基于激光共聚焦扫描显微技术分析激光对扁藻的影响

本文以亚心形扁藻为样品,波长1 341 nm的Nd∶YAP激光为光源,通过激光共聚焦扫描显微技术,研究Nd∶YAP激光辐照亚心形扁藻对亚心形扁藻叶绿体自体荧光强度和叶绿体面积大小的影响。Nd∶YAP激光辐照后的亚心形扁藻通过488 nm Ar^+激光激发获得亚心形扁藻自体荧光图像及其荧光光谱。结果表明,试验中除(10 W,60 s)辐照剂量组外,其余辐照剂量组均提高了亚心形扁藻的自体荧光强度,且所有的辐照剂量组均增大了亚心形扁藻的叶绿体面积。Nd∶YAP激光可刺激亚心形扁藻的叶绿体发育,促进藻细胞的生长,改善叶绿体光合作用的活性。     

Nd:YAP激光辐照对雨生红球藻生理效应的荧光分析

目的：Nd：YAP激光辐照对雨生红球藻生理效应的荧光分析j方法：利用Nd：YAG激光（功率10W,辐照剂量为15s、35s、55s）辐照雨生红球藻,在对辐照细胞进行生长测定以及色素吸收光谱分析的基础上,进一步利用激光共聚焦扫描显微镜（LSCM）,在波长488nm的Ar^＋激发光条件下,对细胞的自体荧光以及吖啶橙染色的细胞核荧光物质进行定位定量分析,获得细胞自发荧光光谱和细胞核荧光物质的荧光光谱。结果：①低剂量的Nd：YAG激光辐照（15s左右）对藻细胞有促长作用,生长速率提高20．3％,色素吸收峰的峰值提高25．3％。较高剂量的Nd：YAG激光辐照（35s、55s）对藻细胞生长有抑制作用,并有明显的致死、致突效应。②对自体荧光的分析结果表明,与对照组相比,低剂量激光辐照组的细胞,在荧光光谱的峰型及峰值上变化不大,在682nm处均有较强的荧光发射,而高剂量激光辐照组的细胞多无明显的荧光发射。③对细胞核荧光物质的分析,雨生红球藻细胞在530nm（DNA）和640nm（RNA）处均有荧光发射峰。与对照组相比,低剂量激光辐照组的DNA荧光发射有所增强,但RNA的荧光发射则有所减弱;高剂量激光辐照组的核物质荧光发射谱,在峰型上与对照组存在差异,荧光强度也明显下降。结论：低剂量的Nd：YAP激光辐照对细胞核的DNA合成与复制有一定的刺激作用,可促进光合色素的合成并提高其对光能的吸收效率,从而增强光合活性,促进细胞的增殖与生长;高剂量激光辐照则对细胞的DNA有损伤作用,是致死率上升并发生突变的可能原因。     

Ar+激光、Nd:YAG激光辐照亚心形扁藻生物学效应初探

采用Ar^＋激光（488 nm,照射时间分别为10 min、20 min、30 min）、Nd：YAG（1064 nm,照射时间为1 min、2 min、3 min）辐照扁藻,通过细胞计数以及扫描色素的吸收光谱研究激光辐照扁藻的生物效应.研究结果表明：非色素吸收峰的激光可以产生激光生物效应;不同波长,不同剂量的激光辐照扁藻可以表现出相类似的生物效应;Ar＋辐照20 min、Nd：YAG辐照2 min可以刺激扁藻生长,明显提高色素吸收光密度值,促进光合作用的进行.文中对不同激光辐照扁藻所产生的生物效应进行了比较和探讨.     

国产Nd:YAP激光组织热损伤效应的比较研究

目的:通过比较1 341 nm Nd:YAP与临床常用激光器脉冲Nd:YAG和连续Nd:YAG对皮肤、肌肉、静脉和肝脏组织的热损伤效应,了解Nd:YAP激光的作用特点,为其临床应用提供实验依据。方法:以大鼠作为实验对象,分为皮肤、肌肉组和静脉、肝脏组。皮肤、肌肉组:Nd:YAP激光、连续和脉冲Nd:YAG选择能量密度1000 J/cm2、2 000 J/cm2,静脉、肝脏组采用Nd:YAP激光和连续Nd:YAG两种激光器,能量密度选择500 J/cm2和1 000 J/cm2,对组织进行非接触式点状照射。照射过程中观察组织变化和损伤直径大小,并于照射后即刻、3天取材做病理切片,光镜下观察病理变化,比较相同能量密度下三种激光器对组织的热损伤和损伤修复情况。结果:皮肤、肌肉组:Nd:YAP激光在1 000 J/cm2条件下对组织的损伤以热凝固效应为主,2 000 J/cm2条件下组织发生明显气化、有一定的切割作用。相同能量密度下,脉冲Nd:YAP激光气化作用较两种Nd:YAG激光显著,而凝固损伤范围较连续Nd:YAG浅,较脉冲Nd:YAG深。肝脏、静脉组:Nd:YAP激光对肝脏的热凝固效应与Nd:YAG激光近似,气化作用明显;Nd:YAP激光对血管及其周围组织的凝固及气化作用均较连续Nd:YAG激光明显。结论:Nd:YAP激光具有良好的凝固与气化作用。     

