Application of the simplified method of optic recording for mapping focuses of the neuronal activity in the somatosensory cortex of the white rats

In rats, a differential signal was used for evoking an optical image in the barrel-field zone which represented a difference between cortical images during the control and the stimulation periods. After a subtraction of averaged sequences of frames, an image of spots reflecting a probable location of activated groups of neurones was obtained. Natural low frequency stimulation of vibrissae is supposed to be the most effective. The method of intrinsic optical activity imaging can be applied for preliminary mapping of cortical zones.     

Laser-guided direct writing of living cells

To perform their myriad functions, tissues use specific cell-cell interactions that depend on the spatial ordering of multiple cell types. Recapitulating this spatial order in vitro will facilitate our understanding of function and failure in native and engineered tissue. One approach to achieving such high placement precision is to use optical forces to deposit cells directly. Toward this end, recent work with optical forces has shown that a wide range of particulate materials can be guided and deposited on surfaces to form arbitrary spatial patterns. Here we report that, when we use the light from a near-infrared diode laser focused through a low numerical aperture lens, individual embryonic chick spinal cord cells can be guided through culture medium and deposited on a glass surface to form small clusters of cells. In addition, we found that the laser light could be coupled into hollow optical fibers and that the cells could be guided inside the fibers over millimeter distances. The demonstration of fiber-based guidance extends by 2 orders of magnitude the distance over which optical manipulation can be performed with living cells. Cells guided into the fiber remained viable, as evidenced by normal cell adhesion and neurite outgrowth after exposure to the laser light. The results indicate that this particle deposition process, which we call "laser-guided direct writing," can be used to construct patterned arrays of tens to hundreds of cells using arbitrary numbers of cell types placed at arbitrary positions with micrometer-scale precision.     

Low temperature trapping method for cytochrome oxidase oxygen intermediates.

The trapping of intermediates in the cytochrome oxidase-oxygen reaction is made possible by a three-step procedure: 1) Oxygenation of the ferrous cytochrome oxidase-carbon monoxide compound at a temperature sufficiently low that no ligand exchange with oxygen occurs (?20 to ?30°C); 2) flash photolysis at a temperature sufficiently low that the subsequent reaction with oxygen can readily be measured with a multichannel spectrophotometer; 3) rapid cooling of the reaction in progress to a sufficiently low temperature that no further reaction takes place and detailed optical or electron paramagnetic resonance spectroscopy can be performed. Since the freezing point of the mitochondrial suspensions during oxygenation can be in the range of ?20°C, only nondeleterious concentrations of ethylene glycol are employed. The reaction kinetics are observed in the solid state in small-diameter circular tubes suitable for rapid trapping. Efficient optical coupling to the sample tube for transmission spectroscopy and efficient laser photolysis are similarly provided by fiber optics coupling. The method is suitable for studying in detail three compounds of cytochrome oxidase and oxygen formed in the temperature range from ?125°C upwards. In addition, the electron transfer processes of the respiratory chain are accurately delineated.     

Integrating photoacoustic microscopy with other imaging technologies for multimodal imaging

As a hybrid optical microscopic imaging technology, photoacoustic microscopy images the optical absorption contrasts and takes advantage of low acoustic scattering of biological tissues to achieve high-resolution anatomical and functional imaging. When combined with other imaging modalities, photoacoustic microscopy-based multimodal technologies can provide complementary contrast mechanisms to reveal complementary information of biological tissues. To achieve intrinsically and precisely registered images in a multimodal photoacoustic microscopy imaging system, either the ultrasonic transducer or the light source can be shared among the different imaging modalities. These technologies are the major focus of this minireview. It also covered the progress of the recently developed penta-modal photoacoustic microscopy imaging system featuring a novel dynamic focusing technique enabled by OCT contour scan.     

