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Thermogenetics is a promising neuromodulation technique based on the use of heat-sensitive ion channels. However, on the way to its clinical application, a number of questions have to be addressed. First, to avoid immune response in future human applications, human ion channels should be studied as thermogenetic actuators. Second, heating levels necessary to activate these channels in vivo in brain tissue should be studied and cytotoxicity of these temperatures addressed. Third, the possibility and safety of chronic neuromodulation has to be demonstrated. In this study, we present a comprehensive framework for thermogenetic neuromodulation in vivo using the thermosensitive human ion channel hTRPV1. By targeting hTRPV1 expression to excitatory neurons of the mouse brain and activating them within a non-harmful temperature range with a fiber-coupled infrared laser, we not only induced neuronal firing and stimulated locomotion in mice, but also demonstrated that thermogenetics can be employed for repeated neuromodulation without causing evident brain tissue injury. Our results lay the foundation for the use of thermogenetic neuromodulation in brain research and therapy of neuropathologies., (© 2024. The Author(s).)

The study of human neural cells, their behaviour and migration are important areas of research in the biomedical field, particularly for potential therapeutic applications. The safety of using neural cells in therapy is still a concern due to a lack of information on long-term changes that may occur. While current methods of cell tracing explore gene manipulations, we elaborate approaches to cell marking with no genetic interference. In this study, we present a novel method for labelling and tracking neural cells using cell-impregnatable photoconvertible polyelectrolyte microcapsules. These capsules demonstrated low cytotoxicity with no effect on the differentiation ability of the neural cells, maintained a high level of fluorescent signal and ability for tracing individual neural cells for over 7 days. The capsules modified with rhodamine- and fluorescein-based dyes were demonstrated to undergo photoconversion by both one- and two-photon lasers while being internalized by neural cells. The finding gives the possibility to select individual capsules inside multicellular structures like spheroids and tissues and alternate their fluorescent appearance. Thus, we can track individual cell paths in complex systems. This new method offers a promising alternative for studying neural cells' long-term behaviour and migration in complex systems such as three-dimensional cellular populations.

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., (© 2023 Wiley-VCH GmbH.)

The influence of the carrier-envelope phase (CEP) of a pump pulse on the multioctave supercontinuum (SC) generation in a gas-filled anti-resonant hollow-core fiber (AR HCF) by soliton self-compression (SSC) has been explored. We have shown an octave-wide third harmonic generation (THG) in the visible-to-near-infrared range during the pulse compression down to a sub-cycle duration. The CEP of a multi-cycle pump pulse provides control of interference between the third harmonic (TH) and the SC that indicates the coherent synthesis of a sub-cycle pulse with a duration of about 0.4 optical cycles and a peak power of more than 2 GW at the fiber output.

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., (© 2022 Wiley-VCH GmbH.)

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

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., (© 2020 Wiley-VCH GmbH.)

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.

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Methods of nonlinear optics provide a vast arsenal of tools for label-free brain imaging, offering a unique combination of chemical specificity, the ability to detect fine morphological features, and an unprecedentedly high, subdiffraction spatial resolution. While these techniques provide a rapidly growing platform for the microscopy of neurons and fine intraneural structures, optical imaging of astroglia still largely relies on filament-protein-antibody staining, subject to limitations and difficulties especially severe in live-brain studies. Once viewed as an ancillary, inert brain scaffold, astroglia are being promoted, as a part of an ongoing paradigm shift in neurosciences, into the role of a key active agent of intercellular communication and information processing, playing a significant role in brain functioning under normal and pathological conditions. Here, we show that methods of nonlinear optics provide a unique resource to address long-standing challenges in label-free astroglia imaging. We demonstrate that, with a suitable beam-focusing geometry and careful driver-pulse compression, microscopy of second-harmonic generation (SHG) can enable a high-resolution label-free imaging of fibrillar structures of astrocytes, most notably astrocyte processes and their endfeet. SHG microscopy of astrocytes is integrated in our approach with nonlinear-optical imaging of red blood cells based on third-harmonic generation (THG) enhanced by a three-photon resonance with the Soret band of hemoglobin. With astroglia and red blood cells providing two physically distinct imaging contrasts in SHG and THG channels, a parallel detection of the second and third harmonics enables a high-contrast, high-resolution, stain-free stereoimaging of gliovascular interfaces in the central nervous system. Transverse scans of the second and third harmonics are shown to resolve an ultrafine texture of blood-vessel walls and astrocyte-process endfeet on gliovascular interfaces with a spatial resolution within 1 μm at focusing depths up to 20 μm inside a brain., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.

