We use transient photoinduced charge-state redistribution of nitrogen-vacancy (NV) centers in diamond as a mechanism for background-robust, all-optical thermometry. Our method requires only a single-wavelength pulsed excitation beam, which induces conversion between the negative (NV−) and neutral (NV0) charge states. At low to moderate excitation intensities, the ensemble evolves from its initial charge-state distribution toward a pump-defined quasi-steady state, with transition rates governed by the ionization and recombination cross sections at the excitation wavelength. NV photoluminescence spectra are recorded in two equal time windows positioned near the beginning and the end of each pump pulse, and the two spectra are then subtracted. The resulting differential spectrum is proportional to the transient change in the NV0/NV− ratio and suppresses background components that do not change on the timescale of a single pulse. Implemented in a fiber-coupled geometry without microwave excitation, magnetic-field control, or auxiliary optical excitation, the method suppresses background-induced temperature bias to <0.1 K even when the background spectral power in the NV zero-phonon line (ZPL) region exceeds the NV0 ZPL signal. The differential spectrum preserves the standard ZPL-based temperature readout and yields a noise-equivalent temperature sensitivity of ≈0.24 K·Hz-0.5. These results show that single-wavelength charge-state modulation can provide an all-optical readout channel for NV ensembles in diamond, usable either as a standalone thermometer or as a background-robust reference measurement for conventional NV-based all-optical thermometry.

Recombinant D-amino acid oxidase (DAAO) is extensively employed for controlled generation of intracellular H2O2. This chemogenetic tool offers unique opportunities for dissecting the contribution of oxidative stress in the occurrence of aging-related degenerative disorders. However, the employment of this tool to induce oxidative stress in the brain in vivo has not yet been tested. Here, we combine viral vector-mediated expression of DAAO and HyPer7, a genetically encoded fluorescent biosensor for H2O2, in hippocampal neurons with real-time in vivo fiber photometry to examine chemogenetic production of H2O2 upon delivery of D-norvaline via intraperitoneal injections or drinking water. Observed changes in the HyPer7 ratiometric signal in response to D-norvaline delivery through both routes confirmed controlled generation of intraneuronal H2O2 in vivo. Raman microspectroscopy revealed a decrease of electron transport chain loading with electrons upon in vivo chemogenetic generation of intraneuronal H2O2. In sum, our findings enable us to suggest that the DAAO-based chemogenetic tool combined with delivery of D-norvaline via either intraperitoneal injections or drinking water is suitable for induction of chronic oxidative stress in the brain in vivo.

Isotropic binary compound semiconductors have been shown to exhibit high optical nonlinearity, which renders them particularly promising for compact optoelectronic and photonic device development through microscale structuring. However, there is a paucity of research tools that provide volumetric imaging of such patterns with high spatial resolution and crystal orientation determination. In this study, the scope of polarization second harmonic generation (SHG) microscopy was expanded to encompass the imaging of single crystalline grains from the deep layers of optically isotropic polycrystalline bulk materials such as chemical vapor deposition zinc selenium (ZnSe). It was demonstrated that focal-plane-localized second harmonic generation can be achieved using a short coherence length and tight focusing, and that grain interfaces provide the majority of the nonlinear signal. The imaging of grain arrangement across a 2 mm-thickness ZnSe sample allowed us to investigate the evolution of spatial resolution with depth provided by a high numerical aperture objective. The developed theoretical approach was used to retrieve the three-dimensional (3D) orientations of individual microcrystallites inside the sample. The present study has expanded the use of the inverse pole figure mapping technique to visualize the crystallographic direction orientations of each grain on the image obtained by polarization SHG microscopy. The demonstrated 3D optical diagnostics method was shown to be a viable investigative tool for isotropic polycrystalline semiconductor materials with a face-centered cubic lattice structure, offering a high spatial resolution and frame rate.

H2O2 is an important signaling molecule and redox regulator of normal cellular metabolism and a major element of oxidative stress. Here we report HyPerFLEX (HyPer with flexible fluorogen excitation), a sensor from the HyPer family designed for high-precision H2O2 monitoring in living cells. HyPerFLEX combines the redox-sensitive OxyR domain from Neisseria meningitidis and circularly permuted fluorogenic protein Y-FAST, yielding oxygen-independent fluorescence upon oxidation of OxyR by H2O2. HyPerFLEX enables imaging H2O2 dynamics in living cells, with tunable spectra from green to far red for multicompartment imaging, even under prolonged hypoxia. It surpasses HyPer7 in detecting ultralow H2O2 concentrations, such as during early glucose-stimulated insulin production, and can measure H2O2 levels in the highly oxidizing endoplasmic reticulum lumen. These advanced features and broad compatibility make HyPerFLEX a powerful tool for studying oxidative stress and cellular signaling.

