私たちのためにMCF-7を準備してくれたP. Yeung氏に感謝します。この作業は、香港特別行政区の研究助成審議会（プロジェクト番号17259316,17207715,17207714,17205215、およびHKU 720112E）、イノベーションおよび技術支援プログラム（ITS / 090/14 ）、およびHKUの大学開発基金。

<ol>
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著者は何も開示することはない。

ATOMシステムのセットアップ中に特別な注意が必要な技術的な詳細がいくつかあります。第1に、非対称/斜めのスペクトル符号化された照明が、吸収コントラストにおける残留位相勾配成分（ すなわち、シャドーイング効果）を導入し、ATOMにおける位相勾配コントラストの増強に影響を及ぼす可能性があることに留意することが重要である。したがって、斜め照明のこの効果は最小限?...

光学イメージングは​​、多くの細胞/亜細胞成分の詳細な空間分布を（ほとんど）非侵襲的に視覚化するための強力なツールおよび細胞ベースのアッセイを提示し、細胞の形態学的、生物物理学的および生体分子の多数の特徴を明らかにする。しかしながら、単一の細胞から高含量の情報を抽出するこの能力は、莫大かつ異種の細胞集団を調査しなければならない場合、一般に損なわれている。これは、測定スループットとコンテンツとの間の細胞ベースアッセイにおける共通のトレードオフを示す。注目すべき例は、フローサイトメトリーにイメージング能力を加えることにより、従来の非イメージングフローサイトメーターに比べて少なくとも1-2桁のスループットのダウンスケーリングがもたらされたことである。それは、標準的なフローサイトメーター1では不可能である複雑な形態学的な単一細胞分析を提供することができるが、イメージングフローサイトメトリーは、一般に、高い統計的信頼性で細胞の異種性を念入りにする。これは、生物学における新たな発見や疾患の病因の理解を得るために必要です。レーザービーム走査（ガルバノメーターミラーなど）、および/または画像センサー（ 例えば、電荷結合素子（CCD）および相補型金属光検出器）によって課される固有の速度限界には、酸化物半導体（CMOS））である。走査速度は、走査ミラーの機械的な慣性によって本質的に制限されるが、CCDまたはCMOSのフレームレートは、撮像速度と感度との間の基本的なトレードオフによって制限される（ すなわち、フレームレートを増加させると、 、 およびその逆）。 全光学的、超高速画像符号化メカニズムに基づいて、光学的時間ストレッチ撮像は、ハイスループット画像フローサイトメトリーのための魅力的なプラットフォームとして実証されている従来の画像センサや機械的なレーザ走査を必要とせずに、タイムストレッチイメージングの動作原理の詳細な説明は、参考文献4,5,6,7に見出すことができる。簡単に説明すると、2つの交換可能なマッピングステップからなる：（i）画像化された標本の空間座標が光パルスビーム8,9のスペクトルにわたって異なる波長にマッピングされるスペクトル符号化（波長空間マッピング）個々のレーザパルスの波長成分が群速度分散（GVD）を経て時間的（波長掃引）波形（ 図1 ）に変換（伸張）される分散フーリエ変換（波長 - 時間マッピング） 9 。重要な時間ストレッチイメージングの特徴は光増幅であり、超高速光検出とGVD損失による感度の低下との戦いにおいて重要な役割を果たし、光検出器のノイズ9によって汚染されることなくイメージ信号対雑音比（SNR）を向上させる。各レーザーパルスは、細胞の一方向の流れに直交する画像化された標本のラインスキャンをコード化するので、有効なライン走査速度は、典型的には10MHzを超えるレーザー反復速度によって決定される。この超高速操作により、10,000〜100,000細胞/秒（ すなわち、従来のイメージングフローサイトメトリーよりも10〜100倍高い）のスループットで、ぼやけのない単一細胞画像捕捉が可能になる。結果として、時間ストレッチイメージングは​​、特に、未知の異種性または珍しい/異常な細胞をかなりの集団（数千〜数百万個）で同定する必要がある場合に、ハイスループット、単一細胞、セル<html>珍しいがん細胞スクリーニング10または微細藻類分類11など）が挙げられる 。 時間ストレッチイメージングは​​、主に、明視野（BF）イメージキャプチャに依存し、そこからイメージコントラストが細胞2,3,10,10,11からの光散乱および吸収によって生成される。このようなラベルフリーの単一細胞イメージング能力は、細胞毒性および光退色などの蛍光標識に関連する有害な影響を回避することができ、さらに、細胞および細胞内テクスチャおよび形態に基づく単一細胞分析に有益な情報を提供する。これらのラベルフリーパラメータは、特に莫大な細胞集団が利用可能な場合に、細胞の深部画像分類に有効であることが証明されている11 。&quot;xref&quot;&gt; 12。しかしながら、多くの場合、BFイメージングは​​、標識を含まない透明細胞の詳細な形態を明らかにするために十分なコントラストを提供することができない。超高速フレームレート13,14,15で撮像コントラストを向上させるために、異なるラベルフリー位相差、時間ストレッチ撮像モダリティが開発されている。これらの技術の中でも、Schlieren撮影と同様の概念に基づく位相勾配（微分干渉コントラスト（DIC）のような）コントラストを明らかにするために、非対称検出時間ストレッチ光学顕微鏡法（ATOM）超高マイクロ流体スピード（最大10m / s）での単一細胞の高コントラスト造影。この効果は、画像符号化されたビーム経路を部分的に遮断するか、または光検出の前にビームを傾斜させることによって、斜めの検出または照明によって容易に生成することができる。 ATOMのもう一つの利点は、逆の方向に沿って2つの位相勾配コントラストを同時に取得する能力。強度減算および2つの反対のコントラスト画像の合計は、同じラインスキャンからのそれぞれ異なる位相勾配コントラストおよび吸収コントラストをもたらす。この作業では、光学セットアップの確立、サンプルの準備、データの取得と可視化を含む、ATOMの実装を記述する詳細なプロトコルを提示します。具体的には、この研究は、ヒト血液細胞、癌細胞、および植物プランクトン（微細藻類）の単一細胞イメージングを用いたATOM操作を実証する。これは、生物医学分野だけでなく、海洋およびバイオ燃料の研究でも、イメージングフローサイトメトリーへのATOMの適用性を強調している17,18 。

