这项工作是支持由LAP（PI）的赠款： 美国国立卫生R21 HD50558，荣获2005-07;国家卫生R01 HD045822研究院颁发的2004-09年;达纳基金会赠款， 荣获2004-06;加拿大创新基金会（CFI“补助金），授予 2008年至2012年，安大略省的研究基金资助，荣获2008年至2012年。

第1部分：在此之前，参与者到达实验室<ol><li>确保该房间是多余的物品，可能分散注意力的参与者。 </li><li>设置和负载fNIRS日立，ETG - 4000系统的实验性协议。 </li><li>设置您的实验范式。实验范式，可以设定不同的演示软件，包括Eprime，简报，Psyscope或一个基于MATLAB的心理工具箱。在这里，我们使用基于MATLAB的心理工具箱。 </li><li>时序数据分析的关键，因此，实验范式，必须完全定时与数据收集。 fNIRS日立，ETG - 4000的触发功能，使实验范式，以触发数据收集或反之亦然。测试演示程序的触发fNIRS日立，ETG - 4000。触发可以使用平行，连续，或USB端口。在这里，我们显示通过并行端口触发。 </li><li>开始fNIRS研究之前，重要的是要进行参与者的背景筛查。在Petitto实验室，我们进行背景审查，参加者或他们的父母填写学习适当的标准化问卷29。 </li></ol>参与者抵达<ol start="6"><li>重要的是进行会议，并以专业的态度对待参与者。在实验开始前，参与者或参与者的父母/法定监护人必须签署一份同意书。至关重要的是要感谢他们的时间，在这些重要的和令人兴奋的的实验的参与者。 </li><li>参与者是坐在舒适的密切fNIRS试验室。婴儿的参加者可坐在父母的腿上。 </li></ol>第2部分：配售Optodes使用10-20系统分析的方法，使一致的数据解释的另一个组成部分是fNIRS录制协议的标准化。这就需要optode安置，参与者定位，并触发刺激演示软件。准确的神经解剖位置的探头和地区的利益（投资回报）的确认是通过使用10-20系统3,4,30。此外，探针阵列的立体定位证实参与者的头骨从Polhemus快速TRAK系统叠加到MRI解剖的合作摆在每个探头的位置3,4与维生素E胶囊进行的参与者登记扫描的3D跟踪信息。配售与会者舒适地在躺椅上，与光纤光学无接触身体或椅子松散的挂，参与最佳参与者定位。 <ol><li>下面的头用卷尺测量和参与者的数据表上写下来： <ul><li>鼻根至枕外隆凸尖周围</li><li>鼻根到Inions超过顶部</li><li>耳耳超过顶部</li></ul></li><li>外科胶带可以用来标记特定的目标位置。在这个实验中，我们将迎来FP，T3/T4，F8/F7 </li><li> Optode阵列放在参与者“与挂靠在10-20分，作为实验的目的具体optodes头。 </li></ol>第4部分：测试Optode阵列<ol><li>日立，ETG - 4000的GUI界面和探针测试。 </li><li>测试信号：一旦optodes参与者的头皮上，信号质量测试。如果一个optode没有一个明确的信号，研究人员轻轻取出optode和头皮的头发。有时optodes可能需要用酒精棉球擦拭。 </li></ol>第5部分：运行实验。 <ol><li>必须至少有两个实验者在房间里总是存在;观察fNIRS日立，ETG - 4000实时读出和其他观测参与者。重点对参与者有一个摄像头，强烈建议事后观察。 fNIRS日立，ETG - 4000的一个优点是，视频和fNIRS信号同步和联合注册。保持一个包含所有相关信息和文件生成的日志。 </li><li>建设血流动力学实验范式有行之有效的方法，即座设计和事件相关的设计。如需更完整的描述，请参阅最近审查文件31。 </li></ol>第6部分：分析参与者一旦所有的数据都已经收集，感谢他们的时间和参与意愿，并离开实验室。分析fNIRS日立，ETG - 4000，，相反，数据分析计算机出口。 <ol><li>从μV的转换血红蛋白浓度。为原料的衰减值在激光强度的衰减（以μV测量）收集，这些值必须被转换成含氧和脱氧血红蛋白有价值ES。这是通过修改后的啤酒 - 兰伯特方程。 </li><li>修改后的啤酒 - 兰伯特的应用是在两个步骤进行。根据散射是在路径的长度，首先衰减的信号计算基准为每个波长（ΔAλ（T））是由比较期间的任务（我的任务 ）的光强度的光密度计算常数（假设我基线 ）。每个波长的ΔA值和采样的时间点（T），解决了修改后的比尔-朗伯方程。 <table border="0"><tbody><tr><td> 公式1 <img style="float:left;" src="/files/ftp_upload/1268/eq1.jpg" alt="式（1）" /></td><td> 公式（2） <img style="float:left;" src="/files/ftp_upload/1268/eq2.jpg" alt="公式（2）" /></td></tr></tbody></table></li></ol> 1λ∈脱氧 ，λ∈ 氧 ，λ∈ 脱氧和2λ∈氧测量输给单位在组织中的浓度距离的吸收光的一小部分的消光系数，常数。由此产生的彗星脱氧和C 氧值是每个 T无氧和含氧血红蛋白的浓度。 第7部分：代表结果血流动力学反应的结果，典型的几个鲜明的特点。在氧血红蛋白反应，首先是有一个特点DIP。此DIP发生，作为一个区域的神经元激活，并耗尽可用氧气。随着血流量的增加，氧合血红蛋白的携带氧血红蛋白的反应高于初始基线水平迅速到一个稳定的状态水平。不再被激活，当该地区，在未来的12-15秒的氧的血红蛋白的反应衰变，慢慢地回落到基线水平。偶尔有一个冲前发生的血流动力学响应返回到初始基线水平。 坏的结果通常是在没有正确固定在头皮上或运动过量optodes的形式。这些类型的噪音 - 被称为“Flatling” - 是很明​​显的信号微伏值饱和，并且不同的渠道，以协调的方式移动氧和脱氧反应。 示范：翻天覆地的光纤。 统计分析：每个通道中提取氧和脱氧血红蛋白值，为每一个参与者，并为每个任务，然后可以提交给传统的统计分析，包括t检验，方差分析，相关性等。

