这项工作得到了国家科学基金会 (NSF) 的资助。IOS 1354956。我们感谢 Dr. 安德鲁 d. 沃伦 (马奎尔昆虫和生物多样性中心, 佛罗里达自然历史博物馆, 佛罗里达大学) 允许使用蝴蝶图像。

1. 昆虫种类、溶液的配制和加料站设置
注: 白菜蝶 (菜青虫菜青虫, 粉蝶) 被选为具有代表性的鳞翅目物种, 因为它们已用于以前的流体摄取能力和口器形态学研究22,23。房子苍蝇 (家蝇, 蝇科) 和蓝瓶苍蝇 (Calliphora vomitoria l., 蝇) 使用, 因为它们经常被观察到多孔表面上的摄食13。
<ol>
	<li>将P. 菜青虫作为来自昆虫供应商的幼虫, 并将它们放在人工饲料中 (见材料表), 直到它们在环境室中蛹和出现时, 设置为23° c 和18升: 6 d 光周期。顺序M。家蝇和C. vomitoria作为蛹, 并在与P. 菜青虫相同的环境条件下将它们后置。在喂食实验之前, 不要喂成年蝶和苍蝇。</li>
	<li>准备一个20% 蔗糖溶液 (控制) 和20% 蔗糖纳米颗粒溶液, 以测试流体摄取量。通过加入荧光磁性纳米微粒 (在直径约 20 nm) 的24到20% 蔗糖溶液 (1 毫克/毫升 dH2O 纳米颗粒与20% 蔗糖) 制备纳米粒子溶液解决方案, 1:1)。准备一个1x 溶液磷酸盐缓冲盐水 (PBS) (10x 稀释到1x 在 dH2O, pH 7.4), 用于解剖。</li>
	<li>设置一个喂食站, 它由一个带有夹具的手动机械手和一个独立的进给工作台 (一个平面平台) (图 1) 组成。在进料台上放置凹滑块, 并有尼龙网过滤器, 其孔径直径为11、20、30、41或60µm, 膜过滤器与孔大小直径为1、5、8或10µm, 用于喂食实验。</li>
</ol>
2. 喂养协议
<ol>
	<li>把昆虫的身体、腿和翅膀裹成一张折叠的纸巾。定位昆虫, 使只有头部和口器暴露。将昆虫的翅膀放在两个显微镜幻灯片之间, 它们由机械手的钳夹在一起, 使昆虫悬浮在喂食台上 (图 1)。</li>
	<li>管理一个30µL 液滴的20% 蔗糖溶液或20% 蔗糖纳米微粒溶液与微的中心凹滑。在凹滑片上放置一个特定孔径的单张滤纸, 使滤纸的中心与纳米微粒溶液的液滴对齐。液滴与滤纸的接触导致溶液沿滤纸扩散, 填充孔隙 (图 1)。 注: 滤纸放在液滴的顶部, 而不是其他的周围, 以尽量减少在昆虫饲料的滤纸上的可能存在的纳米颗粒。</li>
	<li>将昆虫放在机械手的位置, 这样只有口器的远端区域才能接触到进料台上的湿润滤纸 (图 1)。使用昆虫引脚延长口器到滤纸上, 并允许昆虫在四十五年代进食。</li>
	<li>为了尽量减少在滤纸表面可能存在的液体膜上的昆虫, 请放置口器, 使它们与触及幻灯片平坦部分的滤纸的一部分 (i. e) 接触。, 而不是直接在幻灯片的凹部上方)。如果昆虫不表达对哺养的兴趣, 口器可以被拿着对滤纸与昆虫别针在哺养的时间期间。</li>
</ol>
3. 解剖
<ol>
	<li>将 PBS 溶液放在一个50毫米的手表玻璃上, 这样就有足够的解决方法来覆盖昆虫的身体。将手表玻璃置于立体和位置昆虫解剖设备 (弹簧微解剖剪刀, 昆虫针, 细点解剖钳) 旁边的立体。</li>
	<li>喂食后, 将昆虫从纸巾上取出, 并将其用翅膀封住。用弹簧微解剖剪刀将昆虫的头、腿和翅膀移开, 将昆虫放在手表玻璃的 PBS 溶液中 (图 2)。</li>
	<li>如果需要, 在解剖前麻醉昆虫。用钳子把昆虫的表皮靠近腹部的远端。用主手, 使用弹簧微解剖剪刀, 以削减前方向沿侧腹部的角质层, 从后端开始, 直到胸部达到。要特别小心, 确保只有角质层被切断, 并在昆虫的消化道没有损坏 (图 2)。</li>
	<li>用解剖剪刀做更多的角质层切开, 打开腹部, 露出消化道 (图 2)。去除腹部角质层, 脂肪体, 和其他结构的帮助下的昆虫引脚和搬迁他们的手表玻璃以外的后续处置, 只留下胸腔和消化道在手表玻璃。 注意: 解剖将揭示的作物, 这是一个囊状结构 (消化道的延伸) 位于靠近胸腔和腹部的交界处。</li>
	<li>如果作物没有暴露, 用剪刀在胸腔中附加切口直到作物显露出来。一旦作物是可见的, 切去剩下的部分昆虫只留下消化道与作物在手表玻璃 (图 2)。 注: 鳞翅目的作物几乎是透明的和玻璃纸一样的性质, 这可能难以识别, 如果它没有充满液体和扩大或如果它是在解剖切割。</li>
	<li>使用细点解剖钳将作物放置在片 (24 mm x 24 mm) 上进行后续成像 (图 2)。</li>
</ol>
4. 摄入纳米颗粒的测定
<ol>
	<li>将作物放在片上, 用细点解剖钳来防止作物破裂。使用 CY3 通道 (或相衬) 的倒置共焦显微镜成像20X 放大。在解剖后立即对作物进行成像, 以防止其干燥。</li>
	<li>在不控制显微镜操作阶段的手上, 手握一个磁力搅拌杆 (长度为41.3 毫米, 直径8毫米)。</li></ol>在作物附近来回挥动磁性搅拌棒 (距离裁剪大约 10 mm), 使每个来回运动大约需要一秒 (图 2)。
	<li>当磁性搅拌棒被挥动时, 检查庄稼为微粒。在近透明的作物中, 通过显微镜的眼球镜观察, 慢慢地来回移动操作阶段。如果纳米粒子存在于作物中, 表示正向摄食, 它们将响应并与磁性搅拌条 (图 2) 一致地波形。</li>

