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.

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.

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.

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.

We demonstrate a versatile single-laser platform for single-beam dual-color two-photon spectroscopy that combines a short-pulse laser source with a tunable broadband wavelength converter based on a highly nonlinear photonic-crystal fiber (PCF). We show that the short-pulse PCF output can be tailored, via dispersion and nonlinearity management, to deliver a broadband optical driver whose spectral structure is ideally suited for a single-beam two-photon absorption (2PA) spectroscopy of the next-generation genetically encodable fluorescent-protein (FP) sensors of pH and redox-responsive contrast agents. As a promising spectroscopic resource for redox biology, the short-pulse PCF output can be finely sculpted to alternately drive an FP system via a one of its two 2PA-allowed quantum pathways, yielding a high-contrast fluorescence readout for a highly sensitive detection of redox reactions and signaling, pH sensing, and oxidative-stress diagnosis.

We demonstrate single-beam optogenetic multimodal nonlinear-optical microscopy that combines third-harmonic generation (THG) and three-photon-excited fluorescence (3PEF) - two nonlinear-optical processes related to the third- and fifth-order susceptibilities, chi((3)) and chi((5)). A carefully tailored unamplified short-pulse output of mode-locked solid-state lasers is shown to provide an ample parameter space for the optimization of such a single-beam multimodal microscopy, enabling subcellular-resolution, cellspecific imaging using genetically encoded fluorescent-protein-based reporters in a vast variety of biological systems and settings, ranging from HeLa to brain cells. Experiments on brain slices and cell cultures presented in this paper demonstrate the potential of short-pulse 3PEF/THG microscopy for a subcellular-resolution, high-contrast imaging of HeLa-line cancer-cell derivatives, as well as mitochondrial and somatic intracellular structures within deep-brain neurons and astrocytes. As a step beyond the state of the art in optical brain imaging, single-beam subcellular-resolution, cell-specific optogenetic 3PEF/THG imaging of fundamental functional brain units is experimentally demonstrated. One fundamental question that this work brings up for the future nonlinear absorption and Raman/hyper-Raman studies is whether the Herzberg-Teller corrections, needed for an accurate description of two-photon absorption in fluorescent proteins (FPs), would be also sufficient for an adequate treatment of higher-n n-photon absorption in FP-based systems or models that include other quantum pathways would be necessary for the analysis of FP agents for higher-n nonlinear microscopy.

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.

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.

Nonlinear microscopy techniques are widely used in the field of biophotonics thanks to their ability to image in highly scattering tissues such as brain [1]. Techniques that use intrinsic sources of contrast rather than exogenous labels are of great interest, because they are suitable not only for fundamental biological research but also for clinical applications [2]. Neurons are the most studied cells in the central nervous system of mammals. However, in certain parts of brain glial cells called astrocytes outnumber neurons. Historically, astrocytes considered to be simple support cells that structure the brain and control blood brain barrier, but recently were discovered to have great functional diversity and play many roles in healthy tissues as well as in CNS disorders [3].

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.

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 mu m at focusing depths up to 20 mu m inside a brain.

Within the past decade, nonlinear Raman microscopy has earned a well-deserved status of a gold-standard technology for chemically selective imaging. Even though second- and third-harmonic microscopy is much less demanding on a laser source and multifrequency beam arrangement, it is increasingly falling behind nonlinear Raman scattering as a method of bioimaging because it offers no mechanism whereby imaging could be made chemically specific. Here, we show, however, that such a mechanism does exist, helping harmonic-generation microscopy overcome its no-chemical-specificity handicap. We demonstrate that, with the laser wavelength tuned to a three-photon resonance with the Soret band of hemoglobin, third-harmonic generation provides a chemically specific method for a high-contrast imaging of red blood cells in a broad class of biological systems, including live brain. Moreover, third-harmonic generation imaging can be conveniently combined with second-harmonic microscopy on a compact laser platform, providing, as our experiments on rat brain show, a powerful resource for three-dimensional, cell-specific label-free deep-brain imaging.

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.