亚心形扁藻傅里叶变换近红外拉曼光谱

测定并研究了亚心形扁藻的傅里叶变换近红外拉曼光谱。结果表明,采用1 064 nm近红外激光激发,以亚心形扁藻藻液离心所获得的藻泥为测试样品,当激发光功率为360 mW,累加70次,可以获得较清晰的亚心形扁藻近红外拉曼光谱信号。其主要谱峰为：381～432, 552～556, 611～613, 710, 873, 953～964, 1 108～1 119, 1 457, 1 523～1 527, 2 986 cm-1,确认其归属主要为蛋白质、不饱和脂肪酸以及酯类等亚心形扁藻的主要组成物质。该研究为藻类分类以及环境胁迫下藻类的生理生化变化提供一种快速、实时、无损的检测方法。     

双光子阵列点激发同时多维荧光信息的处理

五维同时荧光信息显微成像方法是一种新的荧光信息获取技术，它采用了双光子阵列点激发方式.这一方法可同时获取激发阵列点每点荧光的位置信息、荧光光谱信息和荧光寿命信息，弥补了现有荧光检测技术的不同功能信息不具有同时性的缺陷.给出了从这种技术的复合信息中提取复合光谱几何强度结构图像、不同光谱几何强度结构图像、不同光谱寿命图像的方法.提出了一种激发荧光强度修正系数矩阵方法，消除阵列点激发光强不均匀对激发荧光强弱产生的不利影响，取得明显效果.实验对实际样品做了数据采集和处理，给出图像结果，表明处理的效果良好.对存在的问题也作了讨论. 关键词： 荧光信息处理 双光子 荧光光谱 荧光寿命     

Monte Carlo方法研究支气管组织自体荧光光谱

在实验获得支气管组织显微荧光特性和有关光学特性参量的基础上,将支气管组织简化为三层光学模型,用Monte Carlo方法模拟442nm He-Cd激光在组织模型中的光分布及自体荧光的逃逸函数,计算出各组织层对总体自体荧光的贡献,并模拟出逃逸到组织体表面的自体荧光光谱.进一步实验测量与理论计算的自体荧光光谱比较表明,两者在460～520nm及600～700nm的光谱范围内符合得较好.     

DHL细胞中5-ALA代谢PpIX的双光子荧光光谱

双光子激发生物组织荧光,激发光仅作用于焦点区域,对生物样品的光漂白性和光毒性都很小,因而双光子荧光显微技术已成为细胞生物学研究的一种新技术。文章采用波长为820 nm飞秒激光激发孵育有5-ALA的DHL细胞,在激光扫描显微镜的Lambda模式中获得单个DHL细胞的双光子荧光光谱,并测量DHL细胞内积聚的卟啉九(PpIX)特征荧光值。获得了浓度分别为2, 4和10 mmol·L-1的5-ALA溶液中,细胞代谢的PpIX含量随孵育时间的变化情况。DHL细胞内积聚的PpIX处于动态变化过程,并呈现出两阶段性的特点：细胞内积聚的PpIX含量随着孵育时间增长而增加,在3 h附近达到最大值,随后随着孵育时间增长反而下降。结果表明,基于激光扫描显微的双光子荧光光谱可成为DHL细胞等白血病细胞摄取5-ALA并生成PpIX的动力学研究的有效方法。     

激光偏振性对亚心形扁藻生物效应的影响

实验研究了偏振与非偏振激光对亚心形扁藻的促长作用、叶绿素含量以及色素粗提物吸收光谱等生物学效应的影响.采用488 nm,70 mW输出功率的氩激光,辐照哑心形扁藻,隔天取样进行细胞计数,培养至第6天提取叶绿素,测量叶绿素含量并扫描叶绿素的吸收光谱.实验结果表明,偏振激光与非偏振激光辐照对藻细胞密度、生长速率的促长作用区别不显著;偏振性与否对叶绿素含量的累积没有明显的区别;激光偏振性对色素粗提物的吸收光谱没有影响.激光偏振性与否均可对亚心形扁藻的生物效应产生有效的作用,激光偏振性与激光生物效应关系的研究有待于进一步深入.     

长时程双光子成像技术

双光子成像(Two-Photon Imaging)技术以其优越特性被广泛用于活细胞动态三维成像,但光功率极高的短脉冲光对焦平面荧光分子严重的光漂白极大地影响了双光子长时间成像的图像质量,针对双光子荧光漂白问题,本文提出一种优化光照的双光子(Optimized Lighting-Two Photon,OL-TP)成像技术。通过预扫描获取双光子图像分析高低阈值,以预设的高低阈值为标准优化一幅图像中不同区域的光照时长,利用扫描过程中记录的荧光信息和光照时间信息可以重建OL-TP图像,既保证信噪比又降低荧光漂白。重建的OL-TP图像与传统双光子图像基本一致,信噪比略有降低,但图像并未失真。对110 nm的荧光小球样本分别连续取30幅普通双光子和优化光照的双光子图像,到第30幅图时,重建后的优化光照双光子图像比普通双光子图像荧光漂白降低了28.86%。OL-TP通过优化光照时间大幅降低双光子成像的荧光漂白,使双光子荧光显微镜能够更好地对生物样本进行长时间观测。     

