High-energy self-mode-locked Cr:forsterite laser near the soliton blowup threshold.

At the level of peak powers needed for a Kerr-lens mode-locked operation of solid-state soliton short-pulse lasers, a periodic perturbation induced by spatially localized pulse amplification in a laser cavity can induce soliton instability with respect to resonant dispersive-wave radiation, eventually leading to soliton blowup and pulse splitting of the laser output. Here, we present an experimental study of a high-peak-power self-mode-locking Cr:forsterite laser, showing that, despite its complex, explosion-like buildup dynamics, this soliton blowup can be captured and quantitatively characterized via an accurate cavity-dispersion- and gain-resolved analysis of the laser output. We demonstrate that, with a suitable cavity design and finely tailored balance of gain, dispersion, and nonlinearity, such a laser can be operated in a subcritical mode, right beneath the soliton blowup threshold, providing an efficient source of sub-100-fs 15-20 MHz repetition-rate pulses with energies as high as 33 nJ.

Multimodal label-free imaging of murine hepatocellular carcinoma with a subcellular resolution.

We demonstrate label-free imaging of genetically induced hepatocellular carcinoma (HCC) in a murine model provided by two- and three-photon fluorescence microscopy of endogenous fluorophores excited at the central wavelengths of 790, 980 and 1250 nm and reinforced by second and third harmonic generation microscopy. We show, that autofluorescence imaging presents abundant information about cell arrangement and lipid accumulation in hepatocytes and hepatic stellate cells (HSCs), harmonics generation microscopy provides a versatile tool for fibrogenesis and steatosis study. Multimodal images may be performed by a single ultrafast laser source at 1250 nm falling in tissue transparency window. Various grades of HCC are examined revealing fibrosis, steatosis, liver cell dysplasia, activation of HSCs and hepatocyte necrosis, that shows a great ability of multimodal label-free microscopy to intravital visualization of liver pathology development.

Hyperglycemia exacerbates ischemic stroke not through increased generation of hydrogen peroxide.

Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.

Ultrafast three-dimensional submicrometer-resolution readout of coherent optical-phonon oscillations with shaped unamplified laser pulses at 20 MHz.

An ultrafast three-dimensional readout of coherent optical-phonon oscillations from a diamond film is demonstrated using temporally and spectrally shaped ultrashort laser pulses, delivered by a compact, oscillator-only laser system. This system integrates a long-cavity ytterbium-fiber-laser-pumped 30 fs Cr:forsterite oscillator with a photonic-crystal-fiber soliton frequency shifter and a periodically poled lithium niobate spectrum compressor, providing coherent Raman excitation and time-delayed interrogation of optical phonons in diamond at a 20 MHz repetition rate with a submicrometer spatial resolution.

Broadly wavelength- and pulse width-tunable high-repetition rate light pulses from soliton self-frequency shifting photonic crystal fiber integrated with a frequency doubling crystal.

Soliton self-frequency shift (SSFS) in a photonic crystal fiber (PCF) pumped by a long-cavity mode-locked Cr:forsterite laser is integrated with second harmonic generation (SHG) in a nonlinear crystal to generate ultrashort light pulses tunable within the range of wavelengths from 680 to 1800 nm at a repetition rate of 20 MHz. The pulse width of the second harmonic output is tuned from 70 to 600 fs by varying the thickness of the nonlinear crystal, beam-focusing geometry, and the wavelength of the soliton PCF output. Wavelength-tunable pulses generated through a combination of SSFS and SHG are ideally suited for coherent Raman microspectroscopy at high repetition rates, as verified by experiments on synthetic diamond and polystyrene films.

Polarization instability of ultrashort pulses as a source of vectorial supercontinuum.

Polarization instability (PI) of ultrashort light pulses, giving rise to vectorial supercontinuum generation, is demonstrated using a subwavelength-core, highly birefringent, normally dispersive optical fiber. The evolution of ultrashort pulses in the regime of PI is shown to radically differ from polarization-instability dynamics of cw fields and longer laser pulses. As the peak power of the laser field decreases along the propagation path due to dispersion-induced pulse stretching, the Poincaré-sphere map of field dynamics is shown to evolve from the behavior typical of PI in the highly nonlinear regime toward the beating dynamics of uncoupled polarization modes, characteristic of low field intensities and cw fields.

