Single cell studies of enzymatic hydrolysis of a tetramethylrhodamine labeled triglucoside in yeast

Several hundred molecules of enzyme reaction products were detected in a single spheroplast from yeast cells incubated with a tetramethylrhodamine (TMR) labeled triglucoside, alpha-d-Glc(1-->2)alpha-d-Glc(1-->3)alpha-d-Glc-O(CH2)8CONHCH2- CH2NH- COTMR. Product detection was accomplished using capillary electrophoresis and laser induced fluorescence following the introduction of a single spheroplast into the separation capillary. The in vivo enzymatic hydrolysis of the TMR-trisaccharide involves at least two enzymes, limited by processing alpha-glucosidase I, producing TMR-disaccharide, TMR-monosaccharide, and the free TMR-linking arm. Hydrolysis was reduced by preincubation of the cells with the processing enzyme inhibitor castanospermine. Confocal laser scanning microscopy studies confirmed the uptake and internalization of fluorescent substrate. This single cell analysis methodology can be applied for the in vivo assay of any enzyme with a fluorescent substrate.     

Measuring rotations of a few cross-bridges in skeletal muscle

The ability to measure properties of a single cross-bridge in working muscle is important because it avoids averaging the signal from a large number of molecules and because it probes cross-bridges in their native crowded environment. Because the concentration of myosin in muscle is large, observing the kinetics of a single myosin molecule requires that the signal be collected from small volumes. The introduction of small observational volumes defined by diffraction-limited laser beams and confocal detection has made it possible to limit the observational volume to a femtoliter (10(-15) liter). By restraining labeling to 1 fluorophore per 100 myosin molecules, we were able to follow the kinetics of approximately 400 cross-bridges. To reduce this number further, we used two-photon (2P) microscopy. The focal plane in which the laser power density was high enough to produce 2P absorption was thinner than in confocal microscopy. Using 2P microscopy, we were able to observe approximately 200 cross-bridges during contraction. The novel method of confocal total internal reflection (CTIR) provides a method to reduce the observational volume even further, to approximately 1 attoliter (10(-18) liter), and to measure fluorescence with a high signal-to-noise (S/N) ratio. In this method, the observational volume is made shallow by illuminating the sample with an evanescent field produced by total internal reflection (TIR) of the incident laser beam. To guarantee the small lateral dimensions of the observational volume, a confocal aperture is inserted in the conjugate-image plane of the objective. With a 3.5-mum confocal aperture, we achieved a volume of 1.5 attoliter. Association-dissociation of the myosin head was probed with rhodamine attached at cys707 of the heavy chain of myosin. Signal was contributed by one to five fluorescent myosin molecules. Fluorescence decayed in a series of discrete steps, corresponding to bleaching of individual molecules of rhodamine. The S/N ratio was sufficiently large to make statistically significant comparisons from rigor and contracting myofibrils.     

Imaging and tracking of single GFP molecules in solution

Visualization and tracking of single fluorescent molecules is a recent development in optical microscopy holding great promise for the study of cell biological processes. However, all experimental strategies realized so far confined the observation to extremely thin interfacial layers. The detection and characterization of single molecules in three-dimensionally extended systems such as living cells has yet to be accomplished. We show, here, for the first time that single protein molecules can be visualized and tracked in three-dimensional (3D) samples at room temperature. Using a wide-field fluorescence microscope equipped with an Ar(+)-laser and a low-light-level CCD camera, single molecules of the green fluorescent protein (GFP) were detected in gels and viscous solutions at depths of up to approximately 10 microm from the interface. A time resolution of 5 ms was achieved by a high-speed framing mode. The two-dimensional localization accuracy was determined to be approximately 30 nm. The number of photons emitted by single GFP molecules before photodestruction was found to be < or = 4 * 10(5). Freely diffusing GFP molecules could be tracked over up to nine images acquired at a frame rate of approximately 80 Hz. From the trajectories, the diffusion coefficients of single GFP molecules were derived and found to agree well with expectation and microphotolysis measurements. Our results imply that the visualization and tracking of single molecules in living cells is possible.     

Continuous photobleaching in vesicles and living cells: a measure of diffusion and compartmentation

We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t<0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t>0.1-5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from approximately 10 to 100 microm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 microm2/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 microW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.     