基于自相位调制光谱选择驱动的无标记自发荧光多倍频显微镜系统

由飞秒激光驱动的非线性光学显微镜技术在医学组织成像中具有很多独特的优势,包括多种成像模态、高对比度、高分辨率和无标记的深层光学切片能力等.由于缺乏波长可灵活调谐的飞秒激发光源,导致在多模态成像的同时难以兼顾每种模态的对比度,从而制约了非线性光学显微镜在医学诊断中的广泛应用.本文采用基于自相位调制光谱选择的光纤激光光源,获得了中心波长在990—1110 nm范围内可调谐的高能量飞秒脉冲,并用于驱动非线性光学显微镜.采用990 nm的飞秒脉冲,通过双光子激发荧光和二倍频对胃组织成像,进一步结合图像拼接技术成功获得了胃组织的双模态大视场图像;利用1110 nm的飞秒脉冲,实现了无标记自发荧光多倍频显微镜技术,同时高效激发了胃组织的双光子激发荧光、三光子激发荧光、二倍频和三倍频信号,获得了胃组织的多模态图像.     

双光子激发荧光成像技术对小鼠植入前胚胎的实时观测

双光子激发的蓝移效应可使利用同一束超快激光同时激发多种不同荧光特性的生物荧光染料的设想得以实现。选取锁模飞秒掺钛蓝宝石(Ti-sapphire)激光器输出的730nm激光,分别激发Hoechst33342,Fluo-4,PI和Indo-1四种常用生物荧光染料,分别利用(455±15)nm,(540±15)nm,(580±16)nm和(500±15)nm四种滤光片获得特异性荧光图像。结合双光子激发荧光成像技术穿透深,光损伤小,信噪比好等优势,选取合适的荧光染料组,应用单束激光激发、双染双通道成像方法,对小鼠植入前胚胎内细胞中的钙信号和染色体进行三维、四维实时成像,为探究小鼠植入前胚胎发育规律提供一种全新的多参数观测手段。     

运动训练大鼠的自体荧光研究

首次通过激光诱导荧光光谱技术(LIF)研究运动训练大鼠心脏、肾脏、肝脏、脂肪以及前腿肌和后腿的比目鱼肌和腓肠肌的自体荧光光谱特性。测量所用的激发光波长为250～650 nm而发射波长为300～700 nm。比较参照组和3组不同运动状态组的三维荧光光谱,主要在腓肠肌的光谱中发现了和运动相关且位于激发波长(340±10)nm和发射波长(460±10)nm区域的特有荧光峰。根据这一荧光峰可以判别其对应的荧光物质是NADH(还原态烟酰胺腺嘌呤二核苷酸)。比较3组不同运动模式组的荧光光谱,发现运动模式与其峰强具有明显的相关性。研究结果表明,运动大鼠腓肠肌的能量代谢强于前腿、比目鱼肌及其他脏器,且NADH自体荧光光谱特性是判断肌肉代谢程度的有效的指标之一。     

CdTe量子点-铜酞菁复合体系荧光共振能量转移的研究

以波长为780 nm、重复频率为76 MHz、脉宽为130 fs的飞秒激光作为激发光源, 采用超快时间分辨光谱技术研究了CdTe量子点-铜酞菁复合体系的荧光共振能量转移. 实验结果表明, 在780 nm的双光子激发条件下, 复合体系中CdTe量子点的荧光寿命随着铜酞菁溶液浓度的增加而减少, 荧光共振能量转移效率增加. 同时也研究了激发功率对荧光共振能量转移效率的影响. 结果表明, 随着激发光功率的增加, 复合体系溶液中CdTe量子点的荧光寿命增加, 荧光共振能量转移效率减小, 其物理机理是因为高激发功率下的热效应和由双光子诱导的高阶激发态的跃迁. 当激发光功率为200 mW时, 双光子荧光共振能量转移效率为43.8%. 研究表明CdTe量子点-铜酞菁复合体系是非常有潜力的第三代光敏剂.     

Stationary spectroscopy of biotissues in vivo: Fluorescent studies of some pathological states

The stationary spectra of autofluorescence, along with the reflection coefficient at the wavelength of excitation, are measured in vivo for some stomach tissues in the case of different pathological states (dysplasia, superficial gastritis, and cancer) using a nitrogen laser as the source of excitation (λ_rad=337.1 nm). The fluorescence spectra obtained are decomposed into Gaussian-Lorentzian components. It is found that, in development of dysplasia and tumor processes, at least seven groups of fluorophores can be distinguished that form the entire emission spectrum. The ratio between the fluorescence intensities of flavins and NAD(P)H is determined and the degree of respiratory activity of cells estimated for the states considered. The quantum yields of fluorescence of the biotissues under investigation are estimated.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

超短光脉冲的平均功率和脉宽及锁模状态对双光子显微镜成象的影响

显微镜成象是研究生物活体细胞结构及活动规律的重要技术.飞秒光脉冲是多光子显微镜的主要光源,它的性能(脉宽、平均功率和锁模状态等)严重影响成象的质量.双光子激发荧光强度正比于超短脉冲平均功率的平方,反比于脉宽,锁模稳定状态严重影响成象的清晰度.对用Cameleon转染的HeLa细胞和GFP转基因水稻叶的实验研究结果与理论一致.     