<table><tbody><tr><td>Nikon Plan Fluorite Physiology Objectives 40X</td><td>Nikon</td><td>MRF07420</td><td>Objective lens (Obj2)</td></tr><tr><td>Olympus Plan Fluorite Objective, 0.75 NA, 0.51 mm WD, 40X</td><td>Olympus</td><td>RMS40X-PF</td><td>Objective lens (Obj1)</td></tr><tr><td>N-BK7 Plano-Convex Lenses</td><td>Thorlabs</td><td>LA1145-C</td><td>For relaying spectral shower</td></tr><tr><td>FC/APC Fiber Collimation Package</td><td>Thorlabs</td><td>F220APC-1064</td><td>For outputing and collecting laser pulses</td></tr><tr><td>Pellicle beamsplitter</td><td>Thorlabs</td><td>BP145B3</td><td>For making beam replica</td></tr><tr><td>Protected silver mirror</td><td>Thorlabs</td><td>PF10-03-P01</td><td>For reflecting light</td></tr><tr><td>800-1650nm 12GHz single mode DC-coupled NIR Photoreceiver</td><td>Newport</td><td>1544-B</td><td>For converting light into electrical signal</td></tr><tr><td>Infiniium High-Performance Oscilloscope</td><td>Agilent</td><td>DSOX91604A</td><td>To save the light-converted electrical signals</td></tr><tr><td>HI1060 optical fiber</td><td>Corning</td><td>HI1060</td><td>Optical fiber for time-stretch</td></tr><tr><td>YTTERBIUM DOPED FIBER AMPLIFIER</td><td>Keopsys</td><td>KPS-STD-BT-YFA-37-BO-SM-111-FA-FA</td><td>Optical in-line amplifier</td></tr><tr><td>Holographic grating</td><td>Wasatch Photonics</td><td>020305-6</td><td>Grating</td></tr><tr><td>Infuse/Withdraw Syringe Pumps</td><td>Harvard Apparatus</td><td>PHD 2000</td><td>Syringe pump for sample loading in micro-fluidic channels</td></tr></tbody></table>

microfluidic imaging flow cytometry by asymmetric-detection time-stretch optical microscopy (atom)

