植物幼苗原发性超弱光子成像的研究

采用光子计数成像系统（PIAS）对植物幼苗萌发过程的超弱发光进行观察。结果表明,自发光子长时间积累可形成二维图象;光子计数和采集图象均可得到植物体的自发发光;通过实验探测到幼苗的根,叶在同一平面图象有不同的发光表现;光子成像系统可客观地比较生物自发超弱发光,为进一步研究超弱发光机理提供实验基础。     

Low-light imaging technology in the life sciences

Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence. New high-performance low-light level imaging systems have recently become available for the life science. These systems use advances in camera design and digital image processing and are now being used for a wide range of luminescence applications. They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis. This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening. Improvements in imageprocessing hardware and software have increased the usefulness of these systems in the biosciences. Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays. As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).     

Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells.     

体外循环血栓非侵入在线电阻抗成像方法

目的 在体外循环系统中,血栓的在线检测和可视化具有重要意义。本文提出了基于电阻抗成像（EIT）的体外循环血栓非侵入在线检测方法。方法 首先通过联合仿真研究了传感器尺寸对成像效果的影响。其次,根据仿真结果设计了直径为20 mm的16铜质电极EIT传感器,搭建了循环流动实验平台,并设计了静态和循环流动实验。使用尺寸为3～6 mm的猪血块代替血栓,将血块置于新鲜猪血样本中,采用Tikhonov正则化算法进行成像。将3 mm和5 mm的血块分别置于循环系统中,重建血块在传感器截面的大小和位置图像,并与高速相机拍摄结果进行对比。结果 仿真结果显示当目标物与传感器面积比（AR）不小于0.01时,传感器直径为20 mm和30 mm对应的图像相关系数（IC）均大于0.06,成像效果较好。静态成像结果显示,相对尺寸覆盖率误差（RCR）小于等于0.1。循环流动实验显示,血块经过传感器时,检测到归一化后的相对电导率变化值分别为80和200,结果显示该方法能够检测到循环系统中的血块。结论 该方法具有实时性和非侵入的优点,有望应用于体外血栓的检测。     

Characterization and application of porous gold nanoparticles as 2‐photon luminescence imaging agents: 20‐fold brighter than gold nanorods

Two‐photon nonlinear microscopy with the aid of plasmonic contrast agents is an attractive bioimaging technique capable of generating high‐resolution images in 3 dimensions and facilitating targeted imaging with deep tissue penetration. In this work, physically synthesized gold nanoparticles containing multiple nanopores are used as 2‐photon contrast agents and are reported to emit a 20‐fold brighter 2‐photon luminescence as compared to typical contrast agents, that is, gold nanorods. A successful application of our porous gold nanoparticles is experimentally demonstrated by in vitro nonlinear optical imaging of adipocytes at subcellular level.      

Estimation and correction of Cerenkov-light on luminescence image of water for carbon-ion therapy dosimetry

PurposeThe luminescence images of water during the irradiation of carbon-ions provide useful information such as the ranges and the widths of carbon-ion beams. However, measured luminescence images show higher intensities in shallow depths and wider lateral profiles than those of the dose distributions. These differences prevent the luminescence imaging of water from being applied to a quality assurance for carbon-ion therapy. We assumed that the differences were due to the contaminations of Cerenkov-light from the secondary electrons of carbon-ions as well as the prompt gamma photons in the measured image. In this study, we applied a correction method to a luminescence image of water during the irradiation of carbon-ion beams.MethodsWe estimated the distribution of the Cerenkov-light in water during the irradiation of carbon-ions by Monte Carlo simulation and subtracted the simulated Cerenkov-light from the depth and lateral profiles of the measured luminescence image for 241.5 MeV/u-carbon-ions.ResultsWith these corrections, we successfully obtained depth and lateral profiles whose distributions are almost identical to the dose distributions of carbon-ions. The high intensities in the shallow depth areas decreased and the Bragg peak intensity increased. The beam widths of the measured images approached those of the ionization chamber.ConclusionsThese results indicate that the luminescence imaging of water with our proposed correction has potential to be used for dose distribution measurements for carbon-ion therapy dosimetry.     