Ultra-high resolution imaging by fluorescence photoactivation localization microscopy

Biological structures span many orders of magnitude in size, but far-field visible light microscopy suffers from limited resolution. A new method for fluorescence imaging has been developed that can obtain spatial distributions of large numbers of fluorescent molecules on length scales shorter than the classical diffraction limit. Fluorescence photoactivation localization microscopy (FPALM) analyzes thousands of single fluorophores per acquisition, localizing small numbers of them at a time, at low excitation intensity. To control the number of visible fluorophores in the field of view and ensure that optically active molecules are separated by much more than the width of the point spread function, photoactivatable fluorescent molecules are used, in this case the photoactivatable green fluorescent protein (PA-GFP). For these photoactivatable molecules, the activation rate is controlled by the activation illumination intensity; nonfluorescent inactive molecules are activated by a high-frequency (405-nm) laser and are then fluorescent when excited at a lower frequency. The fluorescence is imaged by a CCD camera, and then the molecules are either reversibly inactivated or irreversibly photobleached to remove them from the field of view. The rate of photobleaching is controlled by the intensity of the laser used to excite the fluorescence, in this case an Ar+ ion laser. Because only a small number of molecules are visible at a given time, their positions can be determined precisely; with only approximately 100 detected photons per molecule, the localization precision can be as much as 10-fold better than the resolution, depending on background levels. Heterogeneities on length scales of the order of tens of nanometers are observed by FPALM of PA-GFP on glass. FPALM images are compared with images of the same molecules by widefield fluorescence. FPALM images of PA-GFP on a terraced sapphire crystal surface were compared with atomic force microscopy and show that the full width at half-maximum of features approximately 86 +/- 4 nm is significantly better than the expected diffraction-limited optical resolution. The number of fluorescent molecules and their brightness distribution have also been determined using FPALM. This new method suggests a means to address a significant number of biological questions that had previously been limited by microscope resolution.     

四种显微技术观测大鼠颌下腺细胞与丝素-壳聚糖体外共培养的形态学特点

目的采用倒置显微镜、扫描电镜（scanning electron microscopy,SEM）、荧光显微镜和激光共聚焦显微镜（（laser scanning confocal microscopy,LSCM））技术对大鼠颌下腺细胞（rat submandibular gland cells,RSMGs）与丝素-壳聚糖（silk fibroin-chitosan,SFCs）的体外复合培养进行形态学观察。为观测、评估种子细胞在三维支架的内部生长情况提供技术支持。方法取0～8 d龄SD大鼠的颌下腺,对大鼠颌下腺细胞进行原代培养、分离纯化并传代;用抗细胞角蛋白单克隆抗体（CK8）及淀粉酶抗体的免疫细胞化学染色鉴定细胞来源。选取传至第二代的对数生长期的RSMGs作为种子细胞,选取SFCs共混膜（5×5×2）mm作为支架材料构建组织工程化涎腺样结构。将种子细胞与支架材料复合培养并分别于倒置显微镜、SEM、荧光显微镜和LSCM下观察二者复合生长情况。结果倒置显微镜可以直接观察活细胞与支架复合生长情况,方法简单易行。SEM可以较精确的展示细胞支架复合生长的表面超微结构。经过荧光染料的着色,荧光显微镜和LSCM都可以观察到支架上锚定的种子细胞。荧光显微镜可见细胞核的荧光信号均匀的分布在支架孔隙内。LSCM通过层扫描及三维重建技术对较厚的标本获取图像;并可以通过旋转图像,从不同角度观察细胞支架复合物的三维剖面或整体结构,得到更为准确的定位信息。结论四种显微技术均可应用于RSMGs与SFCs体外共培养的形态学观测。LSCM的三维重建技术结合荧光染料标记可以较好地获得RSMGs与SFCs复合生长的情况,有着较广泛的应用价值。     

Rapid functional analysis of protein-protein interactions by fluorescent C-terminal labeling and single-molecule imaging

Detection of protein-protein interactions is a fundamental step to understanding gene function. Here we report a sensitive and rapid method for assaying protein-protein interactions at the single-molecule level. Protein molecules were synthesized in a cell-free translation system in the presence of Cy5-puro, a fluorescent puromycin, using mRNA without a stop codon. The interaction of proteins thus prepared was visualized using a single-molecule imaging technique. As a demonstration of this method, a motor protein, kinesin, was labeled with Cy5-puro at an efficiency of about 90%, and the processive movement of kinesin along microtubules was observed by using total internal reflection microscopy. It took only 2 h from the synthesis of proteins to the functional analysis. This method is applicable to the functional analysis of various kinds of proteins.     

Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin.

