This project was supported by Grant Numbers HL96593 from NIH and D56HP20783 from HRSA/ HHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or HRSA / HHS.

注：キャピラリーの画像を取得するための取得プロセスは、以前に公開されていると、対応する画像取得と解析コンピュータプログラムでデジタルスチルカメラを用いて達成される11 21この実験室分析のために静止画像を利用していないビデオ、分析のための画像取得を単純化します。以下は、画像から毛細血管を定量するための新しい技術が記載されています。 1.画像強調処理<ol><li>モノクロデジタルカメラとデジタル画像を取得します。このようなカメラで支配者として知られている長さの物体を撮影することにより、既知の大きさの物体に画像を調整します。このプロセスは、正確に測定し、処理後の毛細血管を計数するためのコンピュータプログラムを可能にします。理想的には最高の精度のために、レチクル（それにエッチング定規とガラスの科学的に製造された作品）を使用する必要があります。 1mm角の画素数を測定しますコンピュータプログラムを使用してボックス。 注：このプロトコルの再現性と標準化の鍵となるのは、主にカウントさ1ミリメートル2ボックスの適切な配置に依存しています。 </li><li>毛細血管を暗くし、毛細血管の可視化を最大化する背景を、軽減するコントラスト強調ツールを使用してください。背景からの毛細血管の初期分化は、後工程での画像の適切なトリミングのために重要です。使用して画像を調整することでこれを行ってくださいにこのクリックを行うには「最適ヒストグラムを。」：キャプチャ、強度、画像ヒストグラム、ベストフィット。 </li><li>新しいイメージとしてキャピラリーカウント用の関心領域（ROI）をトリミング（、関心領域を選択します）。 1ミリメートルに等しい530ピクセルで画像を較正することによって決定された1ミリメートル2ボックスを使用してください。トリミングされた画像は、画像の一番上に毛細血管ループの頂点を配置していることを確認してください。 </li><li>したがって、すべての将来像の調整が均等に適用される画像を平らに画像。で、この表示することができません：、プロセスタブ、2Dフィルタを平らに、「明るい、 &quot;オブジェクトの幅のBG強度が「75」適用します。 </li><li>毛細血管を最大限に可視化されるように画像のコントラストを上げます。これを行うには、クリック：75にコントラストを上げ、タブ、ディスプレイを調整します。 </li><li>プロセス]タブ、2Dフィルタ、スペックル除去、カーネルサイズ7×7をクリックすることにより毛細血管の縁を滑らかに画像をスペックル除去、適用されます。 </li><li>毛細血管が黒で、背景が白になるように画像のコントラストを確定。 「ベストフィット」モデルにヒストグラムを調整することによって、この手順を実行します。 注：処理された画像は、次の手順を実行してどのように見えるかの例については、代表的な結果では、図2を参照してください。 </li></ol>キャピラリーカウント/定量毛細血管密度の実行2. <ol><li>各画像には、手動で「対象オブジェクト」featurを使用して、明確に定義された毛細血管の一部を選択eはプログラムによってカウントされるオブジェクトとして認識されます。そして、カウントアルゴリズムによって無視される必要がある領域を基準として、「バックグラウンド」機能を使用して、背景の小さな部分を選択します。 注：これらのハイライトの組み合わせは、バックグラウンドノイズを無視して、すべての毛細血管が強調表示されます。定量（カウント）プロトコルは、色や形態学に基づいて画像の一部を区別するためにコンピュータ機能を利用します。 </li><li>瞬時に撮像装置を用いて画像内のすべての毛細血管をカウントするカウント機能を使用してください。キャピラリとしてバックグラウンドノイズをカウントしないようにするために5ピクセルのカウントオブジェクトの最小直径を設定します。 </li><li>より信頼性の高い評価を得るために、4つの画像 - 各個人のために、3の平均を数えます。 注：画像は、カウンティング手順の間にどのように見えるかの例については、代表的な結果では図3を参照してください。 </li></ol&gt; 3.作成して画像処理を自動化するマクロの使用注：時間を節約するために、マクロを自動的に一つ又は多数の画像上のプロセスの特定のシーケンスを実行するために作成することができます。これらの配列は、画像の修正を迅速にするためにカスタマイズすることができます。本質的には、これらのマクロは、画像を処理する方法を覚えて、迅速かつユーザー入力ですべての手順を実行します。マクロのない絵あたり約8分とは対照的に、12キャピラリー画像にカウントを実行すると、マクロ（画像ごとに2〜3分）で20〜30分の間、このラボを取ります。したがって、マクロを使用すると、手動で各個々の画像を処理するよりも3〜5倍、より効率的です。 <ol><li>マクロを作成するためには、「マクロの記録」を選択し、一つの画像の上に、上記の手順1と2で説明したようにステップとプロセスは、希望する実行します。今後の参考のために実行された画像処理の手順に基づいて、マクロに名前を付けます。ときUSIを将来の絵のマクロngを、単に「実行マクロ」をクリックして、プログラムが自動的に所望の画像（複数可）に記録された拡張機能が適用されます。 注：このラボでは、数秒のメソッドの第1のステップの一つが、すべてを実行するマクロを使用しています。ユーザー入力を必要とする唯一のステップは、どこで画像をトリミングするために、ステップ1.2を選んでいます。 </li></ol>

