著者らは、H188 cAMP FRETバイオセンサーとケニー・トリン(南アラバマ大学工学部)が独自に開発したプログラミングスクリプトを実行するのに要する時間を短縮するための技術的支援を提供したキース・ジャリンク博士(オランダ癌研究所とファン・レーウェンフック先端顕微鏡センター、アムステルダム、オランダ)を認めたいと考えています。
著者らは、資金源を認めたい:アメリカ心臓協会(16PRE27130004)、国立科学財団。(1725937) NIH, S100D020149, S10RR027535, R01HL058506, P01HL066299).

<ol>
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リーブズリー博士とリッチ博士は、スペクトルイメージング技術を商業化するために設立された新興企業SpectraCyte,LLCに対する金銭的関心を開示しています。しかし、このプロトコルに記載されているすべての手順は、SpectraCyte, LLCに関連しない市販の製品を使用して実施した。

FRETバイオセンサーの開発により、単一細胞における環状ヌクレオチドシグナルの測定と可視化が可能となり、細胞内シグナル伝達事象13,22,37,38を可視化する大きな約束がある。しかし、FRETバイオセンサーの使用は、多くの蛍光タンパク質ベースのFRETレポーターの低いシグナル間特性およびFRETレ?...

環状アデノシン一リン酸(cAMP)は、細胞分裂、カルシウム流入、遺伝子転写、シグナル伝達を含む主要な細胞および生理学的プロセスに関与する第二のメッセンジャーである。証拠の成長体は、シグナル特異性が達成される細胞内のcAMPコンパートメントの存在を示唆しています1,2,3,4,5,6,7.最近まで、cAMP区画化は、異なるG結合受容体アゴニスト8、9、10、11によって誘発される明確な生理学的または細胞的効果に基づいて推測された。より最近では、FRETベース蛍光イメージングプローブは、細胞12、13、14におけるcAMP信号の直接測定および観察のための新しいアプローチを提供している。
フェルスター共鳴エネルギー移動(FRET)は、分子が近接15,16の場合にドナー分子とアクセプター分子間でエネルギー移動が起こる物理的現象である。FRETベース蛍光指標の開発に伴い、この物理現象は、タンパク質相互作用17、タンパク質共局在18、Ca+2シグナル伝達19、遺伝子発現20、細胞分裂21および環状ヌクレオチドシグナル伝達を研究する生物学的応用において使用されてきた。FRET ベースの cAMP 指標は、通常、cAMP 結合ドメイン、ドナーフルオロフォアおよびアクセプターフルオロフォア22から構成されます。例えば、この方法論で使用されるH188 cAMPセンサ12,22は、Epacから得られたcAMP結合ドメインから成り、ターコイズ(ドナー)と金星(アクセプター)フルオロフォアの間に挟まれる。基礎条件(非結合)では、ターコイズとヴィーナスは、蛍光色素間でFRETが発生するような向きである。結合ドメインにcAMPを結合すると、ターコイズとヴィーナスが離れて移動するような立体構造変化が起こり、FRETが減少する。
FRETベースのイメージングアプローチは、細胞内のcAMP信号を調査し、可視化するための有望なツールを提供します。しかし、現在のFRETベースの顕微鏡イメージング技術は、多くの場合、細胞内空間的明瞭度でFRETを測定するのに十分な信号強度を達成するのに部分的にしか成功していない。これは、多くのFRETレポーターの信号強度の制限、FRET効率の変化を正確に定量するために必要な高い精度、および細胞自家蛍光23、24などの交易因子の存在を含むいくつかの要因によるものである。その結果、多くの場合、弱いSNRに悩まされているFRET画像であり、FRETにおける細胞内変化の視覚化は非常に困難になります。さらに、空間的に局在したcAMP信号の調査は、ほぼ2つの空間次元でほぼ独占的に行われ、軸方向のcAMP分布はめったに25と考えられてはなされていない。これは、SNR が低いために cAMP 勾配を 3 つの空間次元で測定および視覚化する機能が妨げられたためです。低SNRを有するFRETセンサの使用の限界を克服するために、我々は、単一細胞25、26、27でFRETを測定するためのハイパースペクトルイメージングおよび分析アプローチを実施した。
ハイパースペクトルイメージングアプローチは、衛星画像28,29に存在する地上の物体を区別するためにNASAによって開発された。これらの技術は、その後、蛍光顕微鏡観察分野30に翻訳され、いくつかの商業的共焦点顕微鏡システムがスペクトル検出器を提供する。従来の(非スペクトル)蛍光イメージングでは、サンプルはバンドパスフィルタまたはレーザーラインを使用して励起され、発光は2番目のバンドパスフィルタを使用して収集され、フッ素フォロアのピーク発光波長に合わせて選択されることがよくあります。対照的に、ハイパースペクトルイメージングアプローチは、特定の波長間隔で蛍光発光26、31、32または励起33、34のいずれかの完全なスペクトルプロファイルをサンプリングすることを求める。我々のこれまでの研究では、ハイパースペクトルイメージングおよび解析アプローチは、従来のフィルタベースのFRETイメージング技術26と比較して、細胞内のFRET信号の定量化を改善できることを示した。ここでは、4次元(x、y、z、λ)ハイパースペクトルFRETイメージングおよび解析を行い、cAMP分布を3つの空間次元で測定および可視化する方法論を提示する。これらのアプローチは、単一細胞25におけるアゴニスト誘発cAMP空間勾配の可視化を可能にした。興味深いことに、アゴニストによっては、cAMP勾配が細胞内で明らかである可能性がある。ここで提示される方法論は、FRET測定の精度を向上させるために、不均一な背景および細胞自家蛍光のスペクトルの非混合を利用する。この方法論は、cAMP FRETバイオセンサーを用いた肺微小血管内皮細胞(PMVEC)で実証されているが、この方法論は、代替FRETレポーターまたは代替細胞株で使用するために容易に変更することができる。