Thermogenetics is a bioengineering technique that enables control of cellular activity via temperature-sensitive ion channels and externally applied heating. We present a fiber-optic platform for infrared laser thermogenetic stimulation combined with the simultaneous optical readout of fluorescent biosensors and temperature in the brain of freely moving animals. Infrared light delivered through a reconnectable double-clad optical fiber enables spatially localized heating of neural tissue, eliciting reproducible behavioral responses via the human transient receptor potential vanilloid 1 (hTRPV1) channel. The same platform supports multimodal optical measurements, including all-optical thermometry based on nitrogen-vacancy (NV) centers in diamonds and dual-wavelength excitation fluorescence detection. The temperature was retrieved from the zero-phonon line (ZPL) of NV − fluorescence, providing an experimentally demonstrated sensitivity of 16 mK·Hz -0.5 , with a shot-noise-limited performance reaching 1.2 mK·Hz -0.5 . This integrated and scalable approach allows for precise thermal neuromodulation while enabling flexible integration with genetically encoded fluorescent sensors.

A method for measuring the group refractive index and dispersion of dielectrics using a pump-probe scheme demonstrated with a ZnSe crystal. In this approach, an intense, ultrashort pump pulse causes ionization of the crystal, leading to scattering and absorption effects on the subsequent broadband probe pulse, which experiences dispersive broadening as it propagates through the crystal. By adjusting the time delay between the pump and probe pulses, we can effectively control the spectrum transmitted through the crystal, enabling the retrieval of both the group refractive index and dispersion characteristics.

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.

Hydrogen peroxide (H2O2) has been identified recently as a meaningful signaling molecule. The ability to perform imaging of H2O2 in complex biological organisms with a high spatio-temporal resolution requires advancements in both biosensors and in vivo visualization methods. In this work, we investigated the potential of fluorescent protein HyPer7, a bright and sensitive indicator of hydrogen peroxide, for multiphoton visualization in cell cultures, acute brain slices and neocortical neurons of anesthetized mice. Employing an alternating interrogation of the reduced and oxidized forms of HyPer7 enables the recording of H2O2 dynamics at a rate of one frame per second. Taking advantage of two-photon ratiometric readout of HyPer7, we monitored the intracellular H2O2 concentration growth in hippocampal neurons under chemogenetics tools handling in acute brain slices. At the final step, we visualized the HyPer7-expressed neurons at depths up to 600µm below the brain surface of the mouse under anesthesia by two- and three-photon excitation microscopy. Autofluorescence, light scattering and out-of-focus background signal reduce the depth limit for HyPer7-imaging by two-photon microscopy, however, these challenges may be overcome by means of three-photon excitation fluorescence by the pulses of the short-wavelength infrared region.

The subject of study is the spectral and nonlinear optical properties of microcrystalline DAST dye powder and the spectral properties of DAST dye solutions. The aim of the study is the investigation of the nonlinear optical properties of microcrystalline DAST dye powder as a promising object for creating new materials and the analysis of the spectral behavior of the DAST dye in solutions of various natures to establish structure-property relationships. Method. The electronic spectra were recorded using spectrophotometry and spectrofluorimetry. The size of the crystal fragment was determined by confocal microscopy. The generation of the second harmonic and excitation of fluorescence in crystalline DAST powder were performed using femtosecond radiation with a wavelength of 1250 nm in an experimental setup. Main results. The spectral properties of the DAST dye in solvents and microcrystalline powder were investigated. The results of second harmonic generation and cascade excitation of luminescence in the DAST powder are presented. The luminescence spectrum of the crystalline DAST with an isolated band at a wavelength of 725 nm was recorded. It is shown that the organic crystals of DAST powder effectively generate the second harmonic under the focusing conditions and parameters of femtosecond radiation at a wavelength of 1250 nm, which are used in microscopy of biological objects and are promising for creating nanosensors and optical elements for converting femtosecond laser radiation with a wavelength of 1250 nm. Practical significance. The DAST dye can be used as a nanomarker in multiphoton microscopy, solution viscosity sensors, as well as optical elements in laser technology and spectroscopy. (c) 2024 Optica Publishing Group

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.

Cardiac arrhythmias are common disorders that can be fatal. Modern methods of treating bradyarrhythmias include the implantation of pacemakers and cardioverters – defibrillators. However, the implantable devices can cause various complications including infectious ones, related to the electrodes installed inside the heart. Less invasive heart rhythm modulation could be beneficial for some cohorts of patients. We present an alternative approach to heart pacing based on thermogenetics. We used adeno-associated viruses to deliver genetic human transient receptor potential subfamily V member 1 (TRPV1), a heat-sensitive cation channel, into isolated cardiomyocytes and the mouse heart. This allowed us to induce action potentials and control contractility using short heat pulses delivered by infrared laser illumination. Using this approach, we demonstrated the thermogenetic pacing of isolated cardiomyocytes in vitro and in the mouse heart in vivo . Our results demonstrate the unique potential of thermogenetics for developing novel therapeutic strategies for heart rhythm modulation.### Competing Interest StatementShemyakin-Ovchinnikov Institute of Bioorganic Chemistry owns patents issued for the distant heating system (RU2802995 on which V.V.B., A.M.N., I.V.K., A.V.B., A.M.Z., and A.A.L. are co-inventors) and for genetic constructions, their method of delivery, and heating of cardiomyocytes by an infrared laser (RU2793182 on which V.V.B., A.M.N., A.V.B., A.A.L., A.B.F., and A.A.Mozh are co-inventors).