<ol>
	<li>Gronenschild, E. H., 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=Semi-automatic+assessment+of+skin+capillary+density:+proof+of+principle+and+validation.">Semi-automatic assessment of skin capillary density: proof of principle and validation.</a> Microvasc Res. 90, 192-198 (2013).</li><li>Allen, J., Howell, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+imaging:+techniques+and+opportunities+for+clinical+physiological+measurements.">Microvascular imaging: techniques and opportunities for clinical physiological measurements.</a> Physiol Meas. 35, R91-R141 (2014).</li><li>Boettcher, M., Gloe, T., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiautomatic+quantification+of+angiogenesis.">Semiautomatic quantification of angiogenesis.</a> J Surg Res. 162, 132-139 (2010).</li><li>Wild, R., Ramakrishnan, S., Sedgewick, J., Griffioen, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+angiogenesis+and+tumor+vessel+architecture+by+computer-assisted+digital+image+analysis:+effects+of+VEGF-toxin+conjugate+on+tumor+microvessel+density.">Quantitative assessment of angiogenesis and tumor vessel architecture by computer-assisted digital image analysis: effects of VEGF-toxin conjugate on tumor microvessel density.</a> Microvasc Res. 59, 368-376 (2000).</li><li>Tresadern, P. A., 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=Simulating+nailfold+capillaroscopy+sequences+to+evaluate+algorithms+for+blood+flow+estimation.">Simulating nailfold capillaroscopy sequences to evaluate algorithms for blood flow estimation.</a> Conf Proc IEEE Eng Med Biol Soc. , 2636-2639 (2013).</li><li>Anderson, M. 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=Computerized+nailfold+video+capillaroscopy--a+new+tool+for+assessment+of+Raynaud's+phenomenon.">Computerized nailfold video capillaroscopy--a new tool for assessment of Raynaud's phenomenon.</a> J Rheumatol. 32, 841-848 (2005).</li><li>Neubauer-Geryk, J., 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=Decreased+reactivity+of+skin+microcirculation+in+response+to+L-arginine+in+later-onset+type+1+diabetes.">Decreased reactivity of skin microcirculation in response to L-arginine in later-onset type 1 diabetes.</a> Diabetes Care. 36, 950-956 (2013).</li><li>Pazos-Moura, C. C., Moura, E. G., Bouskela, E., Torres-Filho, I. P., Breitenbach, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nailfold+capillaroscopy+in+diabetes+mellitus:+morphological+abnormalities+and+relationship+with+microangiopathy.">Nailfold capillaroscopy in diabetes mellitus: morphological abnormalities and relationship with microangiopathy.</a> Braz J Med Biol Res. 20, 777-780 (1987).</li><li>Antonios, T. F., Singer, D. R., Markandu, N. D., Mortimer, P. S., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+skin+capillary+rarefaction+in+essential+hypertension.">Structural skin capillary rarefaction in essential hypertension.</a> Hypertension. 33, 998-1001 (1999).</li><li>Kaiser, S. E., Sanjuliani, A. F., Estato, V., Gomes, M. B., Tibirica, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antihypertensive+treatment+improves+microvascular+rarefaction+and+reactivity+in+low-risk+hypertensive+individuals.">Antihypertensive treatment improves microvascular rarefaction and reactivity in low-risk hypertensive individuals.</a> Microcirculation. 20, 703-716 (2013).</li><li>Cheng, C., Diamond, J. J., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+capillary+rarefaction+in+mild+blood+pressure+elevation.">Functional capillary rarefaction in mild blood pressure elevation.</a> Clinical and Translational Science. 1, 75-79 (2008).