Background-free two-photon fluorescence readout via a three-photon charge-state modulation of nitrogen-vacancy centers in diamond.

We demonstrate that a background-free readout of two-photon fluorescence from nitrogen-vacancy (NV) centers in a strongly fluorescing environment can be accomplished by all-optical means via a multiphoton charge-state modulation of NV centers in a mixture of negatively charged and neutral NV centers. A 100 fs, 1060 nm output of an ytterbium fiber laser is ideally suited for this modality of multiphoton microscopy, providing, as our experiments show, an efficient two-photon excitation of both NV- and NV0 charge states, but keeping the nonlinearity of n-photon ionization needed for NV-/NV0 charge-state modulation to a minimum, n=3.

[Selenium compounds in redox regulation of inflammation and apoptosis]. / Soedineniia selena v redoks-reguliatsii vospaleniia i apoptoza.

Monocytes and macrophages play a key role in the development of inflammation: under the action of lipopolysaccharides (LPS), absorbed from the intestine, monocytes and macrophages form reactive oxygen species (ROS) and cytokines, this leads to the development of oxidative stress, inflammation and/or apoptosis in all types of tissues. In the cells LPS induce an "internal" TLR4-mediated MAP-kinase inflammatory signaling pathway and cytokines through the superfamily of tumor necrosis factor receptor (TNFR) and the "death domain" (DD) initiate an "external" caspase apoptosis cascade or necrosis activation that causes necroptosis. Many of the proteins involved in intracellular signaling cascades (MYD88, ASK1, IKKa/b, NF-kB, AP-1) are redox-sensitive and their activity is regulated by antioxidants thioredoxin, glutaredoxin, nitroredoxin, and glutathione. Oxidation of these signaling proteins induced by ROS enhances the development of inflammation and apoptosis, and their reduction with antioxidants, on the contrary, stabilizes the signaling cascades speed, preventing the vicious circle of oxidative stress, inflammation and apoptosis that follows it. Antioxidant (AO) enzymes thioredoxin reductase (TRXR), glutaredoxin reductase (GLRXR), glutathione reductase (GR) are required for reduction of non-enzymatic antioxidants (thioredoxin, glutaredoxin, nitroredoxin, glutathione), and AO enzymes (SOD, catalase, GPX) are required for ROS deactivation. The key AO enzymes (TRXR and GPX) are selenium-dependent; therefore selenium deficiency leads to a decrease in the body's antioxidant defense, the development of oxidative stress, inflammation, and/or apoptosis in various cell types. Nrf2-Keap1 signaling pathway activated by selenium deficiency and/or oxidative stress is necessary to restore redox homeostasis in the cell. In addition, expression of some genes is changed with selenium deficiency. Consequently, growth and proliferation of cells, their movement, development, death, and survival, as well as the interaction between cells, the redox regulation of intracellular signaling cascades of inflammation and apoptosis, depend on the selenium status of the body. Prophylactic administration of selenium-containing preparations (natural and synthetic (organic and inorganic)) is able to normalize the activity of AO enzymes and the general status of the body. Organic selenium compounds have a high bioavailability and, depending on their concentration, can act both as selenium donors to prevent selenium deficiency and as antitumor drugs due to their toxicity and participation in the regulation of signaling pathways of apoptosis. Known selenorganic compounds diphenyldiselenide and ethaselen share similarity with the Russian organo selenium compound, diacetophenonylselenide (DAPS-25), which serves as a source of bioavailable selenium, exhibits a wide range of biological activity, including antioxidant activity, that governs cell redox balance, inflammation and apoptosis regulation.

Reconnectable fiberscopes for chronic in vivo deep-brain imaging.

Reconnectable bundles consisting of thousands of optical fibers are shown to enable high-quality image transmission, offering a platform for the creation of implantable fiberscopes for minimally invasive in vivo brain imaging. Experiments on various lines of transgenic mice verify the performance of this fiberscope as a powerful tool for chronic in vivo neuroimaging using genetically encoded calcium indicators, neuronal activity markers as well as axon growth regulators and brain-specific protein drivers in deep regions of live brain.

Two-photon imaging of fiber-coupled neurons.

