私たちは、ギャビー·マルティンスと、この作品が始まったとフィジーとImageJのプロジェクトへの貢献者のすべてEMBO2010 3D発達イメージングワークショップの主催者に感謝したいと思います。

1。4Dタイムラプスイメージング実験画像取得のために使用される設定は、使用する機器に応じて異なります。励起波長、ピンホールサイズ、対物レンズの開口数、サンプルが埋め込まれたサンプルおよび媒体の屈折率：光学部の共焦点顕微鏡の能力試料は、多数の要因に依存する。選択された共焦点ピンホールのサイズは、収集された光学部の厚さを決定する。小さなピンホールは、より薄い光学部のz軸の解像度を増加させるしかし、捕捉された光の量を減少を生じる。大きいピンホールは、z軸の解像度を低減しかし捕捉された光の量を増加させる、光学部の厚さを増加させる。 補正前の4Dデータの収集の際に考慮すべき追加的な要因は次のとおりです。 <ol><li>削除するには、スキャン速度を最適化する、または制限、Dドリフトシングル時点の捕獲をuring。そこで画像収集にかかる時間は、タイム点の間隔のごく一部である必要があります。 </li><li>それは、必要な補正を決定することは不可能であるように、完全に反復構造又は格子は、登録のための構造体として適していない。ランダムに分布ビーズや凹凸構造が明確な登録ができるようになります。 </li><li>ドリフトが予想される場合、関心領域は、画像スタック内に留まる確実にするために、走査領域と上下フォーカスリミットを増加させる。 </li><li>確保に加えて、関心対象の構造を解決するため研究されて動的イベントに対する時間分解能を提供するためにサンプリングレートを設定するための適切な空間分解能がある。 注意：画像の時点間の時間がありますので定期的な繰り返しイベントの間に、少なくとも半分の時間間隔も不規則なイベントのために減少するはずである。 </li></ol> 2。共焦点Dを開くataset オープンソースパッケージフィジーは顕微鏡実験から収集されたデータに多数のプロセスを実行するために事前にインストールされているプラ​​グインが含まれているImageJのプログラム、の分布である。このソフトウェアは簡単にプラグインの更新アーキテクチャを提供し、正しい3Dドリフトプラグインは、このプロトコルで使用のコピーが含まれています。ソフトウェアはオープン顕微鏡環境のバイオフォーマットのインポートプラグインを使用することにより、独自の顕微鏡画像フォーマットの広大な配列のインポートをサポートしています。 <ol><li>フィジーのプログラムをインストール（http://fiji.sc/Downloads）。 </li><li> LOCIバイオフォーマットインポータープラグインを使用して取得したデータセットをロードします。 </li><li>データセットは、プログラムに割り当てられたメモリよりも大きい場合は、メモリ管理部内の「使用する仮想スタックオプション」を選択します。 </li></ol> 3。後処理で3Dオブジェクトのドリフトを補正する長時間タイムラプスの過程埋め込まれた場合でも、試料が移動することが実験する。任意の動きを補正し、撮像されたマイグレーション·イベントを分析できるように、画像の後処理を行うことができる。すべての画像後処理は明らかに、この作品から派生した分析の方法論で説明しなければならない。 <ol><li>データセットがロードされると、正しい3Dドリフトプラグインを実行します。 </li><li>複数の画像チャネルがある場合、画像を登録するために使用するチャンネルを選択する。これは理想的ではなく任意の渡り鳥やモバイル要素よりサンプル内の静的な構造を表している必要があります。これが不可能な場合は、少なくとも運動のチャネルが選択されるべきである。 </li><li>この分析に使用されるシステムで使用可能なRAMが元のデータセットの倍未満のサイズであれば、使用する仮想スタック]オプションを選択します。これは、画像シーケンスとしてではなく、RAMにファイルを保存するよりも、登録されhyperstackを格納します。 <ol><li>出力にプラグイン用のフォルダを選択し、個々のIMA修正GESファイル。個別の画像ファイルは、各z位置において各チャンネル毎に作成される。 </li></ol></li><li>プラグインは次いで、データセットに適用される必要な補正を決定するために、各時点の間のペアワイズ位相相関分析を行う。 </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

拡張タイムラプス顕微鏡実験に由来するデータセットのサンプルのドリフトを補正するために後処理ソフトウェアを使用する我々の能力は、多くの要因によって制限される。サンプルの渡り鳥の移動に対するドリフトを識別する能力は、使用される細胞マーカーに依存している。どちら広くサンプル内で発現しているか、画像取得中回遊イベントに関与していない細胞マーカーは、ドリフト?...