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作者没有什么可透露的。

昆虫口器功能是从研究的大体形态学 (e. g, 鳞翅目的喙功能相关的饮水秸秆25,26);然而, 最近的研究, 纳入实验证据, 导致了一个范例的转变, 我们理解昆虫口器和结构-功能关系的复杂性2,12,13,22,27. 虽然现代成像技术, 如扫描电?...

许多昆虫群有口器 (proboscises) 适应在液体哺养, 例如花蜜, 腐烂的果子, 汁液流动 (例如双翅目1, 鳞翅目2, 膜翅目3), 木质部 (半翅目4), 泪花 (鳞翅目5) 和血液 (Phthiraptera6, 蚤7, 双翅目7, 半翅目8, 鳞翅目9)。昆虫在液体中觅食的能力与生态系统健康相关 (如授粉10)、疾病传播4、11、biodiversification2、12, 并研究的收敛演化13。尽管各种各样的食物来源, 一个主题在一些液体哺养的昆虫是能力获取少量液体, 可能被限制到微或纳米微粒、液体影片或者多孔的表面。
考虑到液体喂养昆虫的广泛多样性 (超过20% 的所有动物种类14,15) 和它们在各种食物来源上觅食的能力, 了解它们的摄食行为和流体摄取机制是在许多领域很重要。例如, 昆虫口器功能在仿生技术的发展中扮演了重要的角色,例如, 微流控设备可以执行一些任务, 如使用类似的方法获取少量的流体由昆虫16。然而, 研究流体吸收机制的一个基本问题是, 不仅要确定昆虫如何以液体为食, 而且还要获得支持这种机制的实验证据。仅使用行为 (例如, 用喙12、17进行探测) 作为喂养的指示器是不够的, 因为它不能确认液体的成功摄取, 也不提供一种方法来确定液体通过昆虫传播。此外, 对少量的流体进行实验更好地代表了自然的喂养方案, 其中流体是限制资源2,12。
用 X 射线相衬成像与帝王蝶 (Danaus plexippus L.) 来评估蝴蝶是如何从多孔表面的少量液体中觅食的12。帝王蝶通过表皮投射 (背 legulae) 沿喙间的空间进行毛细管作用, 将液体局限在小孔中进入食物管。进入的流体形成一个影片在食物渠道墙壁生长并且崩溃入一台液体桥梁由高原不稳定12,18, 然后被传送到蝴蝶的内脏由吮泵的行动在头。虽然 x 射线相衬成像是一种优化的工具, 可视化流体在昆虫内部12,19,20,21, 该技术是不容易获得和更方便方法是需要快速评估昆虫的能力, 吸收液体和摄取它们。
要确定D. plexippus的进给机制是否适用于其他鳞翅目和苍蝇 (双翅目) (两组都是从多孔表面上的液体中进食), Lehnert et al.13应用了一种技术, 用于评估昆虫在多孔表面的少量液体中的进食能力, 这里详细报道。虽然这里概述的协议是用于研究, 使用湿润和多孔表面, 方法可以改变的其他研究, 如那些解决池喂养机制。此外, 应用扩展到其他领域, 包括微和生物技术。