Nanoscale Optical Directional Coupler

Ultracompact optical directional coupler is one of the key elements for nanoscale optical networks and highly integrated optical circuits. Although the transverse size has been reduced down to subwavelength by exploiting plasmonic waveguides, the longitudinal size has remained yet on the micrometer-scale, which seems to be a fundamental limitation by the conventional configuration based on cross-talk coupling between two neighboring waveguides. We have proposed a novel conception of optical directional coupler based on loss-overcompensated resonant coupling between two plasmonic waveguides via an in-between gain-assisted nanocavity. The loss-overcompensated state can be achieved by adjusting pumping rate in the nanocavity. The validity of the proposed conception is confirmed by numerical simulations of a physical model with the deep-subwavelength planar footprint of 300 nm × 300 nm, THz bandwidth, and an exceptionally low energy consumption on the order of 0.1 fJ per signal pulse. To our knowledge, it is the first proposed ultrafast nanoscale four-port directional coupler.     

Fano Resonance-Assisted Plasmonic Trapping of Nanoparticles

Plasmonic optical trapping is widely applied in the field of bioscience, microfluidics, and quantum optics. It can play a vital role to extend optical manipulation tools from micrometer to nanometer scale level. Currently, it is a challenge to obtain the highly stable optical trapping with low power and less damage. In this paper, we propose Fano resonance-assisted self-induced back-action (FASIBA) method, through which a single 40-nm gold particle can be trapped in hole-slit nano-aperture milled on metallic film. It is used to achieve ultra-accurate positioning of nanoparticle, metallic nanostructures at wide infrared wavelength range, quite effectively and evidently. The stable plasmonic trapping is achieved by tuning the transmission wavelengths and modifications of nanoslit, indicating that the depth of potential well can be increased from minus 8KT to 12KT, with the input power of 10⁹ W/m². This can be attributed to great modifications in Fano resonance transmissions according to self-induced back-action (SIBA) theory. The results are basically helpful to facilitate the trapping with lower power and less damage to the objects, which enables new scenario for the treatment of undesirable spread of a single nanoscale creature, such as virus.     

Particle detection,number estimation,and feature measurement in gene transfer studies: optical fractionator stereology integrated with digital image processing and analysis

Assessing the efficacy of in vivo gene transfer often requires a quantitative determination of the number, size, shape, or histological visualization characteristics of biological objects. The optical fractionator has become a choice stereological method for estimating the number of objects, such as neurons, in a structure, such as a brain subregion. Digital image processing and analytic methods can increase detection sensitivity and quantify structural and/or spectral features located in histological specimens. We describe a hardware and software system that we have developed for conducting the optical fractionator process. A microscope equipped with a video camera and motorized stage and focus controls is interfaced with a desktop computer. The computer contains a combination live video/computer graphics adapter with a video frame grabber and controls the stage, focus, and video via a commercial imaging software package. Specialized macro programs have been constructed with this software to execute command sequences requisite to the optical fractionator method: defining regions of interest, positioning specimens in a systematic uniform random manner, and stepping through known volumes of tissue for interactive object identification (optical dissectors). The system affords the flexibility to work with count regions that exceed the microscope image field size at low magnifications and to adjust the parameters of the fractionator sampling to best match the demands of particular specimens and object types. Digital image processing can be used to facilitate object detection and identification, and objects that meet criteria for counting can be analyzed for a variety of morphometric and optical properties.     

Bacterial growth properties at low optical densities

A method for accurate quantification of growth rate and yield of bacterial populations at low densities was developed with a modified version of a stepwise linear model for fitting growth curves based on optical density measurements, and adapted to measurements at low optical densities in 96-well microtiter plates. The method can be used for rapid and precise estimates of growth rate and yield, based on optical density measurements of large numbers of cultures of Escherichia coli. E. coli B lines were serially propagated at low glucose concentration during a long-term evolution experiment. Growth rate and yield of populations sampled from each of 12 lines that evolved for 20,000 generations under these conditions and two ancestral clones was measured. Populations were grown at three different glucose concentrations. Consistent with earlier findings, statistical analysis showed that both exponential growth rate and yield per unit of glucose differed significantly between the three glucose concentrations tested. Significant adaptation of the evolved populations to the nutrient conditions in which they evolved for 20,000 generations was observed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tuning FlaSh: redesign of the dynamics,voltage range,and color of the genetically encoded optical sensor of membrane potential