We show using single molecule optical trapping and transient kinetics that the unusually fast Neurospora kinesin is mechanically processive, and we investigate the coupling between ATP turnover and the mechanical actions of the motor. Beads carrying single two-headed Neurospora kinesin molecules move in discrete 8 nm steps, and stall at approximately 5 pN of retroactive force. Using microtubule-activated release of the fluorescent analogue 2'-(3')-O-(N-methylanthraniloyl) adenosine 5'-diphosphate (mantADP) to report microtubule binding, we found that initially only one of the two motor heads binds, and that the binding of the other requires a nucleotide 'chase'. mantADP was released from the second head at 4 s(-1) by an ADP chase, 5 s(-1) by 5'-adenylylimidodiphosphate (AMPPNP), 27 s(-1) by ATPgammaS and 60 s(-1) by ATP. We infer a coordination mechanism for molecular walking, in which ATP hydrolysis on the trailing head accelerates leading head binding at least 15-fold, and leading head binding then accelerates trailing head unbinding at least 6-fold.     

Thermal activation of single kinesin molecules with temperature pulse microscopy

Conventional kinesin is a processive motor protein that keeps "walking" along a microtubule using chemical energy released by ATP hydrolysis. We previously studied the effects of temperature between 15 degrees and 35 degrees C on the moving velocity, force, and processivity of single kinesin molecules using a bead assay [Kawaguchi and Ishiwata, 2000b: Biochem Biophys Res Commun 272:895-899]. However, we could not examine the effects of temperature higher than 35 degrees C because of the thermal damage to proteins. Here, using temperature pulse microscopy (TPM) [Kato et al., 1999: Proc Natl Acad Sci USA 96:9602-9606], we could examine the temperature dependence of the gliding velocity of single kinesin molecules interacting with a microtubule above 35 degrees C up to 50 degrees C (instantaneously, approximately 60 degrees C), where the velocity reached 3.68 microm/s, the highest ever reported. The Arrhenius plot showed no breaks between 15 degrees and 50 degrees C with a unique activation energy of about 50 kJ/mol, suggesting that the molecular mechanism of kinesin motility is common over a broad temperature range including physiological temperature.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

Pattern photobleaching of fluorescent lipid vesicles using polarized laser light.

A burst of linearly polarized laser radiation incident on a spherical lipid vesicle, liposome, or biological cell can produce a well-defined nonuniform distribution of membrane-bound fluorescent molecules, provided the absorption transition dipole moment of the fluorescent label has a nonrandom orientation relative to the membrane surface and can be photobleached by the laser radiation. The return (recovery) of fluorescent membrane-bound molecules to a uniform distribution can be monitored using the same polarized radiation source. Under appropriate conditions this recovery is characterized by a single exponential time constant tau. This time constant is related to the radius R of the vesicle and the lateral diffusion coefficient D of the fluorescent membrane-bound molecules by the equation R2 = 6D tau. In the case of vesicle membranes this result is not limited by diffraction and so should be applicable to vesicles whose radii are less than the wavelength of light. The above considerations are illustrated by the polarized light photobleaching-recovery of lipid vesicles containing a fluorescent lipid, N-4-nitro-benzo-2-oxa,1,3-diazole l-alpha-dimyristoylphosphatidylethanolamine (NBD-DMPE).     

ATP occlusion by P-glycoprotein as a surrogate measure for drug coupling

The multidrug efflux pump P-glycoprotein (Pgp) couples drug transport to ATP hydrolysis. Previously, using a synthetic library of tetramethylrosamine ( TMR) analogues, we observed significant variation in ATPase stimulation ( V m (D)). Concentrations required for half-maximal ATPase stimulation ( K m (D)) correlated with ATP hydrolysis transition-state stabilization and ATP occlusion (EC 50 (D)) at a single site. Herein, we characterize several TMR analogues that elicit modest turnover ( k cat 相似文献   

Motility of single one-headed kinesin molecules along microtubules

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.     

Biased binding of single molecules and continuous movement of multiple molecules of truncated single-headed kinesin

Conventional kinesin has a double-headed structure consisting of two motor domains and moves processively along a microtubule using the two heads cooperatively. The movement of single and multiple truncated heads of Drosophila kinesin was measured using a laser trap and nanometer detecting apparatus. Single molecules of single-headed kinesin bound to the microtubules with a 3.5 nm biased displacement toward the plus end of the microtubule. The position of these single-headed kinesin molecules bound to a microtubule did not change until they had dissociated, indicating that single kinesin heads utilize nonprocessive movement processes. Two molecules of single-headed kinesin moved continuously along a microtubule with a lower velocity and force than that of single molecules of double-headed kinesin. The biased binding of the heads determines the directionality of movement, whereas two molecules of single-headed kinesin move continuously without dissociation from a microtubule.     

