Multiphoton imaging with a direct‐diode pumped femtosecond Ti:sapphire laser

A direct‐diode pumped Ti:sapphire femtosecond oscillator is used to perform multiphoton imaging for the first time.     

Sequential multitrack nonlinear ex vivo imaging of esophageal stroma based on backscattered second-harmonic generation and two-photon autofluorescence

We demonstrate the technique of subsequent multitrack nonlinear imaging based on backscattered second-harmonic generation (B-SHG) and two-photon autofluorescence (TPA) to obtain large-area, high-contrast, submicron-resolution image ex vivo of esophageal stroma. Our findings show that this technique is effective in improving the B-SHG/TPA image contrast. It was found that the method can quantitatively obtain microscopic structural and biochemical information on stroma. Our work suggests that the technique has the potential to provide accurate and comprehensive information in determining the physiological and pathological states of the esophagus.     

基于LD波长调制的环境氧气浓度实时检测技术

氧气在工业生产、环境检测、医疗卫生以及人们的日常生活中都起着非常重要的作用,国内外对高效灵敏的氧气检测手段迫切需求。为了克服传统氧气测量方法的弊端,通过改变温度和电流对激光二极管(LD)发射波长进行调节,利用氧分子在波长760nm附近的A带吸收峰,以实现对实际环境中氧气浓度的快速测量,并拥有较好的精度和线性度。与传统的基于LD调制的氧气浓度测量技术不同,该方法具有测量直接、装置简单等特点,并且适合于测量各种环境下的氧气浓度。     

Morphological alterations of radicular dentine pretreated with different irrigating solutions and irradiated with 980-nm diode laser

Background: The topographical features of intraradicular dentine pretreated with sodium hypochlorite (NaOCl) or ethylenediamine tetraacetic acid (EDTA) followed by diode laser irradiation have not yet been determined. Purpose: To evaluate the alterations of dentine irradiated with 980‐nm diode laser at different parameters after the surface treatment with NaOCl and EDTA. Study design: Roots of 60 canines were biomechanically prepared and irrigated with NaOCl or EDTA. Groups were divided according to the laser parameters: 1.5 W/CW; 1.5 W/100 Hz; 3.0 W/CW; 3.0 W/100 Hz and no irradiation (control). The roots were splited longitudinally and analyzed by scanning electron microscopy (SEM) in a quali‐quatitative way. The scores were submitted to two‐way Kruskal–Wallis and Dunn's tests. Results: The statistical analysis demonstrated that the specimens treated only with NaOCl or EDTA (control groups) were statistically different (P < 0.05) from the laser‐irradiated specimens, regardless of the parameter setting. The specimens treated with NaOCl showed a laser‐modified surface with smear layer, fissures, and no visible tubules. Those treated with EDTA and irradiated by laser presented absence of smear layer, tubules partially exposed and melting areas. Conclusions: The tested parameters of 980‐nm diode laser promoted similar alterations on dentine morphology, dependent to the type of surface pretreatment. Microsc. Res. Tech., 2009. © 2008 Wiley‐Liss, Inc.     

成像标定法测量半导体激光器慢轴远场分布

利用远场成像及标定对比方法,建立了半导体激光器慢轴远场分布的CCD测量系统。介绍了测量的原理和测量系统的组成,并给出了半导体激光器慢轴远场分布的测量结果。实验结果表明,该方法实时性好,测量快速、准确,人为误差因素少,适合仪器化。     

Compact multiphoton/single photon laser scanning microscope for spectral imaging and fluorescence lifetime imaging

An inverted fluorescence microscope was upgraded into a compact three-dimensional laser scanning microscope (LSM) of 65 x 62 x 48 cm dimensions by means of a fast kHz galvoscanner unit, a piezodriven z-stage, and a picosecond (ps) 50 MHz laser diode at 405 nm. In addition, compact turn-key near infrared femtosecond lasers have been employed to perform multiphoton fluorescence and second harmonic generation (SHG) microscopy. To expand the features of the compact LSM, a time-correlated single photon counting unit as well as a Sagnac interferometer have been added to realize fluorescence lifetime imaging (FLIM) and spectral imaging. Using this unique five-dimensional microscope, TauMap, single-photon excited (SPE), and two-photon excited (TPE) cellular fluorescence as well as intratissue autofluorescence of water plant leaves have been investigated with submicron spatial resolution, <270 ps temporal resolution, and 10 nm spectral resolution.     