Optical coupling between a single, individually addressable neuron and a properly designed optical fiber is demonstrated. Two-photon imaging is shown to enable a quantitative in situ analysis of such fiber-single-neuron coupling in the live brain of transgenic mice. Fiber-optic interrogation of single pyramidal neurons in mouse brain cortex is performed with the positioning of the fiber probe relative to the neuron accurately mapped by means of two-photon imaging. These results pave the way for fiber-optic interfaces to single neurons for a stimulation and interrogation of individually addressable brain cells in chronic in vivo studies on freely behaving transgenic animal models, as well as the integration of fiber-optic single-neuron stimulation into the optical imaging framework.

Quantitative cognitive-test characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation.

Cognitive tests on representative groups of freely behaving transgenic mice are shown to enable a quantitative characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation. A systematic analysis of such tests provides a robust quantitative measure for the cognitive effects induced by fiber-optic neurostimulation, validating the performance of fiber-optic neurointerfaces for long-term optogenetic brain stimulations and showing no statistically significant artifacts in the behavior of transgenic mice due to interface implantation.

Three-dimensional fiber-optic readout of single-neuron-resolved fluorescence in living brain of transgenic mice.

A bundle of individually addressable optical fibers is shown to enable three-dimensional optical readout from single neurons in live brain, as well as in intact brain extracted from transgenic mice. With individual fibers in the bundle being only a few microns in diameter, single neurons are readily resolved in brain images transmitted by the fiber bundle. The third dimension is added by scanning the fiber in the longitudinal direction, with the fluorescence return read out from only one of the fibers in the bundle.

Fiber-optic electron-spin-resonance thermometry of single laser-activated neurons.

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 Ca2+ 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 Ca2+ sensors.

Fiber-optic vectorial magnetic-field gradiometry by a spatiotemporal differential optical detection of magnetic resonance in nitrogen-vacancy centers in diamond.

Highly sensitive room-temperature vectorial magnetic-field gradiometry is demonstrated using optically detected magnetic resonance (ODMR) in fiber-coupled nitrogen-vacancy (NV) centers in diamond. With a bulk NV-diamond magnetometer coupled to a pair of optical fibers integrated with a microwave transmission line, the differential ODMR measurements are implemented in both space and time, with magnetic-field gradient measurements supplemented with differential ODMR signal detection in the time domain, allowing efficient noise cancellation and providing a sensitivity of magnetogradiometry at the level of 10-7 nT/(nmHz1/2).

Stimulated fluorescence quenching in nitrogen-vacancy centers of diamond: temperature effects.

Laser-induced fluorescence quenching in nitrogen-vacancy (NV) centers in diamond is studied simultaneously with in situ measurements of the heating-induced shift of the electron spin resonance of NV centers in the presence of a microwave field. These experiments reveal a strong correlation between fluorescence suppression in NV centers and the rise of the local temperature inside the diamond crystal. This finding sheds light on quantum pathways behind stimulated fluorescence quenching in NV centers of diamond and may imply significant limitations on the applications of this effect as a method of superresolving imaging in biological systems.

High-resolution magnetic field imaging with a nitrogen-vacancy diamond sensor integrated with a photonic-crystal fiber.

We demonstrate high-resolution magnetic field imaging with a scanning fiber-optic probe which couples nitrogen-vacancy (NV) centers in diamond to a high-numerical-aperture photonic-crystal fiber integrated with a two-wire microwave transmission line. Magnetic resonance excitation of NV centers driven by the microwave field is read out through optical interrogation through the photonic-crystal fiber to enable high-speed, high-sensitivity magnetic field imaging with sub 30 µm spatial resolution.

Fiber-optic control and thermometry of single-cell thermosensation logic.

Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen--vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels.

Room-temperature magnetic gradiometry with fiber-coupled nitrogen-vacancy centers in diamond.

Differential optical detection of a magnetic resonance induced in nitrogen-vacancy (NV) centers in diamond is shown to enable a high-spatial-resolution room-temperature magnetic-field gradiometry on a fiber platform. An ultracompact design of this fiber-based solid-state magnetic gradiometer is achieved by integrating an NV-diamond magnetic sensor with a two-fiber opto-microwave interface, which couples NV centers to microwave and optical fields, used to resonantly drive and interrogate the spin of NV centers.