Nd：YAP激光的不同功率和时间组合对亚心形扁藻的影响

采用同一剂量不同功率和时间组合的Nd:YAP激光(波长1 341nm)辐照亚心形扁藻,辐照组分为8W 69 s、10 W 55 s和12 W 46 s三个组合,通过藻细胞生长速率和叶绿素含量测定研究激光对扁藻的影响.实验结果表明,即使同一照射剂量,不同的辐照时间与辐照功率的组合,辐照效果仍不相同,本实验中以辐照时间55 s,辐照功率10 W 为最佳组合.     

数学建模用于激光辐照微藻生物学效应的研究

目的：用数学建模模拟激光辐照扁藻细胞的增殖情况,研究激光辐照微藻生物学效应的参数规律。方法：在不同激光参数条件下照射亚心形扁藻,分为不同照射剂量实验组,以各实验组的细胞增殖数目为建立模型数据源。根据单细胞藻类在培养过程中生长及繁殖的规律构造高斯生长曲线,构建线性双目标规划模型,利用逐步回归法从辐照激光参量及三者的衍生量中分别筛选出与细胞增殖数、生长速率相关度较大的参量,并利用模型优化软件得到最优策略。分析结果表明用构建模型对不同激光处理的微藻样本生长曲线的仿真,达较高拟合度,SSE：2．383e-005,R—square：0．9997,验证了模型的正确性。对于给定的激光波长和照射时间,可以预测辐射后扁藻藻种的平均生长速率和生长期的变化趋势。结论：数学建模可以作为寻找激光辐照微藻的参数与其生物学效应之问的规律的分析方法。     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

抗砷细胞荧光染色后的激光共聚焦显微镜检测

目的:应用激光共聚焦显微镜检测活细胞内荧光物质含量.方法:传代培养长期低剂量砷诱导的抗砷细胞,用荧光染料Rhodamine-123对细胞染色30min,实验组与维拉帕米(Verapamil)共同孵育,对照组为单加Rhodamine-123的抗砷细胞.应用激光共聚焦显微镜采集Rhodamine-123的荧光图像动态序列,并且记录不同时间段的细胞内荧光强度.结果:实验组细胞染色12h,24h,36h,48h,60h后,荧光强度依次为(51.567±0.7572)、(46.533±0.7095)、(39.557±0.601)、(38.6±0.6245)和(38.505±0.718),明显高于同时间段对照组的荧光强度,差异均有显著性(P＜0.01).结论:应用激光共聚焦显微成像技术能进行活细胞水平荧光物质实时定量检测.     

三种显微技术对人毛囊蠕形螨的观察和研究

目的 采用荧光显微镜、扫描电镜和激光共聚焦显微镜对人体毛囊形蠕形螨进行形态学观察.方法 选用5 μg/mL碘化丙啶(propidine iodide,PI)对虫体进行荧光染色,避光染色15 min,分别置于荧光显微镜和激光共聚焦显微镜下观察;2.5%戊二醛固定虫体标本,梯度酒精和叔丁醇脱水,金喷镀后扫描电镜观察.结果 荧光显微镜下碘化丙啶对虫体有很强的结合力,虫体荧光信号均匀展示于细胞表面,充分展现虫体形态,扫描电镜更加清楚、细致地展示人体毛囊型蠕形螨的超微结构,激光共聚焦显微镜将虫体分层扫描图片进行三维重建,真实、完全、直观地展露了虫体.结论 三种显微技术均可展示蠕形螨超微形态.PI对虫体有很好的荧光染色作用,使激光扫描共聚焦显微镜能够获得更加精准的超微形态结构,结合三维重建技术有着广泛的应用价值.     

