Name: Video: Video-rate Scanning Confocal Microscopy and Microendoscopy
Uploaded: 2011-10-20T13:00:00
Duration: 14 min 10 s
Description: 27.7K Görüntüleme. Harvard University. Özel, gerçek zamanlı bir konfokal tarama görüntüleme sistemi komple inşaat açıklanmıştır. Video hızı mikroskopi ve mikroendoskopi için kolayca kullanılabilen bu sistem, maliyetinin bir kısmını, standart ticari konfokal sistemlerini kullanarak erişilebilir değil görüntüleme geometrileri ve uygulamaları bir dizi sağlar.

Yazarlar ThorLabs bu projeye destek için teşekkür etmek istiyorum. AJN bir NSF Yüksek Lisans Bursu desteğini kabul etmek istemektedir. Bu çalışma kısmen, NIH Müdürü Yenilikçisi Ödül Programı aracılığıyla, Ulusal Sağlık Enstitüleri, OD007096-01 DP2 hibe sayısı 1 tarafından finanse edildi. Yeni Yenilikçisi Ödülü Programı ile ilgili bilgiler. <a href="http://nihroadmap.nih.gov/newinnovator/">http://nihroadmap.nih.gov/newinnovator/</a> . Yazarlar, Harvard Elektronik laboratuar kullanımı için Tom Hayes teşekkür etmek istiyorum.

<ol>
	<li>Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of biological confocal microscopy. , 985-985 (2006).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nature Reviews Molecular Cell Biology. 2, 444-456 (2001).</li><li>Klonis, N., Rug, M., Harper, I., Wickham, M., Cowman, A., Tilley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+photobleaching+analysis+for+the+study+of+cellular+dynamics.">Fluorescence photobleaching analysis for the study of cellular dynamics.</a> European Biophysics Journal. 31, 36-51 (2002).</li><li>Stephens, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Microscopy+Techniques+for+Live+Cell+Imaging.">Light Microscopy Techniques for Live Cell Imaging.</a> Science. 300, 82-86 (2003).</li><li>McMahon, A., Supatto, W., Fraser, S. E., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Analyses+of+Drosophila+Gastrulation+Provide+Insights+into+Collective+Cell+Migration.">Dynamic Analyses of Drosophila Gastrulation Provide Insights into Collective Cell Migration.</a> Science. 322, 1546-1550 (2008).</li><li>Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dishevelled+controls+cell+polarity+during+Xenopus+gastrulation.">Dishevelled controls cell polarity during Xenopus gastrulation.</a> Nature. 405, 81-85 (2000).</li><li>Laemmel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+Confocal+Fluorescence+Microscopy+(Cell-viZio)+Facilitates+Extended+Imaging+in+the+Field+of+Microcirculation.">Fibered Confocal Fluorescence Microscopy (Cell-viZio) Facilitates Extended Imaging in the Field of Microcirculation.</a> Journal of Vascular Research. 41, 400-411 (2004).</li><li>Moussata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+confocal+laser+endomicroscopy.">The confocal laser endomicroscopy.</a> Acta Endoscopica. 39, 448-451 (2010).</li><li>Dunbar, K., Canto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+endomicroscopy.">Confocal endomicroscopy.</a> Current Opinion in Gastroenterology. 24, 631-637 (2008).</li><li>Udovich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+background+and+transmission+characteristics+of+fiber+optic+imaging+bundles.">Spectral background and transmission characteristics of fiber optic imaging bundles.</a> Applied optics. 47, 4560-4568 (2008).</li></ol>