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The authors have nothing to disclose.

在这项研究中，我们展示了一种新型的，非侵入性fNIRS的脑成像技术来研究有关人类认知和感知人的大脑功能。 fNIRS脑成像技术可能代表的非侵入性的脑成像技术的未来，特别是婴儿和儿童的人口，可能有一天会在实验室广泛使用，医生的办公室，并让医生申请有关的基本的科学发现，在学校系统他们的临床实践中的大脑。

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exploring cognitive functions in babies, children &amp; adults with near infrared spectroscopy

红外光谱（fNIRS）较高的认知过程，如语言 1,2,3,4,5,6,7,8,9,10， 内存 11，皮质活化调查研究， 注意 12附近爆炸功能正在进行全球范围内涉及成人，儿童和婴儿3,4,13,14,15,16,17,18,19典型和非典型的认知20,21,22。当代的认知神经科学fNIRS的挑战是实现系统的数据，例如，他们普遍的解释是 23,24,25,26，从而可能推进人类更高的认知力的基本功能的组织和神经系统的重要科学问题的分析。 现有的神经影像学技术，要么不太可靠的时间或空间分辨率。事件相关电位和磁脑照相的企业资源计划（ERP和MEG）有良好的时间分辨率，而正电子发射断层扫描和功能磁共振成像（PET和fMRI）有更好的空间分辨率。使用非电离光波长在近红外波段（700-1000纳米），优先吸收680 nm和脱氧血红蛋白氧血红蛋白优先吸收830纳米（例如，的确，非常硬连接到波长fNIRS是fNIRS日立，ETG - 400系统这里说明），以及适用于更高的认知力的研究，因为它不使用辐射和良好的空间分辨率（〜4厘米深）既有良好的时间分辨率（〜5秒），并没有要求参与者在一个封闭的结构27,28。与会者皮质活动可以评估，而舒适地坐在一个普通的椅子（成人，儿童），甚至坐在妈妈的腿上（婴儿）。值得注意的是，近红外光谱是独特的便携式（台式计算机的大小），几乎无声，并可以容忍一个微妙的运动参与者。这是特别突出的神经研究人类的语言，这必然有它的嘴在讲话生产或手语的手运动的关键部件之一。 在其中的血流动力学反应是本地化的方式是由激光发射器和探测器阵列。发射器发出的非电离光的强度，而探测器检测到从皮质表面反射回来的金额。紧密联系起来的optodes，空间分辨率更大，而进一步除了optodes，更深入的渗透。对于fNIRS日立，ETG - 4000系统的最优渗透/分辨率optode阵列设置2厘米。 我们的目标是展示我们的采集和分析fNIRS数据，以帮助规范领域，使不同fNIRS实验室在全球范围内都有一个共同的背景的方法。

Watch this Scientific Journal Video about Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy at JoVE.com

Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

在这里，我们介绍了近红外光谱（fNIRS），认知神经科学中使用的一种新型的非侵入性的脑成像系统，功能，特别是在学习孩子大脑发育的数据收集和数据分析方法。这种方法提供了一个重要的数据采集和分析数据的解释和科学发现的普遍标准。

近红外光谱技术在婴儿，儿童及成人的认知功能探索

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

exploring-cognitive-functions-babies-children-adults-with-near

Research

JoVE Journal

Biology

20.4K 次观看.  University of Michigan, Ann Arbor. 在这里，我们介绍了近红外光谱（fNIRS），认知神经科学中使用的一种新型的非侵入性的脑成像系统，功能，特别是在学习孩子大脑发育的数据收集和数据分析方法。这种方法提供了一个重要的数据采集和分析数据的解释和科学发现的普遍标准。