フローサイトメトリーの高度な開発において、多次元データ分析、したがってより高い信頼性の統計結果を可能にする測定可能なパラメータの数をスケーリングすることが、主な傾向であった。注目すべきことに、高解像度イメージング能力を加えることにより、細胞/細胞間構造の複雑な形態学的分析が可能になる。これは標準的なフローサイトメーターでは不可能です。しかし、それは細胞機能の知識を進歩させる上で貴重であり、生命科学の研究、臨床診断、環境モニタリングに役立ちます。フローサイトメトリーへのイメージング能力の組み込みは、主にカメラ技術における速度および感度の制限によるアッセイスループットの低下をもたらす。イメージ品質を維持しながらイメージングフローサイトメトリーに直面するこの速度またはスループットの課題を克服するために、非対称検出時間ストレッチ光学顕微鏡法（ATOM）が、高コントラストの単一細胞イメージを可能にすることが実証されている100,000細胞/秒という高い画像処理スループットで、細胞内分解能を有する。 ATOMは、超高速広帯域レーザパルスの使用による全光学画像の符号化および検索に依存する従来の時間ストレッチ撮像のイメージングコンセプトに基づいて、非標識/非染色細胞の画像コントラストを向上させることによって画像性能をさらに進歩させる。これは、単一ショット広帯域パルスにスペクトル的に符号化されたセルの位相勾配情報にアクセスすることによって達成される。したがって、ATOMは、細胞型、状態、および機能さえ示す単一細胞形態およびテクスチャ情報のハイスループット測定において特に有利である。最終的に、これは細胞の生物物理学的表現型解析のための強力なイメージングフローサイトメトリープラットフォームとなり、現在の最先端の生化学マーカーベースの細胞アッセイを補完することができます。この作業では、ATOMシステムのキーモジュールを確立するためのプロトコルについて説明します（光フロントエンドからデータpロゼイングおよび可視化バックエンド）、ならびにヒト細胞および微細藻類を例として使用して、ATOMに基づくフローサイトメトリーのイメージングのワークフローを提供する。

<html>この研究では、哺乳類細胞（ヒト末梢血単核細胞（PBMC）および乳癌細胞（MCF-7））および植物プランクトン（ScenedesmusおよびChlamydomonas）を有するATOMによる2つの単一細胞イメージング実証が示されている。最初の実験は、血液中の循環腫瘍細胞（CTC）の検出、列挙、および特徴付けのための液体生検への関心の高まりによって動機付けられた23 ?...

Watch this Scientific Journal Video about Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM at JoVE.com

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

このプロトコールは、超高速マイクロ流体流における単一細胞イメージングのための非対称検出時間ストレッチ光学顕微鏡検査システムの実施およびイメージングフローサイトメトリーにおけるその応用を記載する。

非対称検出時間ストレッチ光学顕微鏡（ATOM）によるマイクロ流体イメージングフローサイトメトリー

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

microfluidic-imaging-flow-cytometry-asymmetric-detection-time-stretch

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Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

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10.2K ビュー.  The University of Hong Kong. このプロトコールは、超高速マイクロ流体流における単一細胞イメージングのための非対称検出時間ストレッチ光学顕微鏡検査システムの実施およびイメージングフローサイトメトリーにおけるその応用を記載する。 