<table>
	<tbody>
		<tr>
			<td>Attofluor Cell Chamber</td>
			<td>Invitrogen</td>
			<td>A7816</td>
			<td>Attofluor contains steel cell chambers and a rubber O-ring. Cell chamber holds the coverslip and O-ring provides a lock in mechanism to hold the buffer in cell chamber with out leakage</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td>Solvent used to prepare stock solution forskolin.</td>
		</tr>
		<tr>
			<td>DRAQ5 Fluoroscent Probe Solution</td>
			<td>Thermo Scientific</td>
			<td>62252</td>
			<td>Nuclear label</td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>Contains nutrients and growth factors for the cells to grow and divide in the culture dishes.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F6178</td>
			<td>Growth factor suppliment that is added to culture medium, DMEM</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F3917</td>
			<td>Adenyly cyclase activator.</td>
		</tr>
		<tr>
			<td>H188 Cyclic AMP FRET biosensor</td>
			<td>Netherland Cancer Institute, Dr. K. Jalink</td>
			<td>Gift</td>
			<td>Plasmid encoding Turquoise (donor fluorophore), Venus (acceptor fluorophore), and binding domain obtained from Epac.</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>image.net</td>
			<td>Free download</td>
			<td>Another image processing platform used to extact spectral information and image processing.</td>
		</tr>
		<tr>
			<td>Integrating Sphere</td>
			<td>Ocean Optics</td>
			<td>FOIS-1</td>
			<td>Used to measure illumination intensity of the laser line at different laser intensities (?).</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Invitrogen</td>
			<td>23017-015</td>
			<td>Coverslips are coated with laminin and this helps in cell attachment, growth and motility of the cell.</td>
		</tr>
		<tr>
			<td>Lipofectamine 3000 Transfection Kit</td>
			<td>Invitrogen</td>
			<td>L3000-015</td>
			<td>Transfection reagent used to transfect cells with H188 FRET biosensor</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2019a</td>
			<td>Image processing operations (linear unmixing and FRET efficiency calculations) are performed by writing custom programs in MATLAB programming environment</td>
		</tr>
		<tr>
			<td>Nikon A1R confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>Nikon A1R</td>
			<td>Spectral image acquisition is performed using confocal microscope.</td>
		</tr>
		<tr>
			<td>Nikon Elements Software</td>
			<td>Nikon Instruments</td>
			<td>Software dongle</td>
			<td>used to export and handle nd2 image files (multidimensional image files) that are aquired using Nikon A1R</td>
		</tr>
		<tr>
			<td>NIST-Traceable Calibration Lamp</td>
			<td>Ocean Optics</td>
			<td>LS-1-CAL-INT</td>
			<td>A lamp with a known spectrum for use as a standard</td>
		</tr>
		<tr>
			<td>PBS pH 7.4 (1X)</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td>coomonly used buffer suring cell culture</td>
		</tr>
		<tr>
			<td>Pulmonary Microvascular Endothelial Cells (PMVECs)</td>
			<td>In house - Cell culture core, Univeristy of South Alabama</td>
			<td>Isolated from Rat pulmonary microvasculature</td>
			<td>PMVECs form inner lining of a blood vessel.</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/ml)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>antibiotics are added to culture medum to prevent contamination of the cells.</td>
		</tr>
		<tr>
			<td>Pre-Cleaned Gold Seal Micro Slides</td>
			<td>Clay Adams</td>
			<td>3010</td>
			<td>Microscope slides used for cell fixation</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mounting Media</td>
			<td>Invitrogen</td>
			<td>P36961</td>
			<td>If samples are fixed using antifade mountant, then the later protects fluoroscent dyes and chromophores from fading.</td>
		</tr>
		<tr>
			<td>Spectrometer</td>
			<td>Ocean Optics</td>
			<td>QE65000</td>
			<td>Used to measure spectral response of the light source (?)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Digests the protein-protein bond between the cell and cell matrix and helps to disscociate and lift the cells during cell plating.</td>
		</tr>
		<tr>
			<td>Tyrodes Buffer</td>
			<td>Made in-house</td>
			<td>Made in-house</td>
			<td>Tyrodes buffer is used to make working solutions and to maintain cells in aqueous solution during image acquisition.</td>
		</tr>
		<tr>
			<td>6 Well Cell Culture Plate</td>
			<td>Corning</td>
			<td>3506</td>
			<td>Laminin coated coverslips are placed in 6-well culture dish (one coverlisps/well). Cells along with medium is added into each well.</td>
		</tr>
		<tr>
			<td>25 mm Round Microscope Cover Slips</td>
			<td>Fisher Scientific</td>
			<td>12545102</td>
			<td>Cells were grown on round glass coverslips</td>
		</tr>
		<tr>
			<td>60X Ojective</td>
			<td>Nikon Instruments</td>
			<td>Plan Apo VC 60X/1.2 WI &#8734;/0.15-0.18 WD 0.27</td>
			<td>water immersion and commonly used objective for cells</td>
		</tr>
	</tbody>
</table>