CO2激光辐照氧化矾热像仪的实验

为了研究波段内激光辐照焦平面阵列热像仪时的性能表现.设计了一系列视场内辐照实验.理论上采用了平顶高斯函数和有限个复高斯函数之和模拟激光器输出的平顶型光束以及激光器输出镜和光路中各种光学元件构成的光阑,结合近轴光学系统的柯斯林光束衍射公式计算了激光光束的传递以及变换.在实验中,通过在光路中放置单透镜光学元件以及调节该元件...     

基于新型模糊PID控制单元的LD精密温控研究

为改善温度波动对光通信用半导体激光器性能的影响,设计了基于三维语言变量的高精度跟踪误差温度控制系统。为减小系统成本,利用运算放大器AD620和OP07等器件设计了温度采集系统,并采用最小二乘法拟合温度数据,从而建立温度—电阻关系模型,预测温度变化趋势;加入第三维模糊语言变量,结合窄域论以适当压缩E、EC、ECC的论域,采用模糊规则设定方法,建立新型三维模糊PID规则表并求解得出模糊查询表。结果表明:当预设温度为25℃时,温控系统超调量为0.97℃,最大下冲量出现在第17s,其值为0.69℃;工作51s后,LD系统进入稳定状态,温度保持为25±0.05℃;在第150~210s内,其温度值标准差为0.020 4℃。同时,该系统实现了对半导体激光器0~75℃的大范围精密温控,温控精度为±0.05℃。该系统能够实现对半导体的高效制冷、加热控制,具有响应时间快和系统开销小的优势,能对控制参数实现自整定。     

Second harmonic generation imaging of the deep shade plant Selaginella erythropus using multifunctional two‐photon laser scanning microscopy

Background : Multifunctional two‐photon laser scanning microscopy provides attractive advantages over conventional two‐photon laser scanning microscopy. For the first time, simultaneous measurement of the second harmonic generation (SHG) signals in the forward and backward directions and two photon excitation fluorescence were achieved from the deep shade plant Selaginella erythropus. Results : These measurements show that the S. erythropus leaves produce high SHG signals in both directions and the SHG signals strongly depend on the laser's status of polarization and the orientation of the dipole moment in the molecules that interact with the laser light. The novelty of this work is (1) uncovering the unusual structure of S. erythropus leaves, including diverse chloroplasts, various cell types and micromophology, which are consistent with observations from general electron microscopy; and (2) using the multifunctional two‐photon laser scanning microscopy by combining three platforms of laser scanning microscopy, fluorescence microscopy, harmonic generation microscopy and polarizing microscopy for detecting the SHG signals in the forward and backward directions, as well as two photon excitation fluorescence. Conclusions : With the multifunctional two‐photon laser scanning microscopy, one can use noninvasive SHG imaging to reveal the true architecture of the sample, without photodamage or photobleaching, by utilizing the fact that the SHG is known to leave no energy deposition on the interacting matter because of the SHG virtual energy conservation characteristic.     

基于半导体激光器的乙炔气体光声光谱检测及其定量分析

油中溶解乙炔是变压器等油浸式电气设备早期放电性故障的重要特征气体。基于半导体激光器的光声光谱传感技术具有灵敏度高,选择性好等优点,能很好地应用于微弱气体检测中。论文构建了基于分布反馈半导体激光器光声光谱检测装置,并分析了光声池的特性参数;实验研究了光声信号与激光功率、乙炔气体浓度之间的关系;并借助激光器的波长调制特性,研究了乙炔分子在近红外区第一泛音带1.5μm附近的光声光谱;提出了一种基于最小二乘回归的光声光谱定量分析方法。理论和实验结果为乙炔的光声光谱在线检测及高灵敏度可调谐光声光谱仪的设计提供了参考。     

一种低电压高速激光二极管驱动器

介绍了一种低电压高速激光二极管驱动器 ,可以 +3.3V单电源供电 ,其特殊的高速电流源结构使其满足激光二极管阈值电流的温度特性 ,并可以 62 2 Mbps驱动激光二极管。     

Simultaneous measurement of CO and CO2 at elevated temperatures by diode laser wavelength modulated spectroscopy