用罗丹明B实现微波辐照下细胞水平温度的实时测量

为了探讨用温度敏感性荧光探针罗丹明B(rhodamine B,Rho-B)测量微波辐照下细胞水平温度的方法,利用激光共聚焦显微镜和光纤测温仪测得的数据拟合出罗丹明B荧光探针荧光强度与温度的关系式,并利用该荧光探针对微波辐照过程中细胞水平的温度进行实时测量。结果显示,温度与荧光探针的荧光强度呈现较好的线性关系。利用拟合的温度-相对荧光强度关系式可得到升温过程(25~40℃)中细胞水平的准确温度,这为生物电磁实验中细胞水平的实时温度监测提供了一种较为便捷可行的方法。     

He—Ne激光照射离体鼠巨噬细胞对线粒体跨膜电势的影响

目的：研究Ｈｅ－Ｎｅ激光照射鼠巨噬细胞对线粒体跨膜电势的影响,及其与激光剂量的关系。方法：用亲脂性阳离子荧光染料Ｒｈｏｄａｍｉｎｅ１２３对鼠巨噬细胞线粒体作荧光标记,以不同的激光剂量照射,采用图像分析系统（ＩＡＳ）和荧光显微镜观察线粒体跨膜电势荧光强度的变化。结果：低功率Ｈｅ－Ｎｅ激光照射５,１０,１５ｍｉｎ,激光剂量分别为０．６４９,１．３８８和２．０８２Ｊ／ｃｍ＾２,巨噬细胞线粒体跨膜电势荧光     

He-Ne激光照射对血液及其组分荧光光谱影响的实验研究

为研究弱激光照射对人血液携氧能力的影响及机制,我们用荧光仪分别测量了He-Ne激光照射前后正常血液及其组分（血浆、红细胞）的荧光光谱,研究了激光照射导致的光谱变化,并分析了光谱变化与血液携氧能力改变的关系。实验结果显示：全血液标本在490nm及614nm附近有荧光峰值;血浆的荧光则主要分布在420－500nm之间;红细胞在500nm及614nm附近有荧光。He-Ne激光照射后,全血液及红细胞在614nm处的荧光谱都有较明显的变化,且较相似。由此可得出结论,He-Ne激光照射可影响血液的携氧能力。     

Mechanism of the radioprotective effect of cysteamine

The radioprotective effect of cysteamine combined with the modification of the chromatin state by sodium butyrate has been studied using V-79 and CHEL lines of Chinese hamster cells and HeLa cells. Sodium butyrate enhances the chromatin sensitivity to nucleases and removes the radioprotective effect of cysteamine as measured by the yield of cells with chromosome aberrations. As is indicated by changes in the intensity of fluorescence of the DNA-ethidium bromide complex, measured by laser flow cytometry, the protective agent decreases the binding of the dye with both irradiated and nonirradiated DNA whereas ionizing radiation and sodium butyrate increase thereof. It is concluded that the radioprotective effect of cysteamine depends in its ability to reduce the susceptibility of DNA to nucleases.     

低强度He-Ne激光对红细胞胞浆内游离钙离子浓度的影响

低强度He-Ne激光对红细胞变形性的影响已得到广泛的认可和应用,但其具体调节机制尚不明确,通过研究低强度He-Ne激光对红细胞胞浆内钙离子浓度的影响,探讨其对红细胞变形性影响的机制。采用A23187处理过的红细胞,分别用5 mW和9 mW激光照射后,观察红细胞胞浆内钙离子浓度变化。结果显示:低强度He-Ne激光照射后的红细胞与无照射组的红细胞相比,红细胞胞浆内钙离子浓度显著降低,但在本实验中钙离子浓度的降低与激光照射剂量无显著相关。由此得出结论:降低红细胞胞浆内钙离子浓度可能是低强度He-Ne激光调节红细胞变形性的重要机制。     

