この作品は国立科学財団 (NSF) によってサポートされていた許可なし。IOS 1354956。博士アンドリュー d. ウォーレン (鱗翅目、生物多様性、フロリダ州フロリダ大学自然史博物館・ マクガイア センター) 蝶の画像を使用する許可を感謝します。

1. 昆虫種、ソリューションおよび供給ステーションのセットアップの準備
注: 流体吸収能力、口器形態22,23に関する以前の研究の使用のため、キャベツ蝶 (モンシロチョウL.、シロチョウ科) は代表チョウ目の種として選択されます。家 (イエバエL., イエバエ科) や青いボトル蠅 (オオクロバエ vomitoria L., クロバエ科) は、彼らは多孔性の表面の13の餌、多く観察されるために使用されます。
<ol>
	<li>それら人工のダイエット (材料の表を参照してください) 蛹環境室の大人として浮上するまで昆虫のサプライヤーとリアから幼虫として順序モンシロチョウは 23 ° C、18 L: 6 日長に設定。順序M。イエバエと蛹としてC. vomitoriaとモンシロチョウと同じ環境条件でリアのそれら。彼らは供給実験前蛹から出てくる後は、チョウ類成虫、ハエを給紙されません。</li>
	<li>20% ショ糖液 (コントロール) と流体吸収をテストする 20% ショ糖ナノ粒子溶液を準備します。蛍光磁性ナノ粒子を追加することによってナノ粒子溶液を準備 (鉄酸化物ポリアクリル酸コーティング、約 20 nm 径の) 20% ショ糖液 (1 Mg/ml と dH2O ナノ粒子と 20% スクロースを24ソリューションには、1:1)。リン酸緩衝生理食塩水 (PBS) の 1 x ソリューションの準備 (10 x dH2O、pH 7.4 で 1 倍に希釈)、解剖に使われます。</li>
	<li>手動式マニピュレーターのクランプと別の餌ステージ (平らなプラットフォーム) で構成される供給ステーションの設定 (図 1)。摂食の段階に凹面のスライドを配置、毛穴サイズ直径 1、5、8、11、20、30、41、または 60 μ m、膜フィルターの孔のサイズ径ナイロン ネット フィルターまたは供給実験の近くに 10 μ m。</li>
</ol>
2. 餌のプロトコル
<ol>
	<li>昆虫の体、足と翼を折りたたんだティッシュ ペーパーにラップします。虫の位置を頭と口器のみが公開されます。昆虫、昆虫摂食の段階 (図 1) 上記の中断は、マニピュレーターをクランプによってまとめられる 2 つの顕微鏡のスライド間の翼に配置します。</li>
	<li>凹面のスライドの中央に 20% ショ糖溶液のマイクロ ピペットで 20% ショ糖ナノ粒子液 30 μ L の液滴を管理します。ナノ粒子溶液の液滴とフィルター紙の中心が揃うように、凹面のスライドに特定の細孔径の単一のフィルター紙を置きます。液滴とろ紙の間お問い合わせは満ちる気孔 (図 1)、フィルター紙に沿って広がっているソリューションの結果します。 注: フィルター ペーパーは、昆虫がフィード フィルター紙の上にナノ粒子が存在する可能性を最小限に抑えるため、周りの他の方法ではなく、液滴の上部に配置されます。</li>
	<li>口器の遠位領域のみの摂食の段階 (図 1) 接液部のフィルター ペーパーを連絡することができるように、マニピュレーターを持つ昆虫を配置します。虫ピンを使用してフィルター紙の上に口器を拡張し、45 のフィードに昆虫を許可する s。</li>
	<li>フィルター ペーパーの表面に存在するかもしれない液体の膜にする昆虫のチャンスを最小限に抑えるために口器の位置 (すなわちスライドの平らな部分に触れているろ紙の部分と接触しているので。、スライドの凹の部分の上に直接ではなく)。虫餌に興味を表現していない、口器は餌の時間の期間のろ過紙、虫ピンに保持することができます。</li>
</ol>
3. 解剖
<ol>
	<li>昆虫の体をカバーする十分な解決策があるので、50 ミリメートル時計ガラスに PBS のソリューションを配置します。時計ガラス、万国実体写真の横にある装置 (春マイクロはさみ、昆虫ピン、鉗子を切り裂く細かい点を解剖) を解剖位置と昆虫の下に配置します。</li>
	<li>授乳後ティッシュ ペーパーから昆虫を削除し、翼を閉じた状態でそれを保持します。春の微小解剖はさみで頭、足、および昆虫の翼を取り外し、時計ガラス (図 2) PBS 溶液で昆虫を置きます。</li>
	<li>必要な場合は、解剖前に昆虫を麻酔します。腹部遠位近傍キューティクルによって昆虫を保持するために鉗子を使用します。支配的な手で胸部に達するまで後端から始まる、腹部の側面に沿って前方方向でキューティクルをカットするのにはさみを解離性春マイクロを使用します。表皮だけをカットし、(図 2) が破損している昆虫の中の消化管ではないことを確保するための特別な注意を取る。</li>
	<li>消化管内部 (図 2) を明らかにする腹部を開く解剖はさみでキューティクルの追加カットを作る。昆虫ピンの支援を受けて腹部キューティクル、脂肪体と他の構造を削除し、時計ガラスの胸部と消化管だけを残して後の処分のため時計のガラスの外にそれらを再配置します。 注: 解剖胸部と腹部の合流点近くに位置する嚢のような構造 (消化管の延長) である作物が表示されます。</li>
	<li>作物が公開されていない場合は、作物を明らかにするまで、はさみで胸郭に追加切開を確認します。作物が表示、時計ガラス (図 2) で作物と消化管だけを残して昆虫の残りの部分を切り取る。 注: 鱗翅目昆虫の作物はほぼ透明で流体がないかどうかと、展開を認識することは困難かもしれない性質または解剖中切ってかどうかセロファンのような。</li>
	<li>その後画像 (図 2) の coverslip (24 mm × 24 mm) の上に作物を配置するのに鉗子を切り裂く細かい点を使用します。</li>
</ol>
4. 摂取されたナノ粒子の定量
<ol>
	<li>位置、細かい点を解剖鉗子使用して coverslip の作物は作物の破裂を防ぐために注意します。20 倍の倍率で画像を倒立顕微鏡で CY3 チャネル (または位相) を使用します。乾燥からそれを防ぐために郭清後すぐに作物をイメージします。</li>
	<li>顕微鏡の動作段階の制御ではない手で電磁攪拌バー (41.3 mm の長さと直径 8 mm) を保持します。</li></ol>前後それぞれモーション約 1 秒 (図 2) では、作物 (作物から約 10 mm 距離) の近くに来たり、電磁攪拌棒を振る。
	<li>電磁攪拌棒が振られる間は、ナノ粒子の作物を検査します。ほぼ透明な作物内でナノ粒子の動きのための顕微鏡の接眼レンズをのぞきながら、前後営業ステージをゆっくりと移動します。ナノ粒子が作物である場合肯定的な給餌を示す彼らは応答と波磁気と一体で攪拌棒 (図 2)。</li>