The optical voltage sensor FlaSh, made from a fusion of a GFP "reporter domain" and a voltage-gated Shaker K(+) channel "detector domain," has been mutagenically tuned in both the GFP reporter and channel detector domains. This has produced sensors with improved folding at 37 degrees C, enabling use in mammalian preparations, and yielded variants with distinct spectra, kinetics, and voltage dependence, thus expanding the types of electrical signals that can be detected. The optical readout of FlaSh has also been expanded from single wavelength fluorescence intensity changes to dual wavelength measurements based on both voltage-dependent spectral shifts and changes in FRET. Different versions of FlaSh can now be chosen to optimize the detection of either action potentials or synaptic potentials, to follow high versus low rates of activity, and to best reflect electrical activity in cell types with distinct voltages of operation.     

Graphene Hybrid Surface Plasmon Waveguide with Low Loss Transmission

In this paper, a novel graphene hybrid surface plasmon waveguide structure is designed. Based on the finite element method, the mode characteristics, the quality factor, and the gain threshold of the waveguide structure are analyzed. The results show that the optical field constraint of the designed waveguide can reach a better level of deep sub-wavelength under the optimal parameters of 1550-nm working wavelength. The structure is applied to a laser, and the high quality factor, the low energy loss, the low threshold limit, and the ultra-small effective mode field area are obtained by adjusting waveguide design parameters. Compared with the common waveguide structure, this structure has stronger optical field limiting ability and microcavity binding ability. It provides theoretical and technical support for the development of new high-efficiency nano-laser devices and is expected to be applied to fields such as on-chip interconnects, photonic integrated circuits, optical storage, and optical signal processing.

     

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.     

Estimating Above-Ground Carbon Biomass in a Newly Restored Coastal Plain Wetland Using Remote Sensing

Developing accurate but inexpensive methods for estimating above-ground carbon biomass is an important technical challenge that must be overcome before a carbon offset market can be successfully implemented in the United States. Previous studies have shown that LiDAR (light detection and ranging) is well-suited for modeling above-ground biomass in mature forests; however, there has been little previous research on the ability of LiDAR to model above-ground biomass in areas with young, aggrading vegetation. This study compared the abilities of discrete-return LiDAR and high resolution optical imagery to model above-ground carbon biomass at a young restored forested wetland site in eastern North Carolina. We found that the optical imagery model explained more of the observed variation in carbon biomass than the LiDAR model (adj-R² values of 0.34 and 0.18 respectively; root mean squared errors of 0.14 Mg C/ha and 0.17 Mg C/ha respectively). Optical imagery was also better able to predict high and low biomass extremes than the LiDAR model. Combining both the optical and LiDAR improved upon the optical model but only marginally (adj-R² of 0.37). These results suggest that the ability of discrete-return LiDAR to model above-ground biomass may be rather limited in areas with young, small trees and that high spatial resolution optical imagery may be the better tool in such areas.     

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasi-equilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060, 2005), appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.     

A comparative study of protein immobilization techniques for optical immunosensors.

This paper presents the results of a study of a number of antibody immobilization techniques for application to optical immunosensors. In particular, well-known methods such as covalent binding and physical adsorption have been extended to the Langmiur-Blodgett method in an attempt to improve the density and possibly the uniformity of orientation of monoclonal antibodies on an optical surface. The surface density of active immobilized antibodies was determined from enzyme immunoassay and their thickness and refractive index were deduced from ellipsometry. It is shown that, although high surface densities (500 ng/cm2) of antibody can be obtained, the major obstacle to the detection of low concentrations of antigens or haptens is the non-specific binding of foreign molecules to the sensing surface.     

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (V_m), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both V_m and CaT simultaneously from the same location on the heart^1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping^1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of V_m and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.     

Reduced-stringency DNA reassociation: sequence specific duplex formation.