共焦点イメージングは​​広く形態の細胞の動きや変化に追従するために、細胞および発生生物学で使用されています。異なる焦点面で光一連のセクションをキャプチャすることができ、次いで、3Dデータセットの経時系列を作成することにより、四次元（4D）に拡張することができ、試料の3次元（3D）モデルを生成する。 4Dデータセットの生成は細胞の動きや行動の詳細な測定を可能にします。長期経時実験では、試料の動きを観察することが一般的である。これは、ハードウェア制御の段階と焦点位置のわずかな不正確によって引き起こされる場合があります。他の例では、ドリフトは、サンプルの取り付けメディア内のサンプルの伸びや柔軟性によって誘発される運動の結果ですが。方法はシステムを中心に、ハードウェアの改善を含め、これらの動きを補正するか、制限するために存在し、封入剤の剛性を増加させた。しかしながら、これらのアプローチは、撮像sに多くの場合には適用できないらアップサンプルの維持および増殖に適した条件を提供するために必要。オープンソースのソフトウェア·ソリューションは、ImageJのか、フィジーでSTACKREGとTurboReg（http://bigwww.epfl.ch/thevenaz/stackreg/）5プラグインを使用することで、時間をかけて、2次元の動きを補正するために存在しないが、これらはできない4Dデータセットに適用される。 サンプルドリフトを補正するために、我々はオープンソースの画像処理プラットフォーム、フィジー1を利用するプラグイン（正しい3Dドリフト）を開発した。我々のプラグインは、三次元の時間経過実験において、試料ドリフトの結果として生じる動きを補正する位相相関の登録を行うことができる。位相相関6は、画像間の変換を決定するための計算上効率的な方法である。プラグインは、ここで説明するには、Preibisch らが開発した位相相関ライブラリを利用しています。7。マルチチャンネル実験において、プラグインはdetermiに一つのチャンネルを利用する必要な補正ね。この補正は、次いで、4Dデータセットの登録をもたらす任意の追加のチャネルに適用される。 ゼブラフィッシュモデル系において、多くの時間、あるいは数日の期間にわたって8タイムラプス撮影を行うことができる。ゼブラフィッシュを取り付けるための一般的な方法は、その動きを規制9-11、低融点アガロース（0.8から1.5パーセント）で麻酔し、ライブ胚を埋め込 ​​むことである。移動が規制されながら、試料の成長が依然として位置をずらす視野内の細胞を生じ、発生した。胚内の細胞の移動に追従するためには、まず、試料全体の動きを補正することが必要である。このプロトコルは、ゼブラフィッシュの標本で現像し、画像12体節の開発に利用されているが、任意の共焦点4Dデータセットに適用することができる。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

4次元（4D）、共焦点データセットの生成;時間をかけて3D画像配列からなる;発生過程に関与する細胞の挙動をキャプチャするための優れた方法論を提供します。細胞の動きを追跡し、追跡する能力は、画像取得時に試料又は、場合によっては、成長中に発生するドリフトによる試料の移動によって制限される。ドリフトおよび/または増殖の影響を受けたデータセットで追跡する細胞は、細胞の位置の任意の分析にこれらの動きを組み込む。これは、サンプル内の静的な構造の見かけの動きになることがあります。そのため前細胞追跡するには、任意のサンプルドリフトを修正する必要があります。 ImageJの2,3のオープンソースフィジー分布1と組み込まれた遺伝子座のツール4を使用して、我々は、共焦点データセットに誤ったサンプルの動きを削除するために正しい3Dドリフトプラグインを開発しました。このプロトコルは、効果的に焦点POのサンプル翻訳や変更を補正長時間経過実験にわたって細胞の動きを視覚化し、測定する能力を維持しながら、四次元共焦点データセットの各時点を登録するために、位相相関を利用してsition。

13 -開発ゼブラフィッシュでは、速筋細胞は19時間後に受精（節期20）から多核繊維に融合。核の動き、我々は筋肉の全てを標識するために、骨格α-アクチンプロモーターの制御下で緑色蛍光タンパク質（GFP）で表現したトランスジェニック株を用いて4D共焦点タイムラプス撮影を行った筋細胞との融合を可視化するためにセル14とmCherryが核を標識するために、核局在化配列で?...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

タイムラプス顕微鏡は、発生過程の可視化を可能にする。画像取得中に成長または試料のドリフトが正確に従うと開発中の細胞の動きを測定する能力を低下させる。我々は、時間をかけて、三次元試料ドリフトを補正するためのオープンソースの画像処理ソフトウェアの使用を記載している。

