Optimizing the spatial resolution of Channelrhodopsin-2 activation

Over the past few years, the light-gated cation channel Channelrhodopsin-2 (ChR2) has seen a remarkable diversity of applications in neuroscience. However, commonly used wide-field illumination provides poor spatial selectivity for cell stimulation. We explored the potential of focal laser illumination to map photocurrents of individual neurons in sparsely transfected hippocampal slice cultures. Interestingly, the best spatial resolution of photocurrent induction was obtained at the lowest laser power. By adjusting the light intensity to a neuron's spike threshold, we were able to trigger action potentials with a spatial selectivity of less than 30 microm. Experiments with dissociated hippocampal cells suggested that the main factor limiting the spatial resolution was ChR2 current density rather than scattering of the excitation light. We conclude that subcellular resolution can be achieved only in cells with a high ChR2 expression level and that future improved variants of ChR2 are likely to extend the spatial resolution of photocurrent induction to the level of single dendrites.     

Proteohistography--direct analysis of tissue with high sensitivity and high spatial resolution using ProteinChip technology.

On the proteomic level, all tissues, tissue constituents, or even single cells are heterogeneous, but the biological relevance of this cannot be adequately investigated with any currently available technique. The analysis of proteins of small tissue areas by any proteomic approach is limited by the number of required cells. Increasing the number of cells only serves to lower the spatial resolution of expressed proteins. To enhance sensitivity and spatial resolution we developed Proteohistography. Laser microdissection was used to mark special areas of interest on tissue sections attached to glass slides. These areas were positioned under microscopic control directly on an affinity chromatographic ProteinChip Array so that cells were lysed and their released proteins bound on a spatially defined point. The ProteinChip System, surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), allows the laser to be steered to up to 215 distinct positions across the surface of the spot, enabling a high spatial resolution of measured protein profiles for the analyzed tissue area. Protein profiles of the single positions were visually plotted over the used tissue section to visualize distribution proteohistologically. Results show that the spatial distribution of detectable proteins could be used as a Proteohistogram for a given tissue area. Consequently, this procedure can provide additional information to both a matrix-assisted laser desorption/ionization (MALDI)-based approach and immunohistochemistry, as it is more sensitive, highly quantitative, and no specific antibody is needed. Hence, proteomic heterogeneity can be visualized even if proteins are not known or identified.     

Characterization of a range of fura dyes with two-photon excitation

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.     

Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wavelength of 1.06 micron. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.     

Pulsed laser imaging of rapid Ca2+ gradients in excitable cells.

Excitable cells are thought to respond to action potentials by forming short lived and highly localized Ca2+ gradients near sites of Ca2+ entry or near the site of Ca2+ release by intracellular stores. However, conventional imaging techniques lack the spatial and temporal resolution to capture these gradients. Here we demonstrate the use of pulsed-laser microscopy to measure Ca2+ gradients with submicron spatial resolution and millisecond time resolution in two preparations where the Ca2+ signal is thought to be fast and highly localized: adrenal chromaffin cells, where the entry of Ca2+ through voltage dependent Ca2+ channels triggers exocytotic fusion; and skeletal muscle fibers, where intracellular Ca2+ release from the sarcoplasmic reticulum initiates contraction. In chromaffin cells, Ca2+ gradients developed over 10-100 ms and were initially restricted to discrete submembrane domains, or hot spots, before developing into complete rings of elevated Ca2+ concentration. In frog skeletal muscle large, short-lived (approximately 6 ms) Ca2+ gradients were observed within individual sarcomeres following induction of action potentials. The pulsed laser imaging approach permits, for the first time, the capture and critical examination of rapid Ca2+ signaling events such as those underlying excitation-secretion and excitation-contraction coupling.     

