Spatio-temporal parameters for optical probing of neuronal activity.

The challenge to understand the complex neuronal circuit functions in the mammalian brain has brought about a revolution in light-based neurotechnologies and optogenetic tools. However, while recent seminal works have shown excellent insights on the processing of basic functions such as sensory perception, memory, and navigation, understanding more complex brain functions is still unattainable with current technologies. We are just scratching the surface, both literally and figuratively. Yet, the path towards fully understanding the brain is not totally uncertain. Recent rapid technological advancements have allowed us to analyze the processing of signals within dendritic arborizations of single neurons and within neuronal circuits. Understanding the circuit dynamics in the brain requires a good appreciation of the spatial and temporal properties of neuronal activity. Here, we assess the spatio-temporal parameters of neuronal responses and match them with suitable light-based neurotechnologies as well as photochemical and optogenetic tools. We focus on the spatial range that includes dendrites and certain brain regions (e.g., cortex and hippocampus) that constitute neuronal circuits. We also review some temporal characteristics of some proteins and ion channels responsible for certain neuronal functions. With the aid of the photochemical and optogenetic markers, we can use light to visualize the circuit dynamics of a functioning brain. The challenge to understand how the brain works continue to excite scientists as research questions begin to link macroscopic and microscopic units of brain circuits.

Targeted pruning of a neuron's dendritic tree via femtosecond laser dendrotomy.

Neurons are classified according to action potential firing in response to current injection. While such firing patterns are shaped by the composition and distribution of ion channels, modelling studies suggest that the geometry of dendritic branches also influences temporal firing patterns. Verifying this link is crucial to understanding how neurons transform their inputs to output but has so far been technically challenging. Here, we investigate branching-dependent firing by pruning the dendritic tree of pyramidal neurons. We use a focused ultrafast laser to achieve highly localized and minimally invasive cutting of dendrites, thus keeping the rest of the dendritic tree intact and the neuron functional. We verify successful dendrotomy via two-photon uncaging of neurotransmitters before and after dendrotomy at sites around the cut region and via biocytin staining. Our results show that significantly altering the dendritic arborisation, such as by severing the apical trunk, enhances excitability in layer V cortical pyramidal neurons as predicted by simulations. This method may be applied to the analysis of specific relationships between dendritic structure and neuronal function. The capacity to dynamically manipulate dendritic topology or isolate inputs from various dendritic domains can provide a fresh perspective on the roles they play in shaping neuronal output.

Improved two-photon imaging of living neurons in brain tissue through temporal gating.

We optimize two-photon imaging of living neurons in brain tissue by temporally gating an incident laser to reduce the photon flux while optimizing the maximum fluorescence signal from the acquired images. Temporal gating produces a bunch of ~10 femtosecond pulses and the fluorescence signal is improved by increasing the bunch-pulse energy. Gating is achieved using an acousto-optic modulator with a variable gating frequency determined as integral multiples of the imaging sampling frequency. We hypothesize that reducing the photon flux minimizes the photo-damage to the cells. Our results, however, show that despite producing a high fluorescence signal, cell viability is compromised when the gating and sampling frequencies are equal (or effectively one bunch-pulse per pixel). We found an optimum gating frequency range that maintains the viability of the cells while preserving a pre-set fluorescence signal of the acquired two-photon images. The neurons are imaged while under whole-cell patch, and the cell viability is monitored as a change in the membrane's input resistance.

Four-dimensional multi-site photolysis of caged neurotransmitters.

Neurons receive thousands of synaptic inputs that are distributed in space and time. The systematic study of how neurons process these inputs requires a technique to stimulate multiple yet highly targeted points of interest along the neuron's dendritic tree. Three-dimensional multi-focal patterns produced via holographic projection combined with two-photon photolysis of caged compounds can provide for highly localized release of neurotransmitters within each diffraction-limited focus, and in this way emulate simultaneous synaptic inputs to the neuron. However, this technique so far cannot achieve time-dependent stimulation patterns due to fundamental limitations of the hologram-encoding device and other factors that affect the consistency of controlled synaptic stimulation. Here, we report an advanced technique that enables the design and application of arbitrary spatio-temporal photostimulation patterns that resemble physiological synaptic inputs. By combining holographic projection with a programmable high-speed light-switching array, we have overcome temporal limitations with holographic projection, allowing us to mimic distributed activation of synaptic inputs leading to action potential generation. Our experiments uniquely demonstrate multi-site two-photon glutamate uncaging in three dimensions with submillisecond temporal resolution. Implementing this approach opens up new prospects for studying neuronal synaptic integration in four dimensions.

Isolation of bovine coronavirus (bcoV) in vero cell line and its confirmation by direct FAT and RT-PCR.