Raman detection of cell proliferation probes with antiresonance-guiding hollow fibers.

Antiresonance-guiding hollow-core fibers are shown to enable highly sensitive detection of cell proliferation probes using Raman scattering within the region where the cellular Raman activity is minimal. We demonstrate that such fibers can substantially reduce the level of the background compared to standard index-guiding optical fibers, thus radically improving the sensitivity of Raman detection of DNA synthesis in cells and offering a powerful tool for fiber-based live-cell imaging.

Implantable graded-index fibers for neural-dynamics-resolving brain imaging in awake mice on an air-lifted platform.

We demonstrate a versatile framework for cellular brain imaging in awake mice based on suitably tailored segments of graded-index (GRIN) fiber. Closed-form solutions to ray-path equations for graded-index waveguides are shown to offer important insights into image-transmission properties of GRIN fibers, suggesting useful recipes for optimized GRIN-fiber-based deep-brain imaging. We show that the lengths of GRIN imaging components intended for deep-brain studies in freely moving rodents need to be chosen as a tradeoff among the spatial resolution, the targeted imaging depth and the degree of fiber-probe invasiveness. In the experimental setting that we present in this paper, the head of an awake mouse with a GRIN-fiber implant is fixed under a microscope objective, but the mouse is free to move around an in-house-built flat-floored air-lifted platform, exploring a predesigned environment, configured as an arena for one of standard cognitive tests. We show that cellular-resolution deep-brain imaging can be integrated in this setting with robust cell-specific optical neural recording to enable in vivo studies with minimal physical restraints on animal models. The enhancement of the information capacity of the fluorescence signal, achieved via a suitable filtering of the GRIN-fiber readout, is shown to open routes toward practical imaging modalities whereby the deep-brain neuronal dynamics and axonal connections underpinning the integrative functions of essential brain structures can be studied in awake rodent models.

Real-time fiber-optic recording of acute-ischemic-stroke signatures.

We present an experimental framework and methodology for in vivo studies on rat stroke models that enable a real-time fiber-optic recording of stroke-induced hydrogen peroxide and pH transients in ischemia-affected brain areas. Arrays of reconnectable implantable fiber probes combined with advanced optogenetic fluorescent protein sensors are shown to enable a quantitative multisite time-resolved study of oxidative-stress and acidosis buildup dynamics as the key markers, correlates and possible drivers of ischemic stroke. The fiber probes designed for this work provide a wavelength-multiplex forward-propagation channel for a spatially localized, dual-pathway excitation of genetically encoded fluorescence-protein sensors along with a back-propagation channel for the fluorescence return from optically driven fluorescence sensors. We show that the spectral analysis of the fiber-probe-collected fluorescence return provides means for a high-fidelity autofluorescence background subtraction, thus enhancing the sensitivity of real-time detection of stroke-induced transients and significantly reducing measurement uncertainties in in vivo acute-stroke studies as inherently statistical experiments operating with outcomes of multiply repeated measurements on large populations of individually variable animal stroke models.

Slow light on a printed circuit board.

Slow-light effects induced by stimulated Raman scattering in polymer waveguides on a printed circuit board are shown to enable a widely tunable delay of broadband optical signals, suggesting an advantageous platform for optical information processing and ultrafast optical waveform transformation.

Ionization penalty in nonlinear Raman neuroimaging.

Light-assisted ionization accompanying coherent anti-Stokes Raman scattering (CARS) of ultrashort laser pulses in brain tissue is shown to manifest itself in a detectable blueshift of the anti-Stokes signal. This blueshift can serve as an indicator of ionization processes in CARS-based neuroimaging.

Enhanced-contrast two-photon optogenetic pH sensing and pH-resolved brain imaging.

We present experiments on cell cultures and brain slices that demonstrate two-photon optogenetic pH sensing and pH-resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two-photon fluorescence readout from a high-brightness genetically encoded yellow-fluorescent-protein-based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two-photon-excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways-via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high-contrast optogenetic deep-brain pH sensing and pH-resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.

In vivo dynamics of acidosis and oxidative stress in the acute phase of an ischemic stroke in a rodent model.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo.