Reduced-stringency DNA reassociation conditions allow low stability duplexes to be detected in prokaryotic, plant, fish, avian, mammalian, and primate genomes. Highly diverged families of sequences can be detected in avian, mouse, and human unique sequence dNAs. Such a family has been described among twelve species of birds; based on species specific melting profiles and fractionation of sequences belonging to this family, it was concluded that permissive reassociation conditions did not artifactually produce low stability structures (1). We report S1 nuclease and optical melting experiments, and further fractionation of the diverged family to confirm sequence specific DNA reassociation at 50 degrees in 0.5 M phosphate buffer.     

Real-time label-free immunoassay of interferon-gamma and prostate-specific antigen using a Fiber-Optic Localized Surface Plasmon Resonance sensor

A Fiber-Optic Localized Surface Plasmon Resonance (FO LSPR) sensor was fabricated using spherical gold nanoparticles (Au NPs) on a flattened end-face of the optical fiber. The Au NPs were easily synthesized by the Turkevich method and were immobilized on the end-face of the optical fiber by using a self-assembled monolayer (SAM). In order to examine the possibility of its application as a biosensor for label-free immunoassays, the fabricated FO LSPR sensor was used for the detection of the antibody-antigen reaction of interferon-gamma (IFN-γ) and the limit of detection (LOD) was approximately 2pg/ml. Herein, The antibodies and bovine serum albumins (BSAs) were immobilized on the Au NPs by physisorption. Also, the FO LSPR sensor was used for the detection of a prostate-specific antigen (PSA) and the LOD was 1pg/ml below. The fabricated FO LSPR sensor can be used for real-time label-free immunoassay having fast detection time, high resolution and sensitivity. In addition, the proposed sensor platform has the advantages of low cost, simple optical setup, remote sensing, simple fabrication, real-time detection, low sample volume, and potential application to in-vivo detection systems.     

High quality InP single crystal from Sumitomo electric

InP single crystals are primarily used as the conductive substrates for optical devices such as LEDs and lasers etc, in optoelectronics. Recently, active research for the improvement of MISFET, HEMT, HBT, RHET and OEICs has been demanding the development of a semi-insulating, SI, InP substrate on which these devices can be fabricated. The improvement of quality and reliability of these devices is being vigorously promoted, so high purity and low defect density are required of InP single crystal substrates. At Sumitomo Electric, the LEC method to grow InP single crystals with low defect density has been developed.     

Some applications of chiral liquid affinity chromatography using bovine serum albumin as a stationary phase

The enantioselectivity excerted by many proteins can be utilized for direct optical resolution in liquid chromatographic processes whereby the protein is used as a stationary phase. Bovine serum albumin (BSA), covalently bound to a suitable support, has been shown to act as a chiral discriminator for a variety of racemic organic compounds in aqueous buffers. Columns packed with BSA-silica can be used for determination of enantiomeric composition in aqueous solvents at very low concentrations by HPLC. This technique opens up new possibilities for the preparative isolation of micrograms amounts of enantiomers and for studies of stereoselectivity and mechanisms in enzymatic and microbial reactions.     

Evaluation of the energetic position of the lowest excited singlet state of beta-carotene by NEXAFS and photoemission spectroscopy.

In carotenoids the lowest energetic optical transition belonging to the pi-electron system is forbidden by symmetry, therefore the energetic position of the S(1) (2(1)A(g)) level can hardly be assessed by optical spectroscopy. We introduce a novel experimental approach: For molecules with pi-electron systems the transition C1s-->2p(pi*) from inner-atomic to the lowest unoccupied molecular orbital (LUMO) appears in X-ray absorption near edge spectra (NEXAFS) as an intense, sharp peak a few eV below the carbon K-edge. Whereas the peak position reflects the energy of the first excited singlet state in relation to the ionization potential of the molecule, intensity and width of the transition depend on hybridization and bonding partners of the selected atom. Complementary information can be obtained from ultraviolet photoelectron spectroscopy (UPS): At the low binding energy site of the spectrum a peak related to the highest occupied molecular orbital (HOMO) appears. We have measured NEXAFS and UPS of beta-carotene. Based on these measurements and quantum chemical calculations the HOMO and LUMO energies can be derived.