measurement of 3-dimensional camp distributions in living cells using 4-dimensional (x, y, z, and &lambda;) hyperspectral fret imaging and analysis

サイクリックAMPは、細胞および生理活性の広い範囲に関与している第二のメッセンジャーです。いくつかの研究は、cAMP信号が区画化され、区画化がcAMPシグナル経路内のシグナル特異性に寄与することを示唆している。Fӧrster共鳴エネルギー伝達(FRET)ベースのバイオセンサーの開発は、細胞内のcAMP信号を測定し、可視化する能力を高めています。ただし、これらの測定値は、多くの場合、2 つの空間次元に限定され、データの誤解釈につながる可能性があります。これまで、3空間次元(x、y、z)でのcAMP信号の測定は非常に限られており、本質的に低信号対雑音比(SNR)を示すFRETセンサの使用に関する技術的な制限によりあります。さらに、従来のフィルタベースのイメージング手法は、スペクトルクロストーク、限定的な信号強度、自己蛍光などのさまざまな要因により、局在化した細胞下領域でcAMP信号を正確に測定するのには効果的でないことがよくあります。これらの制限を克服し、FRETベースのバイオセンサーを複数の蛍光ホルで使用できるようにするために、FRET効率を計算するためのスペクトル特異性と、FRET信号を同時交流自発および/または追加の蛍光標識からのシグナルからスペクトル的に分離する能力を提供するハイパースペクトルFRETイメージングおよび分析アプローチを開発しました。ここでは、ハイパースペクトルFRETイメージングを実装するための方法論と、スペクトルアンミックスを行うためにアンダーサンプリングもオーバーサンプリングもしない適切なスペクトルライブラリを構築する必要性を提示する。この方法論は、肺微小血管内皮細胞(PMVEC)における3次元cAMP分布の測定に用いるが、この方法論は、細胞タイプの範囲におけるcAMPの空間分布を研究するために使用することができる。

このプロトコルは、ハイパースペクトルFRETイメージングの使用と、生細胞における3つの空間次元におけるcAMP勾配を測定するための分析アプローチについて説明する。これらの結果の生成には、データの分析と定量を行う際に細心の注意が必要な重要なステップがいくつかあります。これらの主要なステップには、適切なスペクトルライブラリの構築、バックグラウンドスペクトルの非混合?...