Fast non‐negative temporal deconvolution for laser scanning microscopy

Laser scanning microscopy (LSM) is a common technique for high resolution fluorescent imaging. Here we describe a fast algorithm for non‐negative deconvolution and apply it to readout of LSM detector photocurrents. By broadening photon impulses and deconvolving sampled photocurrent, effective quantum efficiency of the imaging system is increased. Using simulation and imaging with a custom‐built two‐photon microscope, we demonstrate improved fidelity of images acquired at short dwell times over a wide range of photon rates. Images formed show increased correlation‐to‐sample equivalent to a 25% increase in photon rate, lower noise, and reduced bleed‐through compared to conventional image generation. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The Covalent Trimethoprim Chemical Tag Facilitates Single Molecule Imaging with Organic Fluorophores

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.     

The Covalent Trimethoprim Chemical Tag Facilitates Single Molecule Imaging with Organic Fluorophores

Chemical tags can be used to selectively label proteins with fluorophores that have high photon outputs. By permitting straightforward single molecule (SM) detection and imaging with organic fluorophores, chemical tags have the potential to advance SM imaging as a routine experimental tool for studying biological mechanism. However, there has been little characterization of the photophysical consequences of using chemical tags with organic fluorophores. Here, we examine the effect the covalent trimethoprim chemical tag (A-TMP-tag) has on the SM imaging performance of the fluorophores, Atto655 and Alexa647, by evaluating the photophysical properties of these fluorophores and their A-TMP-tag conjugates. We measure SM photon flux, survival lifetime, and total photon output under conditions that mimic the live cell environment and demonstrate that the A-TMP-tag complements the advantageous SM imaging properties of Atto655 and Alexa647. We also measure the ensemble properties of quantum yield and photostability lifetime, revealing a correlation between SM and ensemble properties. Taken together, these findings establish a systematic method for evaluating the impact chemical tags have on fluorophores for SM imaging and demonstrate that the A-TMP-tag with Atto655 and Alexa647 are promising reagents for biological imaging.     

Forced Detection Monte Carlo Algorithms for Accelerated Blood Vessel Image Simulations

Two forced detection (FD) variance reduction Monte Carlo algorithms for image simulations of tissue‐embedded objects with matched refractive index are presented. The principle of the algorithms is to force a fraction of the photon weight to the detector at each and every scattering event. The fractional weight is given by the probability for the photon to reach the detector without further interactions. Two imaging setups are applied to a tissue model including blood vessels, where the FD algorithms produce identical results as traditional brute force simulations, while being accelerated with two orders of magnitude. Extending the methods to include refraction mismatches is discussed. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

光合作用-蒸腾作用-气孔导度的耦合模型及C_3植物叶片对环境因子的生理响应(英文)

以叶片的气体传输过程为基础,将蒸腾作用包括在以往光合作用气孔导度的耦合模型中,建立了光合作用蒸腾作用气孔导度的耦合模型。该模型可以模拟边界层导度对生理过程的影响。模拟了Ｃ３植物叶片对环境因子,如光照、温度、湿度、边界层导度和ＣＯ２浓度等的生理响应（光合作用、蒸腾作用、气孔导度）以及Ｃｉ和水分利用效率的变化。在环境因子变化于较大范围的情况下,模拟结果符合许多实验结论。     

皮肤组织模型中吸收和散射系数对单线态氧发光特性的影响

构建由脂肪乳剂和墨水组成的皮肤组织模型,定量研究皮肤组织模型的吸收系数μa和散射系数μs对光敏化单线态氧(singlet oxygen,~1O_2)发光特性的影响。利用~1O_2发光检测系统测量含光敏剂四硫磺基酞菁氯化铝的皮肤组织模型分别在中心波长为1 230,1 270和1 310 nm处的时间分辨发光光谱,对扣除背景信号后的时间分辨~1O_2发光光谱进行积分和拟合,获得~1O_2发光积分强度以及激发三重态寿命τ_T和~1O_2寿命τ_D。结果表明在皮肤组织模型中,~1O_2发光积分强度随着μ_a和μ_s的增大而减小,μ_a对τ_T和τ_D没有影响。τ_T随着μs的增加而增加,τ_D随μ_s的增加先骤降而后缓慢上升。当μ_a1.5 mm~(-1)和μ_s32 cm~(-1)时,~1O_2发光积分强度和τ_T、τ_D均趋于稳定,其中τ_T和τ_D分别为3.4±0.6μs和3.3±0.7μs。     

Simulation of the Physiological Responses of C3 Plant Leaves to Environmental Factors by a Model Which Combines Stomatal Conductance,Photosynthesis and Transpiration