One single semiconductor distributed-feedback (DFB) laser is used to demonstrate the possibility of simultaneous measurements of CO and CO₂ at elevated temperatures. Wavelength modulation spectroscopy with second-harmonic detection is used to improve the detection sensitivity and accuracy. The concentrations of CO and CO₂ are determined from the WMS-1-normalized absorption-based WMS-2f signal peak heights of a proper line pair of CO and CO₂ near 6357.814 cm⁻¹ and 6357.312 cm⁻¹, which are selected using some line-selection criterions for the target temperature range of 300–1000 K. The CO and CO₂ concentrations measurements are within 2.86% and 2.69% of the expected values over the tested temperature range 300–1000 K. The minimum detectable concentrations of CO and CO₂ at 1000 K are 250 ppm m and 280 ppm m respectively.     

Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence

The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.     

低噪声半导体激光器驱动电源的研制

根据半导体激光器的工作特性,研制了一台低噪声、高稳定度半导体激光器的驱动电流源。该电流源特点具有慢启动电路和过流保护电路,提高了半导体激光器抗冲击能力和工作稳定性。     

Single- and two-photon spectral imaging of intrinsic fluorescence of transformed human hepatocytes

Autofluorescence (AF) originating from the cytoplasmic region of mammalian cells has been thoroughly investigated; however, AF from plasma membranes of viable intact cells is less well known, and has been mentioned only in a few older publications. Herein, we report results describing single- and two-photon spectral properties of a strong yellowish-green AF confined to the plasma-membrane region of transformed human hepatocytes (HepG2) grown in vitro as small three-dimensional aggregates or as monolayers. The excitation-emission characteristics of the membrane AF indicate that it may originate from a flavin derivative. Furthermore, the AF was closely associated with the plasma membranes of HepG2 cells, and its presence and intensity were dependent on cell metabolic state, membrane integrity and presence of reducing agents. This AF could be detected both in live intact cells and in formaldehyde-fixed cells.     

基于期望块对数似然的激光吸收光谱层析成像

可调谐二极管激光吸收光谱层析成像(TDLAST)是一种重要的非侵入式燃烧检测技术。然而,TDLAST逆问题的欠定性使得传统算法重建的气体吸收密度存在较大误差。本文将期望块对数似然先验引入TDLAST逆问题的求解,提出基于高斯混合模型(GMM)正则化的温度重建算法(GMMTRT)。该方法利用GMM建模燃烧场气体吸收密度的局部分布特性,采用半二次分裂方法求解引入GMM正则化的TDLAST逆问题。利用火灾动力学模拟器生成的仿真数据与利用TDLAST实验系统获得的实际数据进行的重建实验均表明,GMMTRT重建的温度图像能够准确定位火焰位置,并清晰描述燃烧场感兴趣区域的温度分布。与现有的基于Tikhonov正则化的温度重建算法和同时代数重建算法相比,GMMTRT的重建误差分别能够降低15.42%～36.16%和23.10%～44.79%。     

Ultrastructure and reproduction behaviour of single CHO-K1 cells exposed to near infrared femtosecond laser pulses

In the present work, the authors investigated ultrastructural changes as well as the reproduction behaviour of preselected single CHO-K1 cells exposed to 170 femtosecond laser pulses at different power output levels in comparison with cells outside the illumination volume. The ultrashort laser pulses were provided by an 80 MHz Ti:sapphire laser at 780 nm. The cells were scanned ten times with a scan rate of 1/16 s(-1). Single CHO-K1 cells exposed to low mean power of 2 mW revealed no significant changes in ultrastructure after laser exposure. In some cases, changes of mitochondria with slight disordering of cristae were found. Cytoplasm was filled with vesicles that seemed to be released from Golgi stacks. Cells irradiated with higher powers demonstrated more dramatic changes in ultrastructure. A considerable number of swollen mitochondria in conjunction with loss of cristae was observed. The main event of mitochondrial changes was the formation of electron dense bodies in the mitochondrial matrix. In addition, lumen of endoplasmatic reticulum was enlarged. Highest applied mean laser power of 12.5 mW lead to complete destruction of mitochondria and their transformation to electron dense structures containing membrane material. Compared with cell targets irradiated with 2 mW mean power, the release of vesicles from Golgi stacks seemed to be rather moderate. Cells localised outside the laser beam revealed no ultrastructural changes. Low mean laser power at 2 mW was unable to impair the reproduction behaviour of CHO-K1 cells. At higher laser power output levels, CHO-K1 cells started to delay cell division. At 12.5 mW, no cell division occurred. The obtained results may be helpful in recommending parameters for safe femtosecond laser microscopy of living specimens.     