We present one- and two-photon-absorption fluorescence spectroscopic analysis of biliverdin (BV) chromophore-based single-domain near-infrared fluorescent proteins (iRFPs). The results of these studies are used to estimate the internal electric fields acting on BV inside iRFPs and quantify the electric dipole properties of this chromophore, defining the red shift of excitation and emission spectra of BV-based iRFPs. The iRFP studied in this work is shown to fit well the global diagram of the red-shift tunability of currently available BV-based iRFPs as dictated by the quadratic Stark effect, suggesting the existence of the lower bound for the strongest red shifts attainable within this family of fluorescent proteins. The absolute value of the two-photon absorption (TPA) cross section of a fluorescent calcium sensor based on the studied iRFP is found to be significantly larger than the TPA cross sections of other widely used genetically encodable fluorescent calcium sensors., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polarization maps of high-order harmonics are shown to enable a full vectorial characterization of petahertz electron currents generated in a crystalline solid by an ultrashort laser driver. As a powerful resource of this methodology, analysis of energy-momentum dispersion landscapes, defined by the electron band structure, can help identify, as our analysis shows, special directions within the Brillouin zone that can provide a preferable basis for polarization-sensitive high-harmonic mapping of anisotropic petahertz photocurrents in solids.

Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of single neurons using thermosensitive cation channels and IR stimulation. The main advantage of IR stimulation compared to conventional visible light optogenetics is the depth of penetration (up to millimeters). Due to physiological limitations, thermogenetic molecular tools for mammalian brain stimulation remain poorly developed. Here, we tested the possibility of employment of this new technique for stimulation of neocortical neurons. The method is based on activation gating of TRPV1-L channels selectively expressed in specific cells. Pyramidal neurons of layer 2/3 of neocortex were transfected at an embryonic stage using a pCAG expression vector and electroporation in utero. Depolarization and spiking responses of TRPV1L+ pyramidal neurons to IR radiation were recorded electrophysiologically in acute brain slices of adult animals with help of confocal visualization. As TRPV1L-expressing neurons are not sensitive to visible light, there were no limitations of the use of this technique with conventional fluorescence imaging. Our experiments demonstrated that the TRPV1-L+ pyramidal neurons preserve their electrical excitability in acute brain slices, while IR radiation can be successfully used to induce single neuronal depolarization and spiking at near physiological temperatures. Obtained results provide important information for adaptation of thermogenetic technology to mammalian brain studies in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Quantitative characterization of dispersion near molecular resonances is difficult both conceptually and technically, as it requires systematic measurements of rapidly varying dispersion profiles across the edges of molecular absorption bands. Here, we show that this challenge can be confronted with an ultrafast spectrochronography technique that combines time-resolved four-wave mixing and cross-correlation frequency-resolved optical gating. We demonstrate that, with the spectrum of an ultrashort mid-infrared laser probe stretching across the edge of a molecular absorption band, the Wigner chronocyclic maps of such a probe reveal universal signatures of anomalous dispersion and help quantify such dispersion anomalies.

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 Ca 2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.

Optically detected electron spin resonance in fiber-coupled nitrogen-vacancy (NV) centers of diamond is used to demonstrate a fiber-optic quantum thermometry of individual thermogenetically activated neurons. Laser-induced temperature variations read out from single neurons with the NV-diamond fiber sensor are shown to strongly correlate with the fluorescence of calcium-ion sensors, serving as online indicators of the inward Ca 2+ current across the cell membrane of neurons expressing transient receptor potential (TRP) cation channels. Local laser heating above the TRP-channel activation threshold is shown to reproducibly evoke robust action potentials, visualized by calcium-ion-sensor-aided fluorescence imaging and detected as prominent characteristic waveforms in the time-resolved response of fluorescence Ca 2+ sensors.

Extended coupled-wave analysis of optical parametric chirped-pulse amplification (OPCPA) reveals regimes whereby high-peak-power few-cycle pulses can be generated in the long-wavelength infrared (LWIR) spectral range. Broadband OPCPA in suitable nonlinear crystals pumped at around 2 μm and seeded either through the signal or the idler input is shown to enable the generation of high-power field waveforms with pulse widths shorter than two field cycles within the entire LWIR range.

We demonstrate a robust, all-solid-state approach for the generation of microjoule subcycle pulses in the midinfrared through a cascade of carefully optimized parametric-amplification, difference-frequency-generation, spectral-broadening, and chirp-compensation stages. This method of subcycle waveform generation becomes possible due to an unusual, ionization-assisted solid-state pulse self-compression dynamics, where highly efficient spectral broadening is enabled by ultrabroadband four-wave parametric amplification phase matched near the zero-group-velocity wavelength of the material.

We perform a proof-of-principle demonstration of chemically specific standoff gas sensing, in which a coherent stimulated Raman signal is detected in the direction anticollinear to a two-color laser excitation beam traversing the target volume. The proposed geometry is intrinsically free space as it does not involve back-scattering (reflection) of the signal or excitation beams at or behind the target. A beam carrying an intense mid-IR femtosecond (fs) pulse and a parametrically generated picosecond (ps) UV Stokes pulse is fired in the forward direction. A fs filament, produced by the intense mid-IR pulse, emits a backward-propagating narrowband ps laser pulse at the 337 and 357 nm transitions of excited molecular nitrogen, thus supplying a counter-propagating Raman pump pulse. The scheme is linearly sensitive to species concentration and provides both transverse and longitudinal spatial resolution.