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著者が明らかに何もありません。

昆虫の口器機能は形態学からみた歴史的に (e.g、鱗翅目のテング機能関連飲むわら25,26);。ただし、実験的証拠が組み込まれた最近の研究は、昆虫の口器と構造機能関係2,12,13の複雑さの私達の理解のパラダイム シフトで起因しました。,22,...

多くの昆虫のグループ (proboscises) の口器がある適応流体を供給するため、花の蜜、果実の腐敗など sap フロー (例:双翅目1、鱗翅目2膜翅目3)、木部 (半翅目4)、涙 (鱗翅目5) と血 (目, シラミ目6ノミ7、双翅目7、8半翅目、鱗翅目9)。流体を餌に昆虫の能力は生態系の健全性 (例えば受粉10)、病気伝達4,11生物2,12、および研究に関連します。収斂進化13。多彩な食料源、にもかかわらずいくつかの流体餌昆虫の中でテーマとマイクロやナノ液滴、液膜または多孔性の表面に限定されるが、液体の少量を取得する機能です。
流体食性昆虫 (すべての動物種の14,の15の 20% 以上) と様々 な食料源を餌に能力の膨大な多様性を与えられた彼らの餌を理解動作流体吸収機構の解明、多くの分野で重要であります。昆虫の口器の機能、たとえば、バイオミメティック技術、例えば少量の流体が用いられるものと同様の方法を使用しての取得などのタスクを実行できるマイクロ流体デバイスの開発の役割を果たしています。昆虫16。流体吸収メカニズムの研究における根本的な問題は、流体、昆虫のフィード方法のみを決定するが、メカニズムをサポートして実験的証拠を取得します。のみ動作を使用して (例えば、テング12,17プロービング) 流体の正常な吸収を確認しませんも、それがルートを決定するための手段を提供するため、供給のための指標が十分なこと流体の旅行昆虫を通過するとき。また、少量より流体の実験を実行する流体が制限リソース2,12自然の供給シナリオを表します。
位相コントラスト イメージング多孔性の表面12から液体の少量で蝶がどのようにフィードを評価するオオカバマダラ (オオカバマダラl.) を併用した x 線します。モナルカ蝶フード運河に小さな毛穴に閉じ込められた流体をもたらすテングに沿って毛細管を介して表皮突起 (背 legulae) の間にスペースを使用します。着信の流体は、成長し高原不安定12,18, 頭に吸引ポンプの作用で、蝶の腸を運ばれる液体の橋に崩壊食品管壁のフィルムを形成します。X 線位相コントラスト イメージング昆虫12,19,20,21内の流体の流れを可視化するための最適なツールですが、テクニックはすぐに利用できるより便利メソッドは、昆虫の能力吸収液の迅速な評価のために必要ですし、摂取します。
D. plexippusの供給メカニズム適用他の鱗翅目に、ハエ (双翅目)、(両方のグループが多孔性の表面の液体のフィード) かどうかを決定する Lehnertら13には、ここで詳細に報告されている多孔性の表面の液体の少量を餌に昆虫の能力を評価するための手法が適用されます。ここで説明したプロトコルは接液部を使用して研究と多孔質の表面、プール供給メカニズムのアドレスなど、他の研究方法論を変更できます。また、アプリケーションは、マイクロ流体システムと材料技術など、あらゆる分野に拡張します。

