Search

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.

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.

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.

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 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.

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.

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 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.

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 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.

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 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.

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 demonstrate the generation of very high-order harmonics, up to 250 eV, using argon gas. This extends by 100 eV the highest harmonics previously observed using Ar and exceeds the energies observed using any other medium besides helium. This advance is made possible by using a waveguide geometry to limit plasma-induced laser beam defocusing, making it possible to generate high harmonics from Ar ions. This work shows that high harmonic emission from ions can extend laser-based coherent up-conversion into the soft x-ray region of the spectrum.

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.

A simple fabrication technique to create all silicon/glass microfluidic devices is demonstrated using femtosecond laser ablation and anodic bonding. In a first application, we constructed a cell counting device based on small angle light scattering. The counter featured embedded optical fibers for multiangle excitation and detection of scattered light and/or fluorescence. The performance of the microfluidic cell counter was benchmarked against a commercial fluorescence-activated cell sorter.

Efficient reflection grisms for pulse-compression and material-dispersion compensation have been designed and demonstrated in a 40 fs, 300 microJ, 5 kHz downchirped pulse amplification system for the first time to our knowledge. A grism design for 800 nm femtosecond laser pulse dispersion compensation applications is realized by using standard, commercial diffraction gratings.

Conical emission, redshifted from the atomic resonance, is observed when a ∼2-ps laser beam, blueshifted from resonance, propagates through potassium vapor. In contrast, no cone is visible when a 150-fs pulse is used. The 2-ps-pulse-induced cone has a unique, small, angular spread of Δθ/θ≈0.1, compared with Δθ/θ≈0.3 from other pulsed experiments, which used 25-ps–5-ns-excitation pulses. In addition, the fraction of the 2-ps-pulse power emitted in the cone reached 7.5%, far exceeding this fraction from the longer-pulse experiments. An additional, unexpected result is that the spectral width of the laser pulse is narrower, without significant intensity loss, after propagation through the vapor. The vapor densities and laser detunings that yield cones in the present experiment are similar to those from the longer-pulse experiments. Although the 2-ps-pulse spectrum partially overlaps the resonance, the cone spectrum and cone angles versus density and laser detuning are similar to those previously reported for nanosecond pulses. The fourth (blue-detuned) wave required for satisfying four-wave mixing is absent, again equivalent to what is observed in the nanosecond experiments. The physical origin of cone emission by pulsed excitation of atomic vapors is still largely unknown, and this high efficiency and narrow angular spread of a spectrally broad cone further contradicts existing models, perhaps ultimately providing a clue to a correct explanation.

We demonstrate that high-harmonic generation can be phase-matched in the "water window" region of the spectrum using periodically-modulated hollow fibers. We also observe emission from ions.