<table><tbody><tr><td>20% sucrose solution</td><td>Domino Sugar</td><td></td><td>Sugar needed to produce the sucrose solution with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma-Aldrich</td><td>P5493</td><td>10X concentration diluted to 1X in dH&lt;sub&gt;2&lt;/sub&gt;O for insect dissections</td></tr><tr><td>Single depression concave slide</td><td>AmScope</td><td>BS-C6</td><td>Slide is necessary for feeding stage setup</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY6004700 (60 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY4104700 (41 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY3004700 (30 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY2004700 (20 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>NY1104700 (11 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TCTP04700 (10 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TETP04700 (8 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>TMTP04700 (5 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Filter paper</td><td>EMD Millipore</td><td>RTTP04700 (1 &amp;micro;m)</td><td>Nylon net filters and isopore filters needed to produce a porous surface for insect feeding</td></tr><tr><td>Iris microdissecting scissors</td><td>Carolina Biological Supply Company</td><td>623555</td><td>Scissors used for dissections</td></tr><tr><td>Insect pins (#1)</td><td>Bioquip Products</td><td>1208B1</td><td>Pins used during dissections and feeding trials</td></tr><tr><td>Extra-fine point dissecting forceps</td><td>Carolina Biological Supply Company</td><td>624684</td><td>Dissecting equipment</td></tr><tr><td>Leica M205 C Stereoscope</td><td>Leica Microsystems</td><td>M205 C</td><td>Stereoscope used for dissections</td></tr><tr><td>Inverted confocal microscope</td><td>Olympus</td><td>IX81</td><td>Fluorescent microscope used to detect magnetic nanoparticles</td></tr><tr><td>Fisherbrand PTFE Disposable Stir Bar</td><td>Fisherscientific</td><td>S68067</td><td>Magnet used to detect nanoparticles</td></tr><tr><td>Kimtech Science Kimwipes</td><td>Kimberly-Clark Professional</td><td>34155</td><td>Tissues used to secure insects during feeding trials</td></tr><tr><td>House fly (Musca domestica) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Blue bottle fly (Calliphora vomitoria) pupae</td><td>Mantisplace.com</td><td></td><td>insects for experiments</td></tr><tr><td>Cabbage butterfly (Pieris rapae) larvae</td><td>Carolina Biological Supply Company</td><td>144102</td><td>insects for experiments</td></tr><tr><td>Finnpipette F1&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>4641080N</td><td>micropipette for dispensing liquids</td></tr><tr><td>Finntip 250 pipette tips</td><td>ThermoFisher Scientific</td><td>9400250</td><td>micropipette tips</td></tr><tr><td>Microscope Glass cover slides (=coverslips) (24 x 24 mm)</td><td>AmScope</td><td>CS-S24-100</td><td>coverslips for viewing the insect&#039;s crop on confocal microscope</td></tr></tbody></table>

the ingestion of fluorescent, magnetic nanoparticles for determining fluid-uptake abilities in insects