激光照射对蝗虫诱变杀灭效应的初步研究

为初步研究激光对蝗虫的诱变杀灭效应,将中华剑角蝗成虫、东亚飞蝗成虫和东亚飞蝗幼虫按种群分为照射试验组和对照组,选择采用波长808nm、功率2W的半导体连续单管激光器进行照射试验。分别取距离激光二极管发光面1cm处和2cm处以时间15s、10s、5s和3s进行蝗虫头部眼睛部位和翅膀部位辐照,探索蝗虫不同身体部位对激光的敏感性,比较激光对不同种类和龄期蝗虫的杀灭效应,并对蝗虫的活性进行观察。结果显示:不同强度及辐照时间的激光照射后,蝗虫的活性均有所降低,且照射时间越长、功率密度越大,蝗虫活性下降越显著;随着照射后时间的延长,群体蝗虫的活性降到一个较低的水平;激光对东亚飞蝗的辐照杀灭效应优于中华剑角蝗;蝗蝻较成虫容易被激光灭杀;头部眼睛部位较翅膀部位对激光敏感。     

Direct long-term observation of kinesin processivity at low load

The hand-over-hand stepping mechanism of kinesin at low loads is inadequately understood because the number of molecular steps taken per encounter with the microtubule is difficult to measure: optical traps do not register steps at zero load, while evanescent wave microscopy of single molecules of GFP-kinesin suffers from premature photobleaching. Obtaining low-load data is important because it can efficiently distinguish between alternative proposed mechanisms for molecular walking. We report a novel experiment that records the missing data. We fused kinesin to gelsolin, creating a construct that severs and caps rhodamine-phalloidin actin filaments, setting exactly one kinesin molecule on one end of each fluorescent actin filament. Single kinesin molecules labeled in this way can be tracked easily and definitively using a standard epifluorescence microscope. We use the new system to show that, contrary to a recent report, kinesin run length at low load is independent of ATP concentration in the muM to mM range of ATP concentration. Adding competitor ADP in the presence of saturating ATP decreases both velocity and run length. Based on these data, we propose a simplified model for the mechanism of processive stepping.     

CO2激光预处理对PEG6000胁迫下小麦幼苗根中抗氧化酶活性的影响

用CO2激光对小麦种子分别辐照0、1、3、5min,待其生长至12d时,用10%（W/V）PEG6000胁迫其幼苗,研究激光预处理对PEG6000水分胁迫下小麦幼苗根部脂质过氧化伤害的防护作用。结果表明,CO2激光预处理3min可使水分胁迫的小麦幼苗根部MDA、H2O2含量和O2.-产生速率显著降低（P〈0.05）,可显著提高（P〈0.05）小麦幼苗根部SOD、POD、CAT、APX活性和根长、根干重。激光预处理3min可抑制由水分胁迫引起的小麦幼苗根部脂质过氧化作用。     

Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy

We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-microm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from approximately 30 to approximately 150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.     

Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons

Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.     

Evaluation of a green laser pointer for flow cytometry.

Flow cytometers typically incorporate expensive lasers with high-quality (TEM00) output beam structure and very stable output power, significantly increasing system cost and power requirements. Red diode lasers minimize power consumption and cost, but limit fluorophore selection. Low-cost DPSS laser pointer modules could possibly offer increased wavelength selection but presumed emission instability has limited their use. A $160 DPSS 532 nm laser pointer module was first evaluated for noise characteristics and then used as the excitation light source in a custom-built flow cytometer for the analysis of fluorescent calibration and alignment microspheres. Eight of ten modules tested were very quiet (RMS noise < or = 0.6% between 0 and 5 MHz). With a quiet laser pointer module as the light source in a slow-flow system, fluorescence measurements from alignment microspheres produced CVs of about 3.3%. Furthermore, the use of extended transit times and < or =1 mW of laser power produced both baseline resolution of all 8 peaks in a set of Rainbow microspheres, and a detection limit of <20 phycoerythrin molecules per particle. Data collected with the transit time reduced to 25 micros (in the same instrument but at 2.4 mW laser output) demonstrated a detection limit of approximately 75 phycoerythrin molecules and CVs of about 2.7%. The performance, cost, size, and power consumption of the tested laser pointer module suggests that it may be suitable for use in conventional flow cytometry, particularly if it were coupled with cytometers that support extended transit times.