-ray light can be partially compensated. This makes it possible to use neon gas as the nonlinear medium to coherently convert light up to the water window, illustrating that techniques of nonlinear optics can be applied effectively in the soft x-ray region of the spectrum. These results advance the prospects for compact coherent soft x-ray sources for applications in biomicroscopy and in chemical spectroscopy.

We performed super-resolution imaging of isolated olfactory sensory neurons (OSNs) using a custom-built Stimulated Emission Depletion (STED) microscope. The design for the STED microscope is based on the system developed in the laboratory of Dr. Stefan Hell1. Our system is capable of imaging with sub-diffraction limited resolution simultaneously in two color channels (at Atto 590/Atto 647N wavelengths). A single, pulsed laser source (ALP; Fianium, Inc.) generates all four laser beams, two excitation and two STED. The two STED beams are coupled into one polarization maintaining (PM) fiber and the two excitation beams into another. They are then collimated and both STED beams pass through a vortex phase plate (RPC Photonics) to allow shaping into a donut at the focus of the objective lens. The beams are then combined and sent into an inverted research microscope (IX-71; Olympus Inc.) allowing widefield epifluorescence, brightfield and DIC imaging on the same field of view as STED imaging. A fast piezo stage scans the sample during STED and confocal imaging. The fluorescent signals from the two color channels are detected with two avalanche photodiodes (APD) after appropriate spectral filtering. The resolution of the system was characterized by imaging 40 nm fluorescent beads as ~60 nm (Atto 590) and ~50 nm (Atto 647N). We performed STED imaging on immunolabeled isolated OSNs tagged at the CNGA2 and ANO2 proteins. The STED microscope allows us to resolve ciliary CNGA2 microdomains of ~54 nm that were blurred in confocal.

As time passes, fewer and fewer medical students and house officers know anything about William Osler. And of those who do, about half mispronounce his name. Even worse, almost none of today's medical trainees have ever heard of Francis Weld Peabody. These sad facts reflect the shift of modern medical education from the bedside—where Osler and Peabody excelled—to the laboratory and imaging suite, where numbers rule. Our thanks to Ms Gibson for reminding us where our focus should always be: the patient. Submissions for Peabody's Corner should 1) focus on the interpersonal aspects of a specific patient–doctor experience; 2) be written in storybook fashion; 3) contain no references; and 4) not exceed 5 double-spaced typescript pages.

The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the Schlemm’s canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.

We demonstrate a compact, pulsed diode laser source suitable for multiphoton microscopy of biological samples. The center wavelength is 976 nm, near the peak of the two-photon cross section of common fluorescent markers such as genetically encoded green and yellow fluorescent proteins. The laser repetition rate is electrically tunable between 66.67 kHz and 10 MHz, with 2.3 ps pulse duration and peak powers >1 kW. The laser components are fiber-coupled and scalable to a compact package. We demonstrate >600 μm depth penetration in brain tissue, limited by laser power.

We review multiphoton microscopy (MPM) including two-photon autofluorescence (2PAF), second harmonic generation (SHG), third harmonic generation (THG), fluorescence lifetime (FLIM), and coherent anti-Stokes Raman Scattering (CARS) with relevance to clinical applications in ophthalmology. The different imaging modalities are discussed highlighting the particular strength that each has for functional tissue imaging. MPM is compared with current clinical ophthalmological imaging techniques such as reflectance confocal microscopy, optical coherence tomography, and fluorescence imaging. In addition, we discuss the future prospects for MPM in disease detection and clinical monitoring of disease progression, understanding fundamental disease mechanisms, and real-time monitoring of drug delivery.

We describe a method for integrating fiber optics in substrates by femtosecond laser ablation. In a first demonstration, we fabricate an optically integrated microfluidic device that counts cells by small angle light scattering.

Spectral interferometry coupled to numerical wave propagation is used to extract the spatial and temporal characteristics of an ultraviolet objective used for nonlinear imaging and spectroscopy.

Nonlinear optical effects can be successfully exploited to generate a quantitative metric of molecular, organelle, cellular, and indeed organ-level dynamics as well as the very tools essential for these dynamic nonlinear optical studies.

We demonstrate a simple and efficient grism based stretcher/compression system for the first time. 40 fs, 300 muJ pulses are generated at 5 kHz using this unique amplifier design.