基于高分辨率遥感影像的森林地上生物量估算

以南京市紫金山林区为研究区,利用e-Cognition面向对象分类方法,基于光谱和空间信息融合后的IKONOS影像提取单木树冠阳性冠幅(PoCA, Positive crown area)信息,并结合野外实测的样方生物量数据,分别建立了针叶林和阔叶林地上生物量 (AGB, Aboveground Biomass)的遥感估算模型,并利用实测森林生物量数据对模型进行了验证。结果表明,基于遥感影像提取的PoCA与实测AGB存在较好的非线性相关关系,所建针叶林AGB估算模型的可靠性优于阔叶林模型。对建模样本而言,估算的针叶林和阔叶林AGB与观测数据比较的R2分别为0.62 (P<0.01,n=9) 和0.56(P<0.01,n=16)。验证表明,所建AGB估算模型的可靠性较好,估算的针叶林和阔叶林AGB与观测数据比较的R2分别为0.55(P<0.01,n=6) 和0.52(P<0.01,n=10),但当AGB较低时,模型结果偏高,AGB较低时,模型结果偏低。研究说明通过高分辨率遥感数据的融合、提取树冠信息进行生物量估算是可行性的。     

Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses

For studying the function of specific neurons in their native circuitry, it is desired to precisely control their activity. This often requires dissection to allow accurate electrical stimulation or neurotransmitter application , and it is thus inherently difficult in live animals, especially in small model organisms. Here, we employed channelrhodopsin-2 (ChR2), a directly light-gated cation channel from the green alga Chlamydomonas reinhardtii, in excitable cells of the nematode Caenorhabditis elegans, to trigger specific behaviors, simply by illumination. Channelrhodopsins are 7-transmembrane-helix proteins that resemble the light-driven proton pump bacteriorhodopsin , and they also utilize the chromophore all-trans retinal, but to open an intrinsic cation pore. In muscle cells, light-activated ChR2 evoked strong, simultaneous contractions, which were reduced in the background of mutated L-type, voltage-gated Ca2+-channels (VGCCs) and ryanodine receptors (RyRs). Electrophysiological analysis demonstrated rapid inward currents that persisted as long as the illumination. When ChR2 was expressed in mechanosensory neurons, light evoked withdrawal behaviors that are normally elicited by mechanical stimulation. Furthermore, ChR2 enabled activity of these neurons in mutants lacking the MEC-4/MEC-10 mechanosensory ion channel . Thus, specific neurons or muscles expressing ChR2 can be quickly and reversibly activated by light in live and behaving, as well as dissected, animals.     

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.     

Irradiation of mammalian cultured cells with a collimated heavy-ion microbeam

As the first step for the analysis of the biological effect of heavy charged-particle radiation, we established a method for the irradiation of individual cells with a heavy-ion microbeam apparatus at JAERI-Takasaki. CHO-K1 cells attached on a thin film of an ion track detector, CR-39, were automatically detected under a fluorescence microscope and irradiated individually with an 40Ar13+ ion (11.5 MeV/nucleon, LET 1260 keV/microm) microbeam. Without killing the irradiated cells, trajectories of irradiated ions were visualized as etch pits by treatment of the CR-39 with an alkaline-ethanol solution at 37 degrees C. The exact positions of ion hits were determined by overlaying images of both cells and etch pits. The cells that were irradiated with argon ions showed a reduced growth in postirradiation observations. Moreover, a single hit of an argon ion to the cell nucleus resulted in strong growth inhibition. These results tell us that our verified irradiation method enables us to start a precise study of the effects of high-LET radiation on cells.     

基于高分辨率遥感影像的北亚热带森林生物量反演

以北亚热带湖北省太子山林场为研究对象,基于高空间分辨率GF-2与SPOT-6卫星影像,提取不同窗口大小下的纹理信息与光谱信息,利用随机森林回归算法,并结合野外实测106块样地的生物量数据,建立不同影像下的太子山林场森林生物量反演模型。结果显示：（1） GF-2和SPOT-6虽然空间分辨率有差异,但是从其不同波段反射率的相关系数（0.75、0.78、0.73、0.61）发现,两种影像的波段反射率具有较高的相关性,说明两者的辐射性能相近;（2）通过分析不同纹理特征对生物量模型的影响,发现均值和对比度纹理参数对生物量反演具有很好的效果。（3）高分辨率的遥感数据在生物量反演中具有较好的表现,且GF-2生物量模型精度（R²=0.88,RMSE=27.11 Mg/hm²）与SPOT-6生物量模型的精度（R²=0.89,RMSE=23.93 Mg/hm²）相近。（4）两种影像对不同森林类型的生物量预测值不存在显著差异,都适合对不同林分类型的生物量进行预测。     

Mapping changes in tidal wetland vegetation composition and pattern across a salinity gradient using high spatial resolution imagery