4D共焦点タイムラプスイメージングを、次のサンプルのドリフト補正

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K ビュー.  Monash University. タイムラプス顕微鏡は、発生過程の可視化を可能にする。画像取得中に成長または試料のドリフトが正確に従うと開発中の細胞の動きを測定する能力を低下させる。我々は、時間をかけて、三次元試料ドリフトを補正するためのオープンソースの画像処理ソフトウェアの使用を記載している。 

ビデオ: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

エンジニア心臓組織の移植後免疫反応の評価（EHT）のための生物発光イメージング

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

生物工学

Video-rate Scanning Confocal Microscopy and Microendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical sectioning of complex systems. Confocal microscopy is routinely used, for example, to study specific cellular targets1, monitor dynamics in living cells2-4, and visualize the three dimensional evolution of entire organisms5,6. Extensions of confocal imaging systems, such as confocal microendoscopes, allow for high-resolution imaging in vivo7 and are currently being applied to disease imaging and diagnosis in clinical settings8,9.
Confocal microscopy provides three-dimensional resolution by creating so-called "optical sections" using straightforward geometrical optics. In a standard wide-field microscope, fluorescence generated from a sample is collected by an objective lens and relayed directly to a detector. While acceptable for imaging thin samples, thick samples become blurred by fluorescence generated above and below the objective focal plane. In contrast, confocal microscopy enables virtual, optical sectioning of samples, rejecting out-of-focus light to build high resolution three-dimensional representations of samples.
Confocal microscopes achieve this feat by using a confocal aperture in the detection beam path. The fluorescence collected from a sample by the objective is relayed back through the scanning mirrors and through the primary dichroic mirror, a mirror carefully selected to reflect shorter wavelengths such as the laser excitation beam while passing the longer, Stokes-shifted fluorescence emission. This long-wavelength fluorescence signal is then passed to a pair of lenses on either side of a pinhole that is positioned at a plane exactly conjugate with the focal plane of the objective lens. Photons collected from the focal volume of the object are collimated by the objective lens and are focused by the confocal lenses through the pinhole. Fluorescence generated above or below the focal plane will therefore not be collimated properly, and will not pass through the confocal pinhole1, creating an optical section in which only light from the microscope focus is visible. (Fig 1). Thus the pinhole effectively acts as a virtual aperture in the focal plane, confining the detected emission to only one limited spatial location.
Modern commercial confocal microscopes offer users fully automated operation, making formerly complex imaging procedures relatively straightforward and accessible. Despite the flexibility and power of these systems, commercial confocal microscopes are not well suited for all confocal imaging tasks, such as many in vivo imaging applications. Without the ability to create customized imaging systems to meet their needs, important experiments can remain out of reach to many scientists.
In this article, we provide a step-by-step method for the complete construction of a custom, video-rate confocal imaging system from basic components. The upright microscope will be constructed using a resonant galvanometric mirror to provide the fast scanning axis, while a standard speed resonant galvanometric mirror will scan the slow axis. To create a precise scanned beam in the objective lens focus, these mirrors will be positioned at the so-called telecentric planes using four relay lenses. Confocal detection will be accomplished using a standard, off-the-shelf photomultiplier tube (PMT), and the images will be captured and displayed using a Matrox framegrabber card and the included software.

The complete construction of a custom, real-time confocal scanning imaging system is described. This system, which can be readily used for video-rate microscopy and microendoscopy, allows for an array of imaging geometries and applications not accessible using standard commercial confocal systems, at a fraction of the cost.

ビデオレートでスキャニング共焦点顕微鏡とMicroendoscopy

Confocal microscopy has become an invaluable tool in biology and the biomedical sciences, enabling rapid, high-sensitivity, and high-resolution optical ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

RootChipを使用して、ルート環境の急激な操作によるシロイヌナズナの根の成長のタイムラプス蛍光イメージング

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

3次元細胞培養モデルの質量分析イメージング用試料調製の戦略

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

ICPMSサンプル導入のためのマイクロ流体チップ

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

哺乳類の細胞分裂量的共焦点反射顕微鏡を介して 3 D 行列

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

長期ナノ材料暴露後のインビトロ遺伝子毒性試験のための高度な3D肝モデル

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

タイムラプス顕微鏡による 大腸菌 における生物学的ノイズの連続測定

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

光ファイバー共焦点レーザー微小内視鏡検査を用いた眼内ドラッグデリバリーシステムの時空間的イン ビボ イメージング

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

刺激ラマン散乱顕微鏡によるタイムラプス生細胞イメージングのための柔軟なチャンバー

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...