</li><li>Debbabi, H., Bonnin, P., Levy, B. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+blood+pressure+control+with+perindopril/indapamide+on+the+microcirculation+in+hypertensive+patients.">Effects of blood pressure control with perindopril/indapamide on the microcirculation in hypertensive patients.</a> Am J Hypertens. 23, 1136-1143 (2010).</li><li>He, F. J., Marciniak, M., Markandu, N. D., Antonios, T. F., MacGregor, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+modest+salt+reduction+on+skin+capillary+rarefaction+in+white,+black,+and+Asian+individuals+with+mild+hypertension.">Effect of modest salt reduction on skin capillary rarefaction in white, black, and Asian individuals with mild hypertension.</a> Hypertension. 56, 253-259 (2010).</li><li>Murray, A. K., 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=The+influence+of+measurement+location+on+reliability+of+quantitative+nailfold+videocapillaroscopy+in+patients+with+SSc.">The influence of measurement location on reliability of quantitative nailfold videocapillaroscopy in patients with SSc.</a> Rheumatology (Oxford). 51, 1323-1330 (2012).</li><li>Ingegnoli, F., 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=Feasibility+of+different+capillaroscopic+measures+for+identifying+nailfold+microvascular+alterations.">Feasibility of different capillaroscopic measures for identifying nailfold microvascular alterations.</a> Semin Arthritis Rheum. 38, 289-295 (2009).</li><li>Debbabi, H., 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=Increased+skin+capillary+density+in+treated+essential+hypertensive+patients.">Increased skin capillary density in treated essential hypertensive patients.</a> Am J Hypertens. 19, 477-483 (2006).</li><li>Serne, E. H., 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=Impaired+skin+capillary+recruitment+in+essential+hypertension+is+caused+by+both+functional+and+structural+capillary+rarefaction.">Impaired skin capillary recruitment in essential hypertension is caused by both functional and structural capillary rarefaction.</a> Hypertension. 38, 238-242 (2001).</li><li>Shore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillaroscopy+and+the+measurement+of+capillary+pressure.">Capillaroscopy and the measurement of capillary pressure.</a> Br J Clin Pharmacol. 50, 501-513 (2000).</li><li>Rieder, M. J., O'Drobinak, D. M., Greene, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+method+for+determination+of+microvascular+density.">A computerized method for determination of microvascular density.</a> Microvasc Res. 49, 180-189 (1995).</li><li>Vermeulen, P. B., 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=Quantification+of+angiogenesis+in+solid+human+tumours:+an+international+consensus+on+the+methodology+and+criteria+of+evaluation.">Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.</a> Eur J Cancer. 32A, 2474-2484 (1996).</li><li>Cheng, C., Daskalakis, C., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Assessment+of+Microvascular+and+Endothelial+Function.">Non-invasive Assessment of Microvascular and Endothelial Function.</a> Journal of Visualized Experiments. , (2012).</li><li>Antonios, T. F., 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=Maximization+of+skin+capillaries+during+intravital+video-microscopy+in+essential+hypertension:+comparison+between+venous+congestion,+reactive+hyperaemia+and+core+heat+load+tests.">Maximization of skin capillaries during intravital video-microscopy in essential hypertension: comparison between venous congestion, reactive hyperaemia and core heat load tests.</a> Clin Sci (Lond). 97, 523-528 (1999).</li></ol>