Strongly coupled nonlinear spatiotemporal dynamics of ultrashort mid-infrared pulses undergoing self-focusing simultaneously with soliton self-compression in an anomalously dispersive, highly nonlinear solid semiconductor is shown to enable the generation of multioctave supercontinua with spectra spanning the entire mid-infrared range and compressible to subcycle pulse widths. With 7.9 μm, 150 fs, 2 μJ, 1 kHz pulses used as a driver, 1.2 cycle pulses of mid-infrared supercontinuum radiation with a spectrum spanning the range of wavelengths from 3 to 18 μm were generated in a 5 mm GaAs plate. Further compression of these pulses to subcycle pulse widths is possible through compensation of the residual phase shift.

A physical scenario whereby freely propagating mid-infrared pulses can be compressed to pulse widths close to the field cycle is identified. Generation of tunable few-cycle pulses in the wavelength range from 4.2 to 6.8 μm is demonstrated at a 1-kHz repetition rate through self-focusing-assisted spectral broadening in a normally dispersive, highly nonlinear semiconductor material, followed by pulse compression in the regime of anomalous dispersion, where the dispersion-induced phase shift is finely tuned by adjusting the overall thickness of anomalously dispersive components. Sub-two-cycle pulses with a peak power up to 60 MW are generated in the range of central wavelengths tunable from 5.9 to 6.3 μm.

When coupled to characteristic, fingerprint vibrational and rotational motions of molecules, an electromagnetic field with an appropriate frequency and waveform offers a highly sensitive, highly informative probe, enabling chemically specific studies on a broad class of systems in physics, chemistry, biology, geosciences, and medicine. The frequencies of these signature molecular modes, however, lie in a region where accurate spectroscopic measurements are extremely difficult because of the lack of efficient detectors and spectrometers. Here, we show that, with a combination of advanced ultrafast technologies and nonlinear-optical waveform characterization, time-domain techniques can be advantageously extended to the metrology of fundamental molecular motions in the mid-infrared. In our scheme, the spectral modulation of ultrashort mid-infrared pulses, induced by rovibrational motions of molecules, gives rise to interfering coherent dark waveforms in the time domain. These high-visibility interference patterns can be read out by cross-correlation frequency-resolved gating of the field in the visible generated through ultrabroadband four-wave mixing in a gas phase.

A mode-locked chromium forsterite laser with output power in excess of 0.7 W, a central wavelength of 1.25 μm, a pulse repetition rate of 29 MHz, and an output pulse-width-tunable from 40 to 200 fs is demonstrated. The dynamics behind the buildup of ultrashort light pulses in this laser is shown to involve spectral and temporal breathing due to the interplay of gain, Kerr nonlinearity, and dispersion effects. The pulse-width-tunable 1.25 μm output delivered by the developed laser source suggests a powerful tool for nonlinear-optical bio-imaging and offers an advantageous front end for extreme-power laser technologies.

Unlike spontaneous Raman effect, nonlinear Raman scattering generates fields with a well-defined phase, allowing Raman signals from individual scatterers to add up into a highly directional, high-brightness coherent beam. Here, we show that the phase of coherent Raman scattering can be accurately controlled and finely tuned by using spectrally and temporally tailored optical driver fields. In our experiments, performed with spectrally optimized phase-tunable laser pulses, such a phase control is visualized through the interference of the coherent Raman signal with the field resulting from nonresonant four-wave mixing. This interference gives rise to Fano-type profiles in the overall nonlinear response measured as a function of the delay time between the laser pulses, featuring a well-resolved destructive-interference dip on the dark side of the Raman peak. This phase-control strategy is shown to radically enhance the coherent response from weak Raman modes, thus helping confront long-standing challenges in nonlinear Raman imaging and microspectroscopy.

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.

By combining tunable broadband pulse generation with the technique of nonlinear spectral compression we demonstrate a prototype scheme for highly selective detection of air molecules by backward stimulated Raman scattering. The experimental results allow to extrapolate the laser parameters required for standoff sensing based on the recently demonstrated backward atmospheric lasing.

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.

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., (© 2011 Optical Society of America)

The ultrafast Raman response of C-H vibrations in polymer waveguides on a printed circuit is shown to enable a high-gain amplification and high-order stimulated Raman transformation of ultrashort laser pulses. The pump and Stokes fields coupled by the C-H vibration mode with a vibration cycle of 11 fs give rise to multiple Raman sidebands, suggesting the way toward ultrafast optical data processing and few-cycle optical waveform synthesis on a printed-circuit polymer waveguide platform.

Coherent anti-Stokes Raman scattering (CARS) is used to measure the amplitude, the dephasing lifetime, and parameters of optical nonlinearities of optical phonons in a synthetic diamond film. A compact CARS apparatus demonstrated in this work relies on the use of an unamplified 70 fs 340 mW Cr:forsterite laser output and photonic-crystal fibers optimized for the generation of wavelength-tunable Stokes field and the spectral compression of the probe pulse.