Watch this Scientific Journal Video about Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and ÃŽÂ» Hyperspectral FRET Imaging and Analysis at JoVE.com

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and &lambda; Hyperspectral FRET Imaging and Analysis

Fӧrster共鳴エネルギー伝達(FRET)ベースのセンサの信号対雑音比(SNR)が低いため、cAMP信号の測定は特に3つの空間次元において困難でした。ここでは、3つの空間次元でcAMP分布を測定できるハイパースペクトルFRETイメージングおよび解析方法論について述べる。

4次元(x、y、z、λ)ハイパースペクトルFRETイメージングと解析を用いた生体細胞における3次元cAMP分布の測定

Cyclic AMP is a second messenger that is involved in a wide range of cellular and physiological activities. Several studies suggest that cAMP signals are ...

measurement-3-dimensional-camp-distributions-living-cells-using-4

Research

JoVE Journal

Biology

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis

3.8K ビュー.  University of South Alabama. Fӧrster共鳴エネルギー伝達(FRET)ベースのセンサの信号対雑音比(SNR)が低いため、cAMP信号の測定は特に3つの空間次元において困難でした。ここでは、3つの空間次元でcAMP分布を測定できるハイパースペクトルFRETイメージングおよび解析方法論について述べる。

ビデオ: Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional x, y, z, and λ Hyperspectral FRET Imaging and Analysis

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

生物学

Immunocytochemical Analysis of Human Pluripotent Stem Cells using a Self-Made Cytospin Apparatus

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell sources due to their limitless self-renewal capabilities and their potential to differentiate into multiple cell types. The pluripotent state of hPSCs is typically assessed by techniques such as qPCR, immunocytochemistry, and by other in vitro and in vivo differentiation strategies into multiple cell types. Among these, immunocytochemical techniques have been developed for routine characterization of the undifferentiated state of hPSCs based on analysis of candidate intracellular and cell-surface biomarkers. Given the fact that hPSCs grow as colonies, problems arise in quantifying the expression of these markers at the individual cell level on a routine basis. Flow cytometry analyses serve to address this issue but require cell numbers and use of reagents that are not normally conducive for routine quality control assessment of hPSC cultures. Thus, the development of practical and reproducible means of creating monolayer cell samples with preserved integrity for marker evaluation has many advantages in stem cell research. This greatly benefits immunocytochemical analysis because individual cells from the monolayer can be easily observed and quantified for the expression of specific markers. Towards this goal, a self-made cytospin apparatus was constructed and optimized for use with immunocytochemical staining. Two cell-surface markers (SSEA3/SSEA4) expression were analyzed in a variant BG01 stem cell line for the purpose of this protocol.

Suspension immunocytochemical staining of human pluripotent stem cells (hPSCs) for cell-surface markers (SSEA-3/SSEA-4) was achieved based on use of a self-made cytospin apparatus to create a monolayer of cells for observation and quantification.

セルフメイドサイトスピン装置を用いてヒト多能性幹細胞の免疫細胞化学的解析

Human pluripotent stem cells (hPSCs) that include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are exciting cell ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

髪の再生の間に上皮間葉シグナル伝達の迅速遺伝子解析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

共焦点顕微鏡を用いた生細胞におけるエンドサイトーシスおよび細胞質区画における同時pH測定

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

成体マウス頭蓋冠骨髄中の造血幹細胞および前駆細胞のin vivo 4次元トラッキングで

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

ワンナノメートルの精度で蛍光イメージング（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

FRETベースの共焦点イメージングを使用した培養平滑筋細胞の小胞体内カルシウム変動の測定

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

三次元ハイドロゲルでイメージングをFRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...