Bovine Coronavirus (BCoV) is widespread both in dairy and beef cattle throughout the world. The virus is one of the largest RNA virus and has specific tropism for intestinal and pulmonary epithelial cells. It is responsible for huge economic losses by causing winter dysentery in adult dairy cattle and respiratory and intestinal tract infections leading to pneumo-enteritis in young calves. Isolation of BCoV has been reported to be difficult. Studies regarding epidemiology, virus isolation and molecular detection from India are very few. In the present study Vero cell line was used for isolation of the BCoV from Enzyme Linked Immunosorbent Assay (ELISA) positive samples. Direct florescent antibody technique (dFAT) and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to confirm the isolated virus strains at antigenic and genomic levels, respectively. Out of the 15 positive fecal samples, virus from only seven was able to infect vero cell line. Subsequently BCoV got adapted to the vero cell line upto three passages, which was confirmed both at genomic and antigenic levels by dFAT and RT-PCR testing. It can be concluded that vero cell line can be used for isolation of BCoV, however due to the enormous stain diversity of the virus it is possible that many stains can't grow and get adapt in this cell line. Further studies are required for isolation of different viral strains, finding the susceptible cell lines and also to confirm the variations among the BCoV isolates at antigenic/genomic levels.

Programmable multimode quantum networks.

Entanglement between large numbers of quantum modes is the quintessential resource for future technologies such as the quantum internet. Conventionally, the generation of multimode entanglement in optics requires complex layouts of beamsplitters and phase shifters in order to transform the input modes into entangled modes. Here we report the highly versatile and efficient generation of various multimode entangled states with the ability to switch between different linear optics networks in real time. By defining our modes to be combinations of different spatial regions of one beam, we may use just one pair of multi-pixel detectors in order to measure multiple entangled modes. We programme virtual networks that are fully equivalent to the physical linear optics networks they are emulating. We present results for N=2 up to N=8 entangled modes here, including N=2, 3, 4 cluster states. Our approach introduces the highly sought after attributes of flexibility and scalability to multimode entanglement.

Simultaneous multi-site two-photon photostimulation in three dimensions.

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees.

Optical pattern recognition via adaptive spatial homodyne detection.

We present an experimental demonstration of an optical pattern recognition scheme based on spatial homodyne detection. Our scheme is adaptive, all-optical, utilizes a single-element photo-detector, and provides a single parameter readout to quantify the efficacy of pattern recognition, thereby allowing very fast pattern recognition speeds. The spatial homodyne detector was applied to the identification of one- and two-dimensional phase profiles.

Programmable unitary spatial mode manipulation.

Free space propagation and conventional optical systems such as lenses and mirrors all perform spatial unitary transforms. However, the subset of transforms available through these conventional systems is limited in scope. We present here a unitary programmable mode converter (UPMC) capable of performing any spatial unitary transform of the light field. It is based on a succession of reflections on programmable deformable mirrors and free space propagation. We first show theoretically that a UPMC without limitations on resources can perform perfectly any transform. We then build an experimental implementation of the UPMC and show that, even when limited to three reflections on an array of 12 pixels, the UPMC is capable of performing single mode tranforms with an efficiency greater than 80% for the first four modes of the transverse electromagnetic basis.

Quantum measurements of spatial conjugate variables: displacement and tilt of a Gaussian beam.

We consider the problem of measurement of optical transverse profile parameters and their conjugate variable. Using multimode analysis, we introduce the concept of detection noise modes. For Gaussian beams, displacement and tilt are a pair of transverse-profile conjugate variables. We experimentally demonstrate the optimal encoding and detection of these variables with a spatial homodyning scheme. Using higher-order spatial mode squeezing, we show the sub-shot-noise measurements for the displacement and tilt of a Gaussian beam.

A quantum laser pointer.

The measurement sensitivity of the pointing direction of a laser beam is ultimately limited by the quantum nature of light. To reduce this limit, we have experimentally produced a quantum laser pointer, a beam of light whose direction is measured with a precision greater than that possible for a usual laser beam. The laser pointer is generated by combining three different beams in three orthogonal transverse modes, two of them in a squeezed-vacuum state and one in an intense coherent field. The result provides a demonstration of multichannel spatial squeezing, along with its application to the improvement of beam positioning sensitivity and, more generally, to imaging.

Polarization squeezing of continuous variable stokes parameters.

We report the first direct experimental characterization of continuous variable quantum Stokes parameters. We generate a continuous wave light beam with more than 3 dB of simultaneous squeezing in three of the four Stokes parameters. The polarization squeezed beam is produced by mixing two quadrature squeezed beams on a polarizing beam splitter. Depending on the squeezed quadrature of these two beams the quantum uncertainty volume on the Poincaré sphere becomes a "cigarlike" or "pancakelike" ellipsoid.

[Klinefelter's disease and leydigiomas. A report of one case (author's transl)]. / Klinefelter et leydigiomes. A propos d'une observation.

The authors present the case of a patient with Klinefelter syndrome who, at the age of 36 years, developed a multifocal Leydig cell tumour of the right testicle, presenting as hematuria. After orchidectomy, the patient developed an identical tumour in the remaining testes. This curious case of a patient in whom the clinical and laboratory features of Klinefelterer's syndrome, were masked by the Leydig cell tumour and vice versa, is analyzed. Study of this case, and a review of the literature of the three cases published in the past, offer no definitive solution to the problem of the possible link between the Leydig cell tumour and the Klinefelter syndrome, but a prospective study protocol is suggested with such an aim in mind. More careful clinical and laboratory evaluation of populations of Klinefelter's syndrome and Leydig cell tumours should provide a solution. More routine therapy of Klinefelt syndrome should possibly avoid the development of complications in the form of testicular Leydig cell tumours by causing inhibition of useless and excessive pituitary actions by the administration of androgens immediately after puberty.