FLIM and FCS detection in laser‐scanning microscopes: Increased efficiency by GaAsP hybrid detectors

Photon counting detectors currently used in fluorescence lifetime microscopy have a number of deficiencies that result in less‐than‐ideal signal‐to‐noise ratio of the lifetimes obtained: Either the quantum efficiency is unsatisfactory or the active area is too small, and afterpulsing or tails in the temporal response contribute to overall timing inaccuracy. We have therefore developed a new FLIM detector based on a GaAsP hybrid photomultiplier. Compared with conventional PMTs and SPADs, GaAsP hybrid detectors have a number of advantages: The detection quantum efficiency reaches or surpasses the efficiency of fast SPADs, and the active area is on the order of 5 mm², compared with 2.5 10^?3 mm² for a SPAD. The TCSPC response is clean, without the bumps and the diffusion tails typical for PMTs and SPADs. Most important, the hybrid detector is intrinsically free of afterpulsing. FLIM results are therefore free of signal‐dependent background, and FCS curves are free of the known afterpulsing peak. We demonstrate the performance of the new detector for multiphoton NDD FLIM and for FCS. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Laser‐made 3D Auxetic Metamaterial Scaffolds for Tissue Engineering Applications

Exploiting the unique properties of three‐dimensional (3D) auxetic scaffolds in tissue engineering and regenerative medicine applications provides new impetus to these fields. Herein, the results on the fabrication and characterization of 3D auxetic scaffolds for tissue engineering applications are presented. The scaffolds are based on the well‐known re‐entrant hexagonal geometry (bowtie) and they are fabricated by multiphoton lithography using the organic?inorganic photopolymer SZ2080. In situ scanning electron microscopy–microindentations and nanoindention experiments are employed to characterize the photocurable resin SZ2080 and the scaffolds fabricated with it. Despite SZ2080 being a stiff material with a positive Poisson’s ratio, the scaffolds exhibit a negative Poisson’s ratio and high elasticity due to their architecture. Next, mouse fibroblasts are used to seed the scaffolds, showing that they can readily penetrate them and proliferate in them, adapting the scaffold shape to suit the cells’ requirements. Moreover, the scaffold architecture provides the cells with a predilection to specific directions, an imperative parameter for regenerative medicine in many cell‐based applications. This research paves the way for the utility of 3D auxetic metamaterials as the next‐generation adaptable scaffolds for tissue engineering.     

对氯苯酚的共振增强多光子电离研究

在自行研制的具有恒温加热进样系统的激光质谱仪上实验研究了气相对氯苯酚分子的共振增强多光子电离飞行时间质谱(REMPI-TOFMS),获得了在275～285 nm激发波段的母体离子和主要碎片离子(C6H5O ,C5H5Cl C6H5 ,C5H 5,C4H 3,C3H 3)的(I I)REMPI激发光谱.通过质谱光谱分析,结合分子在中间激发态的能级和结构特征,对主要碎片离子的形成机理进行了研究.结果显示:各个碎片离子与对氯苯酚的母体离子具有相似的光激发谱,并且各个谱峰与S,1态的振动能级是一一对应的.说明各个碎片离子和母体离子的产生经历了相同的共振中间态.分子先吸收一个光子到达中间共振态,然后再吸收一个光子到达电离态电离.根据碎片离子光强指数的阶梯形分布和分子在S,1态的结构特点.判断各个碎片离子经历了母体离子的阶梯式离解,并给出了主要碎片离子的离解通道.     

262～272 nm波段氯苯的激光质谱研究

在自行研制的具有恒温加热进样系统的激光质谱仪上获得了262～272 nm波段气相氯苯的(1 1)共振增强多光子电离/飞行时间质谱(REMPI-TOFMS).研究表明,该波长范围内获得的光激发谱的光谱结构是氯苯第一激发态1B2(S1)振动能级的反映,对其中14条谱线进行了尝试性指认.分析了氯苯在266 nm波长处不同激光能量下的光电离质谱特点,讨论了造成77C4H5 质谱的非对称增宽的原因.实验研究了266 nm波长处氯苯分子离子信号强度与样品浓度之间的关系,估算出氯苯的探测限为ng/L量级.     