Measurement of two‐photon‐absorption spectra through nonlinear fluorescence produced by a line‐shaped excitation beam

We propose an innovative experimental approach to estimate the two‐photon absorption (TPA) spectrum of a fluorescent material. Our method develops the standard indirect fluorescence‐based method for the TPA measurement by employing a line‐shaped excitation beam, generating a line‐shaped fluorescence emission. Such a configuration, which requires a relatively high amount of optical power, permits to have a greatly increased fluorescence signal, thus avoiding the photon counterdetection devices usually used in these measurements, and allowing to employ detectors such as charge‐coupled device (CCD) cameras. The method is finally tested on a fluorescent isothiocyanate sample, whose TPA spectrum, which is measured with the proposed technique, is compared with the TPA spectra reported in the literature, confirming the validity of our experimental approach.     

Noninvasive 3D vital imaging and characterization of notochordal cells of the intervertebral disc by femtosecond near-infrared two-photon laser scanning microscopy and spatial-volume rendering

The central region of the intervertebral disc (IVD) in infant humans is made and maintained by notochordal cells (NCs). These cells disappear during maturation to be replaced by mature chondrocyte-like cells. NCs are completely different morphologically from the mature chondrocyte-like IVD cells and have complex and essential functions but little is known about them. Recently, two-photon laser scanning microscopy (TPLSM) using near-infrared (NIR) femtosecond pulsed lasers has emerged as a promising noninvasive optical technique for observing unfixed living 3D biological specimens in situ and in vitro. Several lines of evidence suggest that compared with conventional laser scanning confocal microscopy (LSCM), femtosecond NIR laser-based TPLSM has any number of advantages including 3D resolution without a spatial filter (confocal pinhole), minimal photobleaching, and photodamage above and below the focal plane, and importantly, greater depth penetration. We have thus taken advantage of these unique features of femtosecond laser-based TPLSM for vital 3D imaging in conjunction with advanced spatial-volume rendering modalities to compare morphologies of NCs/clusters from pig caudal discs with chondrocyte-like IVD cells from bovine caudal discs, both in ex vivo tissue and when isolated and grown in vitro within 3D alginate scaffolds. Our results provide evidence that (a) ex vivo notochordal tissue consists of areas with NC clusters, and those dominated by tubular structures of low cell density (b) within 3D in vitro scaffolds the morphology of NC is heterogeneous and the cells contain distinct cytoplasmic vacuole-like structures occasionally including acidic subinclusions (c) a quantitative determination based on 3D spatial and volumetric-rendering reveals an average NC diameter of 22.05 microm (range 11.96-46.63 microm) and NC volume of 9701 microm(3) (2041-36427 microm(3)) whereas chondrocyte-like cells have a mean volume of 3279 microm(3) and diameter of 12.20 microm. Taken together, this study demonstrates that femtosecond TPLSM has unique advantages over other conventional histological and in particular LSCM for high resolution noninvasive vital characterization of notochordal and chondrocyte-like cells of IVD over extended depths beyond 300-500 microm.     

Two-photon fluorescence and second-harmonic generation imaging of collagen in human tissue based on multiphoton microscopy

Multiphoton microscopic imaging of collagen plays an important role in noninvasive diagnoses of human tissue. In this study, two-photon fluorescence and second-harmonic generation (SHG) imaging of collagen in human skin dermis and submucosa of colon and stomach tissues were investigated based on multiphoton microscopy (MPM). Our results show that multiphoton microscopic image of collagen bundles exhibits apparently different pattern in human tissues. The collagen bundles can simultaneously reveal its SHG and two-photon excited fluorescence images in the submucosa of colon and stomach, whereas it solely emit SHG signal in skin dermis. The intensity spectral information from tissues further demonstrated the above results. This indicates that collagen bundles have completely different space arrangement in these tissues. Our experimental results bring more detailed information of collagen for the application of MPM in human noninvasive imaging.