We discuss the physical processes involved in the generation and optimization of extreme ultraviolet and soft x-ray light though the process of high-order harmonic generation. We show that by manipulating the sub-optical-cycle attosecond dynamics of this process using optimized waveguide structures and pulse shapes, we can control the energy of the emitted photons, the phase matching of the conversion process, and the spatial and temporal coherence of the light. High-order harmonic generation is a useful source of short wavelength light with ultrashort time duration. Thus, optimization and manipulation of high-order harmonic generation demonstrates control of electron dynamics on attosecond time scales.

We demonstrate a simple and efficient grism based stretcher/compression system. 40 fs, 300 µJ pulses are generated at 5 kHz using this unique amplifier design.

We demonstrate the first femtosecond spectroscopy measurements in a microfluidic device. The microfluidic device is characterized using two-photon fluorescence and computer simulations, and pulse propagation through the high N.A. objective is characterized with spectral interferometry.

Two-photon absorption fluorescence imaging is used to quantitatively measure 3D flow and mixing in microfluidics. This is an important characterization tool for developing optimal microfluidic devices for use in the study of biological molecular dynamics.

High harmonic generation (HHG) is a useful source of coherent light in the extreme ultraviolet (EUV) region of the spectrum. However, both the conversion efficiency and the highest achievable photon energy have in the past been limited in the past by the inability to phase-match the frequency conversion process. In this paper, we summarize recent results on the development of new techniques for phase-matching the high-harmonic conversion process. We also summarize finding from three series of experiments that make use of the coherent EUV light generated using HHG: 1) probing of acoustic dynamics in materials; 2) monitoring of chemical dynamics at surfaces using photoelectron spectroscopy; and 3) time-resolved plasma imaging.

We demonstrate temporal compression of intense femtosecond 800-nm pulses in a hollow-core fiber filled with low-pressure argon gas, without any subsequent dispersion compensation. We achieve a final compressed pulse width of ∼ 13 fs

We demonstrate that high-order harmonic generation can be phase-matched in the “water window” region of the spectrum around 300eV using hollow fibers with a periodically modulated diameter.

We present the first demonstration of a new mechanism for temporal compression of ultrashort light pulses that operates at high (i.e., ionizing) intensities. By propagating pulses inside a hollow waveguide filled with low-pressure argon gas, we demonstrate a self-compression from 30 to 13 fs, without the need for any external dispersion compensation. Theoretical models show that 3D spatiotemporal reshaping of the pulse due to a combination of ionization-induced spectral broadening, plasma-induced refraction, and guiding in the hollow waveguide are necessary to explain the compression mechanism.

We demonstrate that high-order harmonic generation can be phase-matched in the water window region of the spectrum around 300eV using hollow fibers with a periodically modulated diameter.

Summary form only given. High-harmonic generation is a convenient method for generating coherent light in the extreme-ultraviolet (EUV) region of the spectrum, using a small-scale ultrafast laser-based apparatus. Recent advances in using hollow waveguides for HHG have demonstrated conversion efficiency of >10/sup -5/ into a well-collimated EUV beam. In this work, we demonstrate that this beam exhibits full spatial coherence to the limits of our measurement capability, making this source the most coherent light source ever in this region of the spectrum. This contrasts with previous measurements of the coherence of HHG radiation generated in a gas-jet geometry, which measured substantially-degraded spatial coherence. We also demonstrate a new and simple technique for measuring the spectrum of this EUV emission by deconvolution of a double-slit interference pattern for the first time.

We have developed a simple design for an in‐line apparatus to measure the spectral and temporal characteristics of an EUV pulse. The apparatus can be used in front of an experiment without disrupting the beam.

The development of technologies to characterize the ocular aqueous outflow system (AOS) is important for the understanding of the pathophysiology of glaucoma. Multiphoton microscopy (MPM) offers the advantage of high-resolution, label-free imaging with intrinsic image contrast because the emitted signals result from the specific biomolecular content of the tissue. Previous attempts to use MPM to image the murine irido-corneal region directly through the sclera have suffered from degradation in image resolution due to scattering of the focused laser light. As a result, transscleral MPM has limited ability to observe fine structures in the AOS. In this work, the porcine irido- corneal angle was successfully imaged through the transparent cornea using a gonioscopic lens to circumvent the highly scattering scleral tissue. The resulting high-resolution images allowed the detailed structures in the trabecular meshwork (TM) to be observed. Multimodal imaging by two-photon autofluorescence and second harmonic generation allowed visualization of different features in the TM without labels and without disruption of the TM or surrounding tissues. MPM gonioscopy is a promising noninvasive imaging tool for high-resolution studies of the AOS, and research continues to explore the potential for future clinical applications in humans. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full