激光光解NO2产物的NO分子之离子谱研究

利用Nd：YAG激光器三倍频，输出波长为λ=355nm的激光光解NO2分子所产生的NO分子，通过共振增强多光子电离及飞行时间质谱技术，获得了振转态分辨的NO(X^2Ⅱ，v“,J“)离子谱NOγ(0，0)，γ(1，0)，γ(1，1)。NO分子的离子信号强度与UV电离激光能量(λ≈226nm)之间的关系能用二次方曲线很好拟合，它表明光解产物NO分子是通过(1 1)多光子吸收过程而被电离的。上述结果对NO2分子光解动力学及其产物NO的电离动力学提供了十分有益的信息。     

半导体量子点材料在Nd:YAG激光辐照下的非线性光学效应

使用10 n,脉冲调Q Nd:YAG激光器Z-scan技术测量了化学合成的无掺杂硫化锌量子点(QDs)以及掺Mn2+硫化锌量子点(QDs)的非线性光学特性,并使用透射电镜技术(TEM)以及X射线衍射法(XRD)表征合成材料的纳米结构.在室温下,分别利用UV-VIS分光光度计和分光荧光计测量了人工合成QDs胶体溶液的线性光学吸收特性以及光致发光的发射特性.样品的吸收特性表明,由于量子限制效应的影响,样品的截止吸收低于硫化锌的截止吸收.样品的光致发光特性显示,掺Mn2+的硫化锌样品显示出明显的光致发光现象,发射峰大约在580 nm;而无掺杂的硫化锌样品在紫外区辐射,发射峰大约在365 nm.对样品的UV-VIS吸收特性分析和TEMXRD分析表明,硫化锌样品的平均粒度(半径)大约为1.2 nm.分析开放光圈(OA) Z-scan技术得到的实验数据,发现在1 064 nm处两种试验样品都会发生四光子吸收(FPA)现象.拟合实验数据得到了两种试验样品的FPA系数以及FPA横截面,结果表明,ZnS QD的FPA横截面的计算值是4.9×1-106cm8·s3·photon-3,比硫化锌的FPA横截面大了5个数量级,而且人工合成的ZnS QD也有光学限制的性质.掺Mn2+离子的样品具有大的FPA横截面和在可见光区有高的发光效率这两个特点,使得该材料适合用于多光子荧光成像.     

Multiphoton Bleaching of Red Fluorescent Proteins and the Ways to Reduce It

Red fluorescent proteins and biosensors built upon them are potentially beneficial for two-photon laser microscopy (TPLM) because they can image deeper layers of tissue, compared to green fluorescent proteins. However, some publications report on their very fast photobleaching, especially upon excitation at 750–800 nm. Here we study the multiphoton bleaching properties of mCherry, mPlum, tdTomato, and jREX-GECO1, measuring power dependences of photobleaching rates K at different excitation wavelengths across the whole two-photon absorption spectrum. Although all these proteins contain the chromophore with the same chemical structure, the mechanisms of their multiphoton bleaching are different. The number of photons required to initiate a photochemical reaction varies, depending on wavelength and power, from 2 (all four proteins) to 3 (jREX-GECO1) to 4 (mCherry, mPlum, tdTomato), and even up to 8 (tdTomato). We found that at sufficiently low excitation power P, the rate K often follows a quadratic power dependence, that turns into higher order dependence (K~P^α with α > 2) when the power surpasses a particular threshold P*. An optimum intensity for TPLM is close to the P*, because it provides the highest signal-to-background ratio and any further reduction of laser intensity would not improve the fluorescence/bleaching rate ratio. Additionally, one should avoid using wavelengths shorter than a particular threshold to avoid fast bleaching due to multiphoton ionization.     

飞行时间质谱法研究Si（CH3）4分子的多光子解离和电离过程

采用超声分子束技术,以飞行时间（TOF）质谱仪,于410－371nm内,在不同能量的激光作用下,着重检测了气相Si(CH3)4分子在15个波长点处的多光子电离（MPI）TOF质谱分布。根据实验结果,讨论了Si(CH3)4可能的MPI机理,得到了Si^ 主要来自于母体分子及中性碎片的多光子解离－硅原子的共振电离、Si(CH3)n^ (n=1,2,3）主要来自于中性碎片Si(CH3)n(n=1,2,3）的自电离、而Si(CH3)4^ 则来自于母体分子的（3＋1）电离的结论。     

飞秒激光在材料微加工中的应用

本文介绍了飞秒激光的特性以及超短脉冲激光与材料相互作用的机理，并着重指出了其与长脉冲激光的区别。飞秒激光可产生超高光强、具有精确的损伤阈值(并且损伤阈值较低较低)、很小的热影响区、几乎可精密加工所有种类材料，并且，加工精度极高，可进行亚微米尺寸的精密加工。通过分析飞秒激光材料微加工的特性，综述超短脉冲激光材料微加工的应用研究现状。