<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

流体食性昆虫を摂取する環境では、プール、映画、または小さな毛穴に閉じ込められた液体の様々 な。口器の構造と機能の関係を評価する液体の獲得の研究が必要とただし、流体の吸収機構の解明は歴史的構造のアーキテクチャは、実験的証拠と時々 一人旅の観察から推論されます。蝶 (鱗翅目) とハエ (双翅目) の液体の少量を使用して流体吸収能力を評価する手法を報告する.昆虫は、特定の細孔径のフィルター ペーパーから蛍光、磁性ナノ粒子と混合 20% ショ糖液が供給されます。作物 (流体を格納するために使用される内部構造) は、昆虫から削除され、共焦点顕微鏡上に配置。磁石は昆虫が水分を摂取することができるかどうかを示すナノ粒子の存在を確認するための作物で手を振った。この手法を使用して、機構の解明、広範な供給 (毛細管と液体ブリッジの形成) 多孔性の表面からを供給するとき可能性のある鱗翅目、双翅目の間で共有されます。さらに、このメソッドは液餌昆虫、病気の感染、バイオミメティックス、ナノまたはマイクロ導管を含む可能性のある他の研究で重要なを含むさまざまな機能の研究使用できます、液体の輸送には、検証が必要です。

液餌昆虫の中で流体吸収能力のパターンの研究には、餌が発生したときの決意が必要です。ここで説明したプロトコルを使用して、鱗翅目、双翅目の13の間制限の細孔サイズ仮説をテストしています。制限の細孔サイズ仮説は、細孔径が供給管12の直径より小さい場合は、液体充填孔から液餌昆虫を与えることができない状態します...

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

流体食性昆虫は、多孔性の表面の液体の微量を取得する能力を持っています。このプロトコルでは、直接蛍光、磁性ナノ粒子供給溶液を用いた多孔質表面から液体を摂取する昆虫の能力を決定する方法について説明します。

蛍光灯、摂取昆虫における流体吸収能力を決定するための磁気ナノ粒子

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 ビュー.  Kent State University at Stark. 流体食性昆虫は、多孔性の表面の液体の微量を取得する能力を持っています。このプロトコルでは、直接蛍光、磁性ナノ粒子供給溶液を用いた多孔質表面から液体を摂取する昆虫の能力を決定する方法について説明します。

ビデオ: 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.

生体内イオン濃度の測定のための蛍光ナノ粒子

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

生物工学

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.

マルチスペクトルイメージングフローサイトメトリーによるナノ粒子と細菌の細胞内在化を分析する

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.

光シートベースの蛍光生活の顕微鏡または固定し、染色<em&gt;モドキTribolium castaneum</em&gt;胚

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

発生生物学

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.

定量的食物摂取の試金を組み合わせると強制的にショウジョウバエの食欲を勉強するニューロンを活性化

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

行動学

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.

生物発光カルシウムインジケーター、GFPエクオリンに基づいて生体内脳機能イメージングのアプローチで

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 ...

神経科学

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.

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

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

免疫・感染学

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).

磁気測定を用いて細胞の組み込みとそれに続く化学的合成酸化鉄ナノ粒子の生分解をモニタリングする

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.

ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

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.

マイクロ流体デバイスを用いた磁性ナノ粒子を用いたイムノアッセイ

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

免疫学

抗体ベースの技術

抗体ベースのイメージングと検出方法

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.

蛍光灯、摂取昆虫における流体吸収能力を決定するための磁気ナノ粒子

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