The authors have no conflicts of interest.

Nailfold capillaroscopyは、様々な腫瘍学、心臓血管、およびリウマチ疾患の用途のために、将来的に臨床的に有用なツールとして有望です。画像取得プロセスは、研究者の間でかなり一貫性のある、まだ画像処理及び分析のための多数の方法が現在存在します。メソッドは、現在のコンピュータと手動キャピラリー数が含まれています。彼らは時間がかかり、ユーザの主観と疲労にさらされるよ?...

微小血管イメージングは、多くの潜在的な臨床用途に急速に成長している分野である。2例えば、癌専門医は、可能な治療の選択肢への腫瘍と洞察力の状態に関する貴重な情報が得られる、腫瘍血管新生の程度を決定するために微小血管イメージングを使用しています。3 4しかし、 nailfoldのcapillaroscopyは、おそらく微小血管イメージングの最もコスト効率的で広く適用可能な形態です。研究者らは、血流量を研究し、毛細血管の形態を調査するために、ビデオnailfoldのcapillaroscopyを使用しています。5 6両方のビデオと静止画nailfoldのcapillaroscopyをレイノー現象や、全身性硬化症などの様々な結合組織疾患の診断と治療の世話をするために補助剤です。2 Nailfold capillaroscopyは、同様に、様々な潜在的な心血管系の用途を有します。 nailfold capillaroscopyを使用して現在の研究は示唆していますその糖尿病1型および2型の患者が異常な毛細管形態の有病率が高いを示し、まだ非糖尿病個体と比較して変わらない毛細血管密度を有する。7-8 Capillaroscopyも高血圧で実験的に研究されてきました。減少毛細血管密度につながる構造毛細管希薄非高血圧個体と比較して、高血圧の個体において実証されている。9-10これらの古い高血圧患者とは対照的に、構造的な希薄化を示し、より最近の研究はことを示した（40および上記の平均年齢）若い高血圧患者は（40歳未満の平均年齢）構造的希薄化することなく、機能的な希薄化を持っている。11これは、機能的な希薄化を前に発生し、構造的な希薄化に時間をかけて進行し得ることを示唆しています。 興味深いことに、このようなペリンドプリル/インダパミドなどの特定の降圧薬で処置された高血圧患者はノルムを表示しましたアル毛細血管密度および治療 ​​後内皮機能、ACE（アンジオテンシン変換酵素）阻害剤または利尿剤で処置したものは、同程度の血圧制御にもかかわらず、低毛細血管密度を維持しながら12は、これは、いくつかの降圧薬は、毛細管を逆にすることによって毛細血管密度を正規化することを示唆しています高血圧によって生じる希薄化。また、他の研究者は、食塩摂取量の減少は高血圧の個体の両方の機能的および構造的な毛細管希薄の逆転につながることが示されている。13 この技術の様々な潜在的な臨床応用にもかかわらず、ほとんどの標準化は、毛細血管密度画像を定量するための技術であります。2日に、研究者は毛細血管密度の結果のみが正確な場合、イントラオブザーバーと観察者間の両方の観点から、再現性があることを発見しました同じ領域が毎回カウントされている。1,14 15 </sup&gt;注目すべきは、以前の研究者は、主に遅く、主観的なプロセスである裸眼、9 16 17 18を使用して手動でキャピラリーカウントを行いました。 毛細血管画像の定量のための標準化された、コンピュータベースのアルゴリズムは、理論的にcapillaroscopyの臨床応用を促進する、より少ない主観で、より効率的かつ再現可能なデータ分析を提供しています。一部の研究者は、確かにnailfold capillaroscopic写真からデータを定量するために、コンピュータベースのプログラムを使用していた。1,6 19 20しかし、現在までの唯一の出版物は、capillaroscopyデータを定量化するために利用可能な複雑なソフトウェアプログラムの信頼性を説明し1、このプログラムは次のように複雑です以前にまったく同じ視野をカウントするための要件によって上記の。ここでは、することが可能標準化されたアルゴリズムを使用して、毛細血管を定量するための簡単​​な、信頼性の高いプロトコルを提示複数の視覚的なフィールドの使用。複数の視野を使用すると、手続きを簡素化するだけでなく、毛細血管数の正常な生物学的変化の評価を可能にするだけでなく。 本研究の目的は、毛細管定量プロセスを標準化し、再現性、効率的なコンピュータベースのアルゴリズムを記述することです。これらの方法は完全に自動化されていないが、彼らは非常に少ないユーザー入力を必要とし、絵の迅速かつ信頼性の高い定量を提供します。