液体喂养的昆虫摄取各种液体, 它们存在于环境中, 如水池、薄膜, 或局限于小孔隙。液体采集的研究需要评估口器结构和功能关系;然而, 流体吸收机制的历史推断从结构结构的观察, 有时无人陪伴的实验证据。在这里, 我们报告了一种新的方法, 以评估液体摄取能力与蝴蝶 (鳞翅目) 和苍蝇 (双翅目) 使用少量的液体。昆虫用20% 的蔗糖溶液与荧光、磁性纳米微粒混合, 从特定孔径的滤纸中过滤。该作物 (用于储存液体的内部结构) 从昆虫中移除并放在共聚焦显微镜上。一个磁铁是由作物挥舞, 以确定存在的纳米颗粒, 这表明, 如果昆虫能够摄取液体。这种方法是用来揭示一个广泛的喂养机制 (毛细管作用和液体桥梁形成), 可能共享之间的鳞翅目和双翅目时, 从多孔表面喂养。此外, 该方法可用于研究各种供液昆虫的摄食机制, 包括那些在疾病传播和仿生学中重要的, 以及可能涉及纳米或微米导管的其他研究液体运输需要验证。

研究液体喂养昆虫的吸水能力的模式需要确定进食发生的时间。此处所述的协议用于测试鳞翅目和双翅目中的极限孔径假设13。限制性孔隙大小假说指出, 如果孔径直径小于进料管道的直径12, 则液体喂养的昆虫无法从充液的孔隙中进食。从多孔表面进入的流体必须形成一个稳定的液体桥梁, 通过高原不稳定18, 以克服毛?...

Watch this Scientific Journal Video about The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects at JoVE.com

The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

液体喂养的昆虫有能力从多孔表面获取少量液体。该协议描述了一种方法, 直接确定昆虫的能力, 从多孔表面摄取液体使用荧光, 磁性纳米微粒的喂养解决方案。

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

Fluid-feeding insects ingest a variety of liquids, which are present in the environment as pools, films, or confined to small pores. Studies of liquid ...

the-ingestion-fluorescent-magnetic-nanoparticles-for-determining

Research

JoVE Journal

Biology

6.3K 次观看.  Kent State University at Stark. 液体喂养的昆虫有能力从多孔表面获取少量液体。该协议描述了一种方法, 直接确定昆虫的能力, 从多孔表面摄取液体使用荧光, 磁性纳米微粒的喂养解决方案。

视频: The Ingestion of Fluorescent, Magnetic Nanoparticles for Determining Fluid-uptake Abilities in Insects

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

在生物系统中的离子浓度测量荧光纳米粒子

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

科研

生物工程

Analyzing Cellular Internalization of Nanoparticles and Bacteria by Multi-spectral Imaging Flow Cytometry

Nanoparticulate systems have emerged as valuable tools in vaccine delivery through their ability to efficiently deliver cargo, including proteins, to antigen presenting cells1-5. Internalization of nanoparticles (NP) by antigen presenting cells is a critical step in generating an effective immune response to the encapsulated antigen. To determine how changes in nanoparticle formulation impact function, we sought to develop a high throughput, quantitative experimental protocol that was compatible with detecting internalized nanoparticles as well as bacteria. To date, two independent techniques, microscopy and flow cytometry, have been the methods used to study the phagocytosis of nanoparticles. The high throughput nature of flow cytometry generates robust statistical data. However, due to low resolution, it fails to accurately quantify internalized versus cell bound nanoparticles. Microscopy generates images with high spatial resolution; however, it is time consuming and involves small sample sizes6-8. Multi-spectral imaging flow cytometry (MIFC) is a new technology that incorporates aspects of both microscopy and flow cytometry that performs multi-color spectral fluorescence and bright field imaging simultaneously through a laminar core. This capability provides an accurate analysis of fluorescent signal intensities and spatial relationships between different structures and cellular features at high speed.
	Herein, we describe a method utilizing MIFC to characterize the cell populations that have internalized polyanhydride nanoparticles or Salmonella enterica serovar Typhimurium. We also describe the preparation of nanoparticle suspensions, cell labeling, acquisition on an ImageStreamX system and analysis of the data using the IDEAS application. We also demonstrate the application of a technique that can be used to differentiate the internalization pathways for nanoparticles and bacteria by using cytochalasin-D as an inhibitor of actin-mediated phagocytosis.