Toluene laser-induced fluorescence for in-cylinder temperature imaging in internal combustion engines

A single-laser single-camera imaging technique was demonstrated for in-cylinder temperature distribution measurements in a direct-injection internal combustion engine. The single excitation wavelength two-color detection technique is based on toluene laser-induced fluorescence (LIF). Toluene-LIF emission spectra show a red-shift with increasing temperature. Temperature can thus be determined from the ratio of the signal measured in two separate wavelength ranges independent of the local tracer concentration, laser pulse energy, and the intensity distribution. An image doubling and filtering system is used for the simultaneous imaging of two wavelength ranges of toluene LIF onto the chip of a single camera upon excitation at 248 nm. The measurements were performed in a spark-ignition engine with homogeneous charge and yielded temperature images with a single-shot precision of approximately ±?6%.     

荧光光谱用于光动力疗法中光敏剂光漂白特性研究

应用荧光光谱技术研究溶液中血卟啉单甲醚(HMME)的光漂白与光产物生成。以532 nm倍频Nd∶YAG激光器照射样品,功率密度为100 mW·cm-2,以光学多通道分析仪(OMA)采集荧光光谱。照光过程与荧光光谱采集同步进行。通过构建基本光谱与最小二乘拟合,由单条实测光谱中分解求得HMME荧光(613 nm)、光产物荧光(639 nm)及自体荧光的强度。HMME初始浓度不超过10 μg·mL-1时符合荧光-浓度线性函数关系。对照光过程的荧光光谱监测同时观察到HMME漂白、光产物生成与漂白,以及样品光学特性变化引起的自体荧光强度起伏。光产物漂白后的二次产物引起样品光学特性显著改变。所建立的荧光光谱探测系统与光谱分析方法可满足光敏剂漂白特性体外研究的需要,并为光动力治疗的剂量学在体监测提供有效研究方法。     

Nonlinear Excitation of Tryptophan Emission Enhanced by Silver Nanoparticles

We measured and analyzed the behavior of the fluorescence of tryptophan water solutions with and without silver nanoparticles, excited by one, two and three photon processes. Two different colloids with silver nanoparticles with distinct diameters (0.65 nm and 9 nm) were used in the experiments. Fluorescence quenching was observed with one and two photon excitation. However, upon three-photon excitation, significant fluorescence enhancement was observed in the colloid. In this case excitation of the amino acid is assisted by the nonlinear absorption of infrared light by the silver nanoparticles. In this paper we are proposing a new way to explore metallic nanoparticles to enhance autofluorescence of biomolecules.     

Sensitive detection of optical discrete absorption and lasing of fused silica by the depopulation of the ground state

Ultraviolet light induced high resolution optical absorption spectra and resonance coherent fluorescence of spectroscopically pure fused silica have been studied, due to their potential applications in optoelectronics and laser flash spectroscopy for laboratory use, for optical fibers and for power optics for lasers. Ultraviolet discrete absorption spectra and resonance coherent fluorescence were recorded photographically between 200 and 260 nm. Resonance discrete and coherent fluorescence (i.e. lasing effect) were observed in the 250–255 nm band at room temperature by high photon flux excitation spectroscopy.     

Laser-Induced Fluorescence at 488 nm Excitation for Detecting Benign and Malignant Lesions in Stomach Mucosa

This work aims the detection of the histopathologic alterations of in vitro human gastric mucosa using spectral informations from laser-induced fluorescence spectroscopy (LIFS) technique with excitation at 488 nm (argon laser). A total of 108 biopsies with endoscopic diagnosis of gastritis and gastric cancer were obtained at the antral gastric region, from 35 patients with dyspeptic digestive complaints. The biopsies were collected during the endoscopic examination. On each biopsy fragment the autofluorescence spectrum was collected in two random points, through a fiber-optic catheter coupled to the excitation laser. The fluorescence emission spectra collected by the fibers were directed to the spectrograph and detected by the CCD camera. The spectra were then separated in groups (N, normal; LI, light inflammation; MI, moderated inflammation; CA, adenocarcinoma), based on the histopathology. The ratio between the emission wavelengths 550 and 600 nm was used as a diagnostic parameter. Analysis of fluorescence spectra was able to identify the normal tissue from adenocarcinoma lesions with 100% of sensibility and specificity. The ratio intensities between inflammation (light and moderated), although presented significantly statistical differences when compared to the normal mucosa, do not furnish enough sensibility and specificity for use as an identification method due to high variations. LIFS, with excitation of 488 nm, could be used in the differentiation of normal tissue and neoplasic lesions, assisting a less invasive diagnosis.