Two- and three-photon absorption cross-section characterization for high-brightness, cell-specific multiphoton fluorescence brain imaging.

We demonstrate an accurate quantitative characterization of absolute two- and three-photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high-brightness, cell-specific two- and three-photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two-photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep-tissue experiments.

Tailoring the soliton output of a photonic crystal fiber for enhanced two-photon excited luminescence response from fluorescent protein biomarkers and neuron activity reporters.

Dual-cladding photonic crystal fibers (PCFs) with two zero-dispersion points are used to enhance the two-photon excited luminescence (TPL) response from fluorescent protein biomarkers and neuron activity reporters in dye-cell experiments and in in vivo work on transgenic mice and tadpoles. The soliton transmission of ultrashort pulses through a PCF suppresses dispersion-induced temporal pulse spreading, maintaining a high level of field intensity needed for efficient TPL excitation. The soliton self-frequency shift, stabilized against laser power fluctuations by a specific PCF dispersion design, is employed to accurately match the wavelength of the soliton PCF output with the two-photon absorption spectrum of dye or fluorescent protein biomarker molecules, enhancing their TPL response and allowing the laser damage of biotissues to be avoided.

Enhancement of guided-wave two-photon-excited luminescence response with a photonic-crystal fiber.

Photonic-crystal fiber (PCF) is shown to substantially increase the guided-wave luminescent response from molecules excited through two-photon absorption (TPA) by femtosecond near-infrared laser pulses. With only a few nanoliters of TPA-excited molecules filling airholes in a specifically designed PCF, the guided-wave two-photon-excited luminescence (TPL) signal is enhanced by more than 2 orders of magnitude relative to the maximum TPL signal attainable from a cell with the same dye excited and collected by the same PCF. Biophotonic implications of this waveguide TPL-response enhancement include fiber-format solutions for online monitoring of drug delivery and drug activation, interrogation of neural activity, biosensing, endoscopy, and locally controlled singlet oxygen generation in photodynamic therapy.

Thermogenetic neurostimulation with single-cell resolution.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen-vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Highly birefringent silicate glass photonic-crystal fiber with polarization-controlled frequency-shifted output: A promising fiber light source for nonlinear Raman microspectroscopy.

A highly birefringent silicate glass photonic-crystal fiber (PCF) is employed for polarization-controlled nonlinear-optical frequency conversion of femtosecond Cr: forsterite laser pulses with a central wavelength of 1.24 mum to the 530--720-nm wavelength range through soliton dispersion-wave emission. The fiber exhibits a modal birefringence of 1.2.10(-3) at the wavelength of 1.24 mum due to a strong form anisotropy of its core, allowing polarization switching of the central wavelength of its blue-shifted output by 75 nm. Polarization properties and the beam quality of the blue-shifted PCF output are shown to be ideally suited for polarization-sensitive nonlinear Raman microspectroscopy.

Hollow-core photonic-crystal fibres for laser dentistry.

Hollow-core photonic-crystal fibres (PCFs) for the delivery of high-fluence laser radiation capable of ablating tooth enamel are developed. Sequences of picosecond pulses of 1.06 microm Nd:YAG-laser radiation with a total energy of about 2 mJ are transmitted through a hollow-core photonic-crystal fibre with a core diameter of approximately 14 microm and are focused on a tooth surface in vitro to ablate dental tissue. The hollow-core PCF is shown to support the single-fundamental-mode regime for 1.06 microm laser radiation, serving as a spatial filter and allowing the laser beam quality to be substantially improved. The same fibre is used to transmit emission from plasmas produced by laser pulses on the tooth surface in the backward direction for detection and optical diagnostics.

Fiber-optic probes for in vivo depth-resolved neuron-activity mapping.

Specialty fiber probes are used for in vivo depth-resolved mapping of neuron activity through the optical detection of fluorescent-protein reporters expressed inside the living brain of anesthetized transgenic mice. Supercontinuum radiation produced by highly nonlinear photonic-crystal fibers is employed to demonstrate a simultaneous multicolor interrogation of several biomarkers in a model aqueous solution system, thus suggesting the way toward a multiplex mapping of various types of neuron dynamics inside the living brain.