Using fluorescence spectroscopy, the two-photon absorption cross sections of aqueous solutions of the styryl dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DASPI) and its inclusion complexes with cucurbit[n]urils (CB[n] n = 6–8) have been measured. A nonmonotonic dependence of the cross section on the excitation wavelength and on the cavitand cavity size has been revealed. Compared to the free dye, a sevenfold increase in the two-photon absorption cross section has been observed in DASPI inclusion complexes with CB[8] at an excitation wavelength of 980 nm.

The generation of spectral components sensitive to the carrier-envelope phase of a laser pulse in a thin zinc selenide film has been experimentally demonstrated and confirmed by a numerical simulation. A pump–probe scheme has been implemented so that a pump pulse with a duration of about 1.5 field cycles, a central wavelength of 1.7 μm, and a stabilized carrier-envelope phase induces photoionization in a thin zinc selenide film. The probe pulse is scattered by the plasma, generating new phase-sensitive spectral components at the edges of its spectrum. The theoretical analysis has confirmed plasma nonlinearity as a mechanism for generating these components. The observed effect can be used to characterize the carrier-envelope phase of ultrashort pulses during the generation of high-order harmonics and sequences of attosecond pulses.

The paper presents a technique for broadband two-dimensional infrared spectroscopy with signal detection in visible range by nonlinear chirped-pulse upconversion. This approach helps to avoid direct measurement of the mid-infrared signal that requires cryogenic technology, and instead uses low-cost high-sensitivity multichannel silicon linear arrays. This leads to a reduction by two orders of magnitude of the measurement time of a single two-dimensional spectrum, which makes it possible to observe the fast dynamics of complex molecular compounds. The use of a quasi-phase-cycling achieved by sub-cycle delay modulation suppresses scattering background by almost two orders of magnitude and increases the measurement speed twice compared to optical chopping. Numerical simulation using the density matrix formalism and analysis of its evolution based on the solution of the Bloch–Redfield equation effectively reproduces the features of the two-dimensional infrared spectrum of inorganic octacarbonyl dicobalt compound.

We present a compact laser system for quantitative two-photon excitation spectra measurements and ratiometric two-photon imaging of fluorescent protein indicators. The fundamental of the system is a short segment of photonic crystal fiber (PCF), which supports a nonlinear transformation of low-power ultrashort pulses by preserving temporal coherence, and this generates an ultrafast almost octave-spanning supercontinuum (SC). Accurate sculpting of the SC by its amplitude and phase modulation provides implementation of the spectroscopic and microscopic modalities. The spectroscopic one was exhibited by two-photon action cross section spectra measuring for the genetically encoded fluorescent sensing proteins of the vital biochemical parameters: acidity (SypHer3s), concentration of hydrogen peroxide (HyPer3 and HyPer7), redox status of NADH and glutathione (RexYFP and Grx1-roGFP2), hypohalous acids and their derivatives (Hypocrates). For the microscopy, we investigated and optimized the intensity pump pulse profiles under the high numerical objective by dispersion scan technique. We conducted real-time monitoring of the dynamics of hydrogen peroxide in HeLa cells with subcellular spatial resolution by means of ratiometric two-photon imaging of Hyper7 sensors. The presented hybrid laser system provides an ideal optical toolbox in order to develop ratiometric fluorescent sensors, which can be visualized in vivo using two-photon microscopy.

ABSTRACT Virtually all major processes in cells and tissues are regulated by calcium ions (Ca 2+ ). Understanding the influence of Ca 2+ on cell function requires technologies that allow for non-invasive manipulation of intracellular calcium levels including the formation of calcium patterns, ideally in a way that is expandable to intact organisms. The currently existing tools for optical and optogenetic Ca 2+ manipulation are limited with respect to response time, and tissue penetration depth. Here we present G enetically E ncoded C alcium Co ntroller ( GECCO ), a system for thermogenetic Ca 2+ manipulation based on snake TRP channels optically controlled by infrared illumination. GECCO is functional in animal and plant cells and allows studying how cells decode different profiles of Ca 2+ signals. GECCO enabled the shaping of insulin release from β-cells, the identification of drugs that potentiate Ca 2+ -induced insulin release, and the generation of synthetic Ca 2+ signatures in plants.

В работе проведен анализ модового состава широкополосного излучения со спектром, простирающимся в диапазоне от 200 до 2500 нм, полученного в процессе солитонной самокомпрессии лазерного импульса в полом антирезонансном волноводе. Продемонстрировано, что наиболее энергоемкая инфракрасная часть излучения от 1300 до 2500 нм, формирующая предельно короткий импульс, локализована в основной пространственной моде LP01 . Выполнение условий фазового согласования в видимой части суперконтинуума приводит к генерации третьей гармоники вблизи 620 нм в высших волноводных модах и модах капилляров. Было показано, что локализованное в сердцевине световода излучениетретьей гармоники на длине волны ~ 700 нм позволяет измерять фазу поля относительно огибающейпредельно коротких импульсов на выходе из волновода.

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.