Ultrahigh-contrast imaging by temporally modulated stimulated emission depletion.

Stimulated emission depletion (STED) is the key optical technology enabling super-resolution microscopy below the diffraction limit. Here, we demonstrate that modulation of STED in the time domain, combined with properly designed lock-in detection, can radically enhance the contrast of fluorescent images of strongly autofluorescent biotissues. In our experiments, the temporally modulated STED technique, implemented with low-intensity continuous-wave laser sources, is shown to provide an efficient all-optical suppression of a broadband fluorescent background, allowing the contrast of fluorescent images of mammal brain tissues tagged with nitrogen-vacancy diamond to be increased by five orders of magnitude.

Fiber-optic magnetic-field imaging.

We demonstrate a scanning fiber-optic probe for magnetic-field imaging where nitrogen-vacancy (NV) centers are coupled to an optical fiber integrated with a two-wire microwave transmission line. The electron spin of NV centers in a diamond microcrystal attached to the tip of the fiber probe is manipulated by a frequency-modulated microwave field and is initialized by laser radiation transmitted through the optical tract of the fiber probe. The two-dimensional profile of the magnetic field is imaged with a high speed and high sensitivity using the photoluminescence spin-readout return from NV centers, captured and delivered by the same optical fiber.

Fiber-optic magnetometry with randomly oriented spins.

We demonstrate fiber-optic magnetometry using a random ensemble of nitrogen-vacancy (NV) centers in nanodiamond coupled to a tapered optical fiber, which provides a waveguide delivery of optical fields for the initialization, polarization, and readout of the electron spin in NV centers.

Electron spin manipulation and readout through an optical fiber.

The electron spin of nitrogen--vacancy (NV) centers in diamond offers a solid-state quantum bit and enables high-precision magnetic-field sensing on the nanoscale. Implementation of these approaches in a fiber format would offer unique opportunities for a broad range of technologies ranging from quantum information to neuroscience and bioimaging. Here, we demonstrate an ultracompact fiber-optic probe where a diamond microcrystal with a well-defined orientation of spin quantization NV axes is attached to the fiber tip, allowing the electron spins of NV centers to be manipulated, polarized, and read out through a fiber-optic waveguide integrated with a two-wire microwave transmission line. The microwave field transmitted through this line is used to manipulate the orientation of electron spins in NV centers through the electron-spin resonance tuned by an external magnetic field. The electron spin is then optically initialized and read out, with the initializing laser radiation and the photoluminescence spin-readout return from NV centers delivered by the same optical fiber.

Implantable fiber-optic interface for parallel multisite long-term optical dynamic brain interrogation in freely moving mice.

Seeing the big picture of functional responses within large neural networks in a freely functioning brain is crucial for understanding the cellular mechanisms behind the higher nervous activity, including the most complex brain functions, such as cognition and memory. As a breakthrough toward meeting this challenge, implantable fiber-optic interfaces integrating advanced optogenetic technologies and cutting-edge fiber-optic solutions have been demonstrated, enabling a long-term optogenetic manipulation of neural circuits in freely moving mice. Here, we show that a specifically designed implantable fiber-optic interface provides a powerful tool for parallel long-term optical interrogation of distinctly separate, functionally different sites in the brain of freely moving mice. This interface allows the same groups of neurons lying deeply in the brain of a freely behaving mouse to be reproducibly accessed and optically interrogated over many weeks, providing a long-term dynamic detection of genome activity in response to a broad variety of pharmacological and physiological stimuli.

The phase-controlled Raman effect.

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.

Stimulated Raman amplification and high-order Raman sideband generation in a polymer waveguide on a printed circuit.

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.

Spectral interference of frequency-shifted solitons in a photonic-crystal fiber.

Frequency-shifted solitons in a highly nonlinear photonic-crystal fiber (PCF) are shown to give rise to high-visibility interference fringes in PCF output spectra, indicating flat spectral phase profiles of individual solitons in the PCF output. This experimental finding, supported by numerical simulations, suggests a promising method of fiber-format pulse shaping and an attractive technology for few-cycle pulse synthesis through a coherent addition of frequency-shifted solitons generated in a highly nonlinear fiber.