ビデオ: Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

生物工学

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

複数の異なった株を勉強するためのマイクロ流体デバイス

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

携帯電話上の広視野蛍光顕微鏡と蛍光イメージングフローサイトメトリー

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

流体デバイスと顕微鏡とフローサイトメトリーを組み合わせて、空間スケールを越えた多孔質培地における微生物輸送を研究

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

環境学

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

構造化照明顕微鏡データの時空間画像相関分光法により定量化したT細胞における皮質アクチンの流れ

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

免疫・感染学

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

磁気ナノ粒子を用いたイムノアッセイのための千鳥制限を有するマイクロ流体アクリルデバイスのコンピュータ数値制御マイクロミリング

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

工学

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

1分子研究のためのデュアル光ピンセットとマイクロ流体工学の使用

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

生化学

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, appearance of defects in microcirculation can be a primary indicator in the development of multiple pathologies. Advances in optical imaging have made intravital microscopy (IVM) a practical approach, permitting imaging at the cellular and subcellular level in live animals at high-speed over time. Yet, despite the importance of maintaining adequate tissue perfusion, spatial and temporal variability in capillary flow is seldom documented. In the standard approach, a small number of capillary segments are chosen for imaging over a limited time. To comprehensively quantify capillary flow in an unbiased way we developed Spatial Temporal Analysis of Fieldwise Flow (STAFF), a macro for FIJI open-source&#160;image analysis software. Using high-speed image sequences of full fields of blood flow within capillaries, STAFF produces&#160;images that represent motion over time called kymographs for every time interval for every vascular segment. From the kymographs STAFF calculates velocities from the distance that red blood cells move over time, and outputs the velocity data as a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses. In normal mouse livers, STAFF analyses quantified profound differences in flow velocity between pericentral and periportal regions within lobules. Even more unexpected are the differences in flow velocity seen between sinusoids&#160;that are side by side and fluctuations seen within individual vascular segments over seconds. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of capillary flow.

To quantify microvascular flow from high speed capillary flow image sequences, we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow) software. Across the full image field and over time, STAFF evaluates flow velocities and generates a sequence of color-coded spatial maps for visualization and tabular output for quantitative analyses.

微小血管系におけるフィールドワイズフローの空間時間解析

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

<meta charset="utf8"></head><body>シングルセルサイトカイン分泌解析のためのマイクロ流体液滴アッセイ

Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. This response involves numerous immune cells secreting various cytokines to orchestrate the immune reaction. However, the secretion dynamics, amounts, and co-occurrence of the different cytokines by various cell subtypes remain poorly understood due to a lack of appropriate tools to study them. Here, we describe a protocol using a microfluidic droplet platform that allows the time-resolved quantitative measurement of secretion dynamics for several cytokines in parallel on the single-cell level. This is enabled by the encapsulation of individual cells into microfluidic droplets together with a multiplexed immunoassay for parallel quantification of cytokine concentrations, their immobilization for dynamic fluorescent imaging, and the analysis of the respective images to derive secreted quantities and dynamics. The protocol describes the preparation of functionalized magnetic nanoparticles, calibration experiments, cell preparation, and the encapsulation of the cells and nanoparticles into droplets for fluorescent imaging and subsequent image and data analysis using the example of lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The presented platform identified distinct cytokine secretion behavior for single and co-secreting cells, characterizing the expected phenotypic heterogeneity in the measured cell sample. Furthermore, the modular nature of the assay allows its adaptation and application to study a variety of proteins, cytokines, and cell samples, potentially leading to a deeper understanding of the interplay between different immune cell types and the role of the different cytokines secreted dynamically to shape the tightly regulated immune response. These new insights could be particularly interesting in the studies of immune dysregulations or in identifying target populations in therapy and drug development.

<meta charset="utf8"></head><body>著者スポットライト:免疫応答における多機能性と不均一性の解明

The protocol describes an advanced microfluidic platform to quantitatively measure cytokine secretion dynamics of individual human peripheral blood mononuclear cells. The platform measures up to three cytokines in parallel (IL-6, TNF&#945;, and IL-1&#946;) for each individual cell stimulated with lipopolysaccharide as an example.

個々の多機能細胞の同時および逐次サイトカイン分泌を解明するためのマイクロ流体アプローチ 

Infections, autoimmune diseases, desired and adverse immunological responses to treatment can lead to a complex and dynamic cytokine response in vivo. ...