Transpiration element is included in the integrated stomatal conductance-photosynthesis model by considering gaseous transfer processes, so the present model is capable to simulate the influence of boundary layer conductance. Leuning in his revised Ball' s model replaced relative humidity with VPDs(the vapor pressure deficit from stomatal pore to leaf surface) and thereby made the relation with transpiration more straightforward, and made it possible for the regulation of transpiration and the influence of boundary layer conductance to be integrated into the combined model. If the differences in water vapor and CO2 concentration between leaf and ambient air are considered, VPDs, the evaporative demand, is influenced by stomatal and boundary layer conductance. The physiological responses of photosynthesis, transpiration, and stomatal function, and the changes of intercellular CO2 and water use efficiency to environmental factors, such as wind speed, photon flux density, leaf temperature and ambient CO2, are analyzed. It is shown that ff the boundary layer conductance drops to a level comparable with stomatal conductance, the results of simulation by the model presented here differ significantly from those by the previous model, and, in some cases, are more realistic than the latter.     

Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency

One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.     

A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples

A combination of a two-dimensional photon detector (double-microchannel plate) with single-photon sensitivity and an optical projection system that allows space-resolved quantitation of luminescent emissions from spatially extended objects is described. A "luminescent image" of the object focused onto the detector is accumulated over a preset time and stored in a digital frame memory from which photon counts over areas of interest can be read. In this study, the object consisted of a microtiter plate containing luminescent samples which was placed below a projecting lens (2.0/21 mm, 36 X 24-mm format camera lens) at a distance of 38.5 cm. Although geometry substantially limited photon collection, the sensitivity achieved was only 10X less than that obtained with a dedicated photon-counting luminometer. A slightly diminished photon collection from peripheral wells was apparently caused by the projection system and could be corrected arithmetically. Both chemically generated luminescence (ATP bioluminescence) and cell-derived, superoxide-dependent luminescence (with lucigenin as chemilumigenic probe) were detected with excellent spatial resolution and linearity of response over a wide range.     

Monte Carlo kilovoltage X-ray tube simulation: A statistical analysis and compact simulation method

IntroductionMonte Carlo (MC) simulations are a powerful tool for improving image quality in X-ray based imaging modalities. An accurate X-ray source model is essential to MC modeling for CBCT but can be difficult to implement on a GPU while maintaining efficiency and memory limitations. A statistical analysis of the photon distribution from a MC X-ray tube simulation is conducted in hopes of building a compact source model.Materials & methodsMC simulations of an X-ray tube were carried out using BEAMnrc. The resulting photons were sorted into four categories: primary, scatter, off-focal radiation (OFR), and both (scatter and OFR). A statistical analysis of the photon components (energy, position, direction) was completed. A novel method for a compact (memory efficient) representation of the PHSP data was implemented and tested using different statistical based linear transformations (PCA, ZCA, ICA), as well as a geometrical transformation.ResultsThe statistical analysis showed all photon groupings had strong correlations between position and direction, with the largest correlation in the primary data. The novel method was successful in compactly representing the primary (error < 2%) and scatter (error < 6%) photon groupings by reducing the component correlations.Discussion & conclusionStatistical linear transforms provide a method of reducing the memory required to accurately simulate an X-ray source in a GPU MC system. If all photon types are required, the proposed method reduces the memory requirements by 3.8 times. When only primary and scatter data is needed, the memory requirement is reduced from gigabytes to kilobytes.     

Portable Optical Epidural Needle-A CMOS-Based System Solution and Its Circuit Design

Epidural anesthesia is a common anesthesia method yet up to 10% of procedures fail to provide adequate analgesia. This is usually due to misinterpreting the tactile information derived from the advancing needle through the complex tissue planes. Incorrect placement also can cause dural puncture and neural injury. We developed an optic system capable of reliably identifying tissue planes surrounding the epidural space. However the new technology was too large and cumbersome for practical clinical use. We present a miniaturized version of our optic system using chip technology (first generation CMOS-based system) for logic functions. The new system was connected to an alarm that was triggered once the optic properties of the epidural were identified. The aims of this study were to test our miniaturized system in a porcine model and describe the technology to build this new clinical tool. Our system was tested in a porcine model and identified the epidural space in the lumbar, low and high thoracic regions of the spine. The new technology identified the epidural space in all but 1 of 46 attempts. Experimental results from our fabricated integrated circuit and animal study show the new tool has future clinical potential.