Assessment of fibroblast cells submitted to ultrasonic irradiation

Physiotherapists consider ultrasound an indispensable tool, which is commonly employed in clinical practice as a treatment aid for musculoskeletal dysfunctions. The aim of our study has been to analyze fibroblast cell structures following low-intensity pulsed ultrasonic irradiation. Fibroblast cell cultures irradiated with ultrasound were analyzed through electron microscopy to determine an ideal irradiation beam that preserved cell morphology and integrity. Analysis by fluorescence microscopy and transmission electron microscopy was used to follow morphological changes of the nucleus and cytoskeleton following different ultrasound irradiation intensities. According to the parameters used in the pulsed irradiation of fibroblast cultures, control over the intensity employed is fundamental to the optimal use of therapeutic ultrasound. Cell cultures submitted to low-intensity pulsed ultrasonic irradiation (0.2-0.6 W/cm2) at 10% (1:9 duty cycle) and 20% (2:8 duty cycle) maintained shape and cellular integrity, with little damage. In the group irradiated with an intensity of 0.8 W/cm2, a loss of adhesion was observed along with an alteration in the morphology of some cells at an intensity of 1.0 W/cm2, which resulted in the presence of cellular fragments and a decrease of adhering cells. In cells irradiated at 2.0 W/cm2, there was a complete loss of adhesion and aggregation of cellular fragments. The present study confirms that biophysical properties of pulsed ultrasound may accelerate proliferation processes in different biological tissues.     

Measuring fast calcium fluxes in cardiomyocytes

Cardiomyocytes have multiple Ca(2+) fluxes of varying duration that work together to optimize function (1,2). Changes in Ca(2+) activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gα(q;) pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine (3,4). We have recently found that plasma membrane protein domains called caveolae(5,6) can entrap activated Gα(q;)(7). This entrapment has the effect of stabilizing the activated state of Gα(q;) and resulting in prolonged Ca(2+) signals in cardiomyocytes and other cell types(8). We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca(2+) indicator. In our studies, we used Ca(2+) Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca(2+) responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca(2+) waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca(2+) waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca(2+)fluxes in the cells, and to determine their impact on various cellular events.     

Single-cell flux measurement by continuous fluorescence microphotolysis

Continuous fluorescence microphotolysis (CFM) was adapted to flux measurements in single cells. The principle of the method is simple: Cells are equilibrated with a fluorescent solute, an individual cell is continuously irradiated by a laser beam focussed down to approximately the diameter of the cell, and fluorescence originating from the irradiated cell is monitored. In this procedure irradiation irreversibly photolyzes chromophores in the cell while fresh chromophores enter the cell by membrane transport (flux). The resulting fluorescence decay can be analyzed for the rate constants of both membrane transport and photolysis. As an experimental test of the new method the band-3 mediated transport of the fluorescent anion N-(7-nitrobenzofuranzan-4-yl)-taurine (NBD-taurine) across the erythrocyte membrane was measured. For various experimental conditions good agreement between values obtained by CFM and by fluorescence microphotolysis (FM) was observed. By measurements on single ghosts it was furthermore found that photolysis of NBD-taurine is first-order with respect to the power of irradiation. On this basis a stepped-intensity procedure was worked out that facilitates data evaluation in flux measurements. Also, by analysing the relations between CFM and FM flux measurements a method was devised by which FM data can be corrected for (inevitable) photolysis.     

紫外激光消融主动脉斑块和感生荧光光谱观测

利用XeCI准分子激光辐照主动脉血管正常组织和斑块组织,观察到组织被激光消融。消融所产生的凹坑与辐射时间呈对数直线关系。308nm激光感生的血管壁荧光光谱在可见波段出现二个荧光极大值,用二极值的相对强度之比可以判断正常或斑块组织。     

Changes in Intensity and Spectral Distribution of Fluorescence: Effect of Light Treatment on Normal and DCMU*-Poisoned Anacystis nidulans‡

The intensity of the "steady-state" fluorescence of "aerobic" Anacystis nidulans is variable under prolonged illumination with orange (590 mmu) or blue (440 mmu) light for both normally photosynthesizing and DCMU-poisoned cells. In general, orange light illumination causes an increase of the fluorescence intensity followed by a decrease, while blue light causes an increase until a steady level is reached. Poisoned Anacystis cells show four to eight times larger changes in fluorescence intensity than the normal cells; the detailed time course of fluorescence changes is also different in poisoned and normal cells. When algae are cooled to -196 degrees C in light, the light-induced changes in the "steady-state" fluorescence disappear in both types of cells. Difference fluorescence spectra, constructed by subtracting the fluorescence spectra taken after 5-15 min of illumination from those after 60-90 min of illumination, show a doublet structure of the difference band with a major peak coinciding with the Anacystis emission maximum (685 mmu) and a minor peak located at about 693 mmu.     

紫外激光消融主动脉斑块和感生荧光光谱观测

利用XeCI准分子激光辐照主动脉血管正常组织和斑块组织,观察到组织被激光消融。消融所产生的凹坑与辐射时间呈对数直线关系。308nm激光感生的血管壁荧光光谱在可见波段出现二个荧光极大值,用二极值的相对强度之比可以判断正常或斑块组织。