In this article, we describe a method utilizing multi-spectral imaging flow cytometry to quantify the internalization of polyanhydride nanoparticles or bacteria by RAW 264.7 cells.

由多光谱成像流式细胞仪分析细胞内化纳米粒子和细菌

Nanoparticulate  systems have emerged as valuable tools in vaccine delivery through their  ability to efficiently deliver cargo, including proteins, to ...

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

发育生物学

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

行为学

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

神经科学

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

免疫与感染

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles&#8217; integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Iron oxide nanoparticles are synthesized via a nonaqueous sol gel procedure and coated with anionic short molecules or polymer. The use of magnetometry for monitoring the incorporation and biotransformations of magnetic nanoparticles inside human stem cells is demonstrated using a vibrating sample magnetometer (VSM).

使用磁力学监测化学合成氧化铁纳米粒子的细胞合并和后续生物降解

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are the main branches of innate immunity, and many of the factors regulating these responses are evolutionarily conserved between invertebrates and mammals. Phagocytosis, the central component of cellular innate immunity, is carried out by specialized blood cells of the immune system. The fruit fly, Drosophila melanogaster, has emerged as a powerful genetic model to investigate the molecular mechanisms and physiological impacts of phagocytosis in whole animals. Here we demonstrate an injection-based in vivo phagocytosis assay to quantify the particle uptake and destruction by Drosophila blood cells, hemocytes. The procedure allows researchers to precisely control the particle concentration and dose, making it possible to obtain highly reproducible results in a short amount of time. The experiment is quantitative, easy to perform, and can be applied to screen for host factors that influence pathogen recognition, uptake, and clearance.

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

This protocol describes an in vivo phagocytosis assay in adult Drosophila melanogaster to quantify phagocyte recognition and clearance of microbial infections.

果蝇、果蝇细胞免疫应答的评估

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

Source: Hern&#225;ndez-Ortiz, J. A., et al. <a href="https://app.jove.com/v/63899">Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays.</a> J. Vis. Exp. (2022).
This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic substrate emitting fluorescence upon catalysis by the peroxidases, the captured immunocomplexes are detected under a microscope.

使用微流控设备的基于磁性纳米粒子的免疫测定

1. Device preparation

	Fill the channels with distilled water using a syringe. Make sure there are no leaks or resistance to flow. Immerse the device in ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Detecting Bacterial Contamination Using Magneto-fluorescent Nanosensors

In order to prepare solutions for reading in the magnetic relaxometry, 300 microliters of PBS is first pipetted into an eppendorf tube. Then, a sample of bacterial stock is added, followed by the addition of nanosensors. The solution is then transferred to a glass tube, and a piece of parafilm is placed on top in order to prevent evaporation.
The glass tube is then placed inside a larger NMR tube and inserted into the magnetic relaxometer. A baseline solution containing no bacteria and only nanosensor in PBS is used to get a baseline T2 reading as shown. Then, solutions containing various concentrations of bacteria are inserted into the magnetic relaxometer for analysis, and the changes in T2 values are caused by the binding between the nanosensors and the bacteria.
As shown, the presence of as few as one bacterial CFU can be detected within minutes using this modality. However, as the bacterial concentration increases, the MR readings are less quantifiable, which is why the use of fluorescence emission data is also tantamount to an accurate bacterial quantification. Before fluorescence data can be collected, the sample must first be centrifuged.
The solution is transferred from the glass tube to an eppendorf tube, and then centrifuged. This separates the bacteria and the nanosensors bound to it from free-floating nanosensors in solution. The supernatant is discarded and the bacterial pellet is resuspended in PBS. Finally, the sample may be analyzed via fluorescence emission. The strength of the emission will be relative to the amount of nanosensors remaining in solution, and therefore, also to the amount of bacteria present.

吸收荧光、磁性纳米微粒测定昆虫的吸液能力

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