Detailed vegetation mapping of wetlands, both natural and restored, can offer valuable information about vegetation diversity and community structure and provides the means for examining vegetation change over time. We mapped vegetation at six tidal marshes (two natural, four restored) in the San Francisco Estuary, CA, USA, between 2003 and 2004 using detailed vegetation field surveys and high spatial-resolution color-infrared aerial photography. Vegetation classes were determined by performing hierarchical agglomerative clustering on the field data collected from each tidal marsh. Supervised classification of the CIR photography resulted in vegetation class mapping accuracies ranging from 70 to 92%; 10 out of 12 classification accuracies were above 80%, demonstrating the potential to map emergent wetland vegetation. The number of vegetation classes decreased with salinity, and increased with size and age. In general, landscape diversity, as measured by the Shannon’s diversity index, also decreased with salinity, with an exception for the most saline site, a newly restored marsh. Vegetation change between years is evident, but the differences across sites in composition and pattern were larger than change within sites over two growing seasons.     

滩涂湿地入侵种互花米草植被覆盖度的高空间分辨率遥感估算

互花米草是沿海滩涂生态系统的重要入侵物种,其分布状况和覆盖度是湿地生态研究的重要参数和基础。以宁德三沙湾(三都澳)滩涂湿地为研究区,以SPOT6 6 m空间分辨率卫星影像为数据源,对互花米草分布和植被覆盖度进行研究,并与同期10 cm空间分辨率无人机影像进行比较验证。结果表明,影像覆盖区域内互花米草总面积为20.19 km~2,其中蕉城区互花米草分布较广,面积为9.63 km~2,占研究区互花米草总面积的47.70%。互花米草植被覆盖度整体上以40%-60%和60%-80%的中、高覆盖度分布为主,其分布面积分别为5.44 km~2和4.95 km~2,占互花米草总分布面积的26.92%和24.52%,而40%以下的低覆盖度和80%以上较高覆盖度分布相对较少。SPOT6遥感影像估算得到的互花米草植被覆盖度具有较好的精度,与无人机影像值之间的均方根误差RMSE为0.117,线性回归决定系数R~2为0.918,可用于滩涂湿地植被覆盖度分析。     

Electrical properties of a light-addressable microelectrode chip with high electrode density for extracellular stimulation and recording of excitable cells

A light-addressable microelectrode chip with 3600 TiN electrodes was fabricated. Amorphous silicon (a-Si:H) serves as a photo conductor. The electrodes on the chip are addressed by a laser spot and electrical properties of the system are determined. DC measurements show a dark to bright dynamic of 10(6)-10(7). The AC impedance dynamic @ 1 kHz/100 mV and thus the signal-to-noise-ratio is determined to 60. This value is quite sufficient for electrophysiological measurements. For the first time, recordings from cardiac myocytes are reported using the principle of light-addressing. Measurements were done with a standard laser scan microscope (Zeiss LSM 410).     

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique

Transgenic cell lines of loblolly pine （Pinus taeda L.） were analyzed by a compact laser-tweezers-Raman-spectroscopy （LTRS） system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.     

Inactivation of alpha VEE virus using a high intensity laser with UV-radiation

Inactivation of VEE virus with laser UV impulses of nano- and picosecond duration was investigated. It has been shown that in both cases there is a decrease of the inactivation cross-section with the rise of irradiation intensity. It points to the fact that the major lethal photoproduct in VEE is formed by a single-quantum mechanism.     

Effect of near-infrared and blue laser light on vero E6 cells SARS-CoV-2 infection model

Photobiomodulation therapy (PBMT) employing laser light has been emerging as a safe strategy to challenge viruses. In this study the effect of blue and near-infrared (NIR) laser light was assessed in an in vitro model of SARS-CoV-2 infection. PBMT at blue wavelength inhibited viral amplification when the virus was directly irradiated and then transferred to cell culture and when cells already infected were treated. The NIR wavelength resulted less efficacious showing a minor effect on the reduction of the viral load. The cells receiving the irradiated virus or directly irradiated rescued their viability to level comparable to not treated cells. Virion integrity and antigenicity were preserved after blue and NIR irradiation, suggesting that the PBMT antiviral effect was not correlated to viral lipidic envelope disruption. Our results suggested that PBMT can be considered a valid strategy to counteract SARS-CoV-2 infection, at least in vitro.