<table><tbody><tr><td>Image-Pro Premier</td><td>Media Cybernetics, Inc</td><td>9.1</td><td>Image processing software</td></tr></tbody></table>

a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy

Capillaroscopyは、効率的で、直接微小循環を可視化するための方法論を学ぶために、比較的安価で容易な非侵襲的です。 capillaroscopy技術は、潜在的に価値のある皮膚科、眼科、リウマチおよび心血管臨床のさまざまなアプリケーションにつながる、患者の微小血管の健康への洞察を提供することができます。さらに、腫瘍増殖は、腫瘍内微小血管密度を測定することによって定量することができる血管形成に依存してもよいです。しかし、技術の無い標準化に現在ほとんどありませんし、現在まで唯一の出版物はcapillaroscopyデータを定量するための現在利用可能な、複雑なコンピュータベースのアルゴリズムの信頼性を報告します。1本論文では、アルゴリズムをカウント新しい、より簡単な、信頼性の高い、標準化された毛細血管を説明nailfold capillaroscopyデータを定量します。このような単純な、再現性のあるコンピュータ化されたcapillaroscopyアルゴリズムは、より多くのを容易にするであろう研究者や臨床医の間で技術の普及。多くの研究者は、現在の結果のユーザ疲労や主観を促進し、手でcapillaroscopy画像を解析。本稿では、再現性、半自動計数アルゴリズムに加えて新たな、簡単に使用できる自動化された画像処理アルゴリズムを説明します。このアルゴリズムは、主観を削減しながら、数分で画像の解析を可能にします。トレーニング時間の最小量は、（我々の経験では、1時間未満）が技術を習得するために必要とされます。

この画像処理手順の目的は、それらが正確に定量することができるように、背景画像から毛細血管を区別することです。不完全な画像処理や過度の画像処理の両方が毛細血管を定量化するためのプログラムの機能に有害です。 図3に見られるように、不完全な画像処理は、バックグラウンドから区別する毛細血管を困難にします。これは、ユーザーが、上記のカウント方法が正確...

Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Nailfold Capillaroscopyを使用して、毛細血管密度の定量のための再現性のコンピュータ化された方法

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

a-reproducible-computerized-method-for-quantitation-capillary-density

Research

JoVE Journal

Medicine

8.9K ビュー.  Thomas Jefferson University. Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm. 

ビデオ: A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

定量的な光学顕微鏡：細胞生物物理の測定は、標準的な光学顕微鏡で特徴

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

生物工学

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

微小血管と内皮機能の非侵襲的評価

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

医学

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

in vitroでカルシウム沈着を定量するための半自動化された再現性のある生物学的ベースの方法

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

生化学

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

キャピラリー配列で複数の核酸の視覚的検出

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

核酸の光度計微量定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

生物学

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィル テスト

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

神経科学

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

光干渉断層撮影血管造影法で測定した黄斑灌流密度に対する毛細血管および他の血管寄与の評価

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

Nailfold Capillaroscopyを使用して、毛細血管密度の定量のための再現性のコンピュータ化された方法

要約

さらに動画を探す

この動画の章

定量的な光学顕微鏡：細胞生物物理の測定は、標準的な光学顕微鏡で特徴

微小血管と内皮機能の非侵襲的評価

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

in vitroでカルシウム沈着を定量するための半自動化された再現性のある生物学的ベースの方法

キャピラリー配列で複数の核酸の視覚的検出

核酸の光度計微量定量

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィルテスト

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

光干渉断層撮影血管造影法で測定した黄斑灌流密度に対する毛細血管および他の血管寄与の評価

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

Nailfold Capillaroscopyを使用して、毛細血管密度の定量のための再現性のコンピュータ化された方法

要約

さらに動画を探す

この動画の章

定量的な光学顕微鏡：細胞生物物理の測定は、標準的な光学顕微鏡で特徴

微小血管と内皮機能の非侵襲的評価

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

in vitroでカルシウム沈着を定量するための半自動化された再現性のある生物学的ベースの方法

キャピラリー配列で複数の核酸の視覚的検出

核酸の光度計微量定量

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィル テスト

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

光干渉断層撮影血管造影法で測定した黄斑灌流密度に対する毛細血管および他の血管寄与の評価

ImageJのプラグインを使用して細胞の計数手順の自動定量化と分析

拡散反射率分光: 取得 1 つの親指の下の毛細血管リフィルテスト