视频: Exploring Cognitive Functions in Babies, Children & Adults with Near Infrared Spectroscopy

Recordings of Neural Circuit Activation in Freely Behaving Animals

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. Traditional non-invasive methods require at least partially restrained research subjects, and they only allow identification of large numbers of simultaneously activated neurons. On the other hand, small ensembles of neurons or individual neurons can only be measured using single-cell recordings obtained from largely reduced preparations. Since the expression of natural behavior is limited in restrained and dissected animals, the underlying neural mechanisms that control such behavior are difficult to identify. 
Here, I present a non-invasive physiological technique that allows measuring neural circuit activation in freely behaving animals. Using a pair of wire electrodes inside a water-filled chamber, the bath electrodes record neural and muscular field potentials generated by juvenile crayfish during natural or experimentally evoked escape responses. The primary escape responses of crayfish are mediated by three different types of tail-flips which move the animals away from the point of stimulation. Each type of tail-flip is controlled by its own neural circuit; the two fastest and most powerful escape responses require activation of different sets of large &#8220;command&#8221; neurons. In combination with behavioral observations, the bath electrode recordings allow unambiguous identification of these neurons and the associated neural circuits. Thus activity of neural circuitry underlying naturally occurring behavior can be measured in unrestrained animals and in different behavioral contexts.

Non-invasive measurements of neural activity patterns in freely behaving animals are obtained by combining neurophysiological recordings with high speed videography.

自由表现动物的神经回路活化的录音

The relationship between patterns of neural activity and corresponding behavioral expression is difficult to establish in unrestrained animals. ...

科研

生物学

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

大会，调整和使用蛋白成像Apertureless近场红外显微镜

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

在学前儿童Neuromagnetic的脑功能自定义尺寸的MEG测量

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

对于波长和红/近红外光治疗对氧化应激强度的范围影响的评估方法<em&gt;在体外</em

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

Near Infrared NIr Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

近红外（NIR）光增加线粒体功能的标志物的表达，在鼠标前庭上皮细胞

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

纯化野生型和突变CFTR蛋白的功能重建和通道活性测定

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

该芯片外型方法：确定蛋白质-DNA相互作用与近基部精密配对

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving immunization coverage worldwide. Here, a novel injectable, ultraviolet- curable and delayed burst release- enabling vaccine delivery platform called polybubbles is introduced. Cargo was injected into polyester-based polybubbles that were formed in 10% carboxymethycellulose -based aqueous solution. This paper includes protocols to maintain spherical shape of the polybubbles and optimize cargo placement and retention to maximize the amount of cargo within the polybubbles. To ensure safety, chlorinated solvent content within the polybubbles were analyzed using neutron activation analysis. Release studies were conducted with small molecules as cargo within the polybubble to confirm delayed burst release. To further show the potential for on-demand delivery of the cargo, gold nanorods were mixed within the polymer shell to enable near-infrared laser activation.

This article describes the protocols used to produce a novel vaccine delivery platform, &#34;polybubbles,&#34; to enable delayed burst release. Polyesters including poly(lactic-co-glycolic acid) and polycaprolactone were used to form the polybubbles and small molecules and antigen were used as cargo.

生产近红外敏感、核心壳疫苗输送平台

Vaccine delivery strategies that can limit the exposure of cargo to organic solvent while enabling novel release profiles are crucial for improving ...

Exploring Adipose Tissue Structure by Methylsalicylate Clearing and 3D Imaging

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity is characterized by an increase in adipose tissue (AT) mass due to adipocyte hyperplasia and/or hypertrophia, leading to profound remodeling of its three-dimensional structure. Indeed, the maximal capacity of AT to expand during obesity is pivotal to the development of obesity-associated pathologies. This AT expansion is an important homeostatic mechanism to enable adaptation to an excess of energy intake and to avoid deleterious lipid spillover to other metabolic organs, such as muscle and liver. Therefore, understanding the structural remodeling that leads to the failure of AT expansion is a fundamental question with high clinical applicability. In this article, we describe a simple and fast clearing method that is routinely used in our laboratory to explore the morphology of mouse and human white adipose tissue by fluorescent imaging. This optimized AT clearing method is easily performed in any standard laboratory equipped with a chemical hood, a temperature-controlled orbital shaker and a fluorescent microscope. Moreover, the chemical compounds used are readily available. Importantly, this method allows one to resolve the 3D AT structure by staining various markers to specifically visualize the adipocytes, the neuronal and vascular networks, and the innate and adaptive immune cells distribution.

Here, we describe a simple, inexpensive and fast clearing method to resolve the 3D structure of both mouse and human white adipose tissue using a combination of markers to visualize vasculature, nuclei, immune cells, neurons, and lipid-droplet coat proteins by fluorescent imaging.

通过甲基异硫清除和三D成像探索脂肪组织结构

Obesity is a major worldwide public health issue that increases the risk to develop cardiovascular diseases, type-2 diabetes, and liver diseases. Obesity ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

超快力钳谱分析合成性肌球蛋白的机械酶学特性

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...