Bu video Bu video Üretimi Thorlabs, Inc tarafından sponsor oldu

Bu video-rate görüntüleme sistemi, yaklaşık 8 kHz de bir rezonans galvanometric ayna işletim kullanımı yapar. Rezonans aynalar tam güçle çalıştırıldığında oldukça yüksek sesle, ve yüksek perde yeterli pozlama süreleri az rahatsız edici ve hatta tehlikeli olabilir. Burada gösterilen olmasa da, bu sistem birimi ve / veya kulak tıkacı gibi uygun işitme koruyucu giysiler giymek önemli ölçüde azaltmak için, şeffaf bir çantasının içinde yankılanan galvanometric ayna korumak için tavsiye edilir. Rezonans galvanometric ayna sinüzoidal bir desen tarar. Ancak, Framegrabber kartı kartları, sinyal, yatay ve dikey yönde hem de tamamen doğrusal bir tarama hızı varsayarak okuyun. Sinüzoidal bir süpürme tarama kenarlarında yavaşlar, görüntü sıkıştırma eserler hızlı (yatay) görüntü ekseni boyunca görülebilir. Bu sorunu en aza indirmek için bir yolu bilerek rezonans galvo ayna tarama aralığı sürücü için önemli ölçüde daha büyükröle objektif çapı. Bunu yaparken, sadece sinüzoidal tarama desen neredeyse doğrusal merkezi süpürme görüntü distorsiyonları en aza indirmek, örnek hareket edecektir. Diğer bir yaklaşım, hızlı bir eksen linearize işlem sonrası toplanan görüntüler olacaktır. Bu bilinen bir floresan paterni (bir ızgara gibi) görüntüleme ile başarılı ve bilinen desen ölçüleri kullanılarak toplanan görüntüleri unwarps bir işlem komut dosyası oluşturmak için olabilir. Bu özel tarama sistemi genellikle dik odaklı bir video hızı mikroskop gerektirir in vivo görüntüleme amacı ile dizayn edilmiştir. Hücresel görüntüleme deneyleri için, ters mikroskoplar genellikle kullanılır. Böyle bir inverted mikroskop kolayca oluşturmak için burada sunulan tasarım değişti; gerekli olduğunu, son 2 &quot;çap ayna bir dönme. Ayna ayna aşağı tarama ışını doğrudan yönlendirme yerine, yukarıya doğru ışın yönlendirebilirsiniz. Objektif lens yerleştirilmesit, örnek bir aşaması ile birlikte ayna aynı mesafede ters bir geometri görüntüleme için izin verecek. Görüntüleme sistemi, sadece microendoscopic görüntüleme için inşa edilmekte ise, tüm dikey mikroskop tasarım &quot;kat&quot; için hiçbir sebep yoktur. Bunun yerine, tüm tarama sistemi ile tek bir objektif lens optik tabloya yönelik paralel yatay breadboard üzerinde inşa edilebilir. En ticari konfokal mikroskoplar bulunabilir gibi bu daha çok yönlü bir sistem isteyen kullanıcılar, bir değişken iğne deliği içeren düşünebilirsiniz, büyük inşa sadelik ve uyum kolaylığı sağlarken; bu yapı mikroskop sabit bir iğne deliği yapılandırmasını kullanır. Kullanıcı emisyon yoğunluğu değişik örnekler için telafi etmek için iğne deliği boyutunu ayarlamak için izin vererek, bu kullanıcı belirli bir numune daha iyi sinyal gücü ve çözünürlük arasındaki tradeoff optimize edilmesine olanak tanır. Chmikroskop için seçilen görüntü, fiber OICE önemlidir. Biz onların yakın fiber çekirdek aralığı ve düşük bağıl otofloresans nedeniyle Sumitomo tutarlı bir görüntü lifleri kullanmanızı öneririz. Fujikura tarafından üretilen liflerin bir örnek zayıf floresan sinyalleri bastıran ve mikroendoskop nihai hassasiyet sınırı otofloresans 10 yüksek miktarda olduğu tespit edilmiştir. Bu özel kurulum kullanılan 8-30N Sumitomo üretilen liflerin, kendi Fujikura muadillerine göre çok daha düşük otofloresans seviyeleri var. Leeched lif demetleri mikroendoskopi için çekici olarak kabul edilebilir olsa da, tasarım genellikle fiber çekirdek seyrek örnek nesneler, potansiyel ilgi önemli bölgelerinde dışarıda bırakarak, bunun anlamı, çok uzak bireysel lif çekirdek yerleştirir. Son olarak, mikroskop burada açıklanan, in vitro ve in vivo Uyg çeşitli yararlı olacağını belirtmek gerekirEklentiler ve tam özellikli bir ticari sistem maliyetinin bir kısmı için oluşturulabilir, iletilen ışık algılama, izleme için bir mercek, ya da bir ışın yolu olmayan konfokal widefield Epifloresans gibi özelliklere sahip değildir. Sıfırdan bu özelliklere sahip bir sistem inşa etmek mümkün olsa da, böyle bir sistem elde etmek isteyen okuyucuların kendi ihtiyaçlarını karşılamak yerine tamamen yeni inşa başlatmak için mevcut bir ticari sistemi değiştirmek isteyebilirsiniz.

<table border="1"><tbody><tr><td> Parça Adı </td><td> Üretici </td><td> Ürün Numarası </td><td> Özellikler </td><td> Miktar </td></tr><tr><td> 515 nm Band Geçiren Filtre </td><td> Renk parlaklığı </td><td> HQ515/50M </td><td> 46 FWHM </td><td> 1 </td></tr><tr><td> Akromatik İkili Lens 25,4 Dia. x 50mm FL, MgF2 Kaplama </td><td> Edmund Optik </td><td> NT49-766 </td><td></td><td> 1 </td></tr><tr><td> Akromatik İkili Lens 25,4 Dia. x 76,2 mm, FL MgF2 Kaplama </td><td> Edmund Optik </td><td> NT49-768 </td><td></td><td> 1 </td></tr><tr><td> Akromatik İkili Lens 25,4 Dia. x 88.9mm FL, MgF2 Kaplama </td><td> Edmund Optik </td><td> NT49-769 </td><td></td><td> 2 </td></tr><tr><td> Akromatik İkili Lens 50mm Dia. x 300mm FL, MgF2 Kaplama </td><td> Edmund Optik </td><td> NT45-179 </td&gt; <td></td><td> 1 </td></tr><tr><td> 8 kHz R Yüksek Frekanslı Optik Tarayıcı </td><td> Elektro-Optik Ürünler Şirketi (EOPC) </td><td> SC-30 </td><td> 8 kHz </td><td> 1 </td></tr><tr><td> AGC Sürücü </td><td> Elektro-Optik Ürünler Şirketi (EOPC) </td><td> ACG: 8K </td><td></td><td></td></tr><tr><td> H7422-PA Fotosensör Modülü </td><td> Hamamatsu </td><td> H7422-PA </td><td> Şu tavsiye sınırlayıcı </td><td> 1 </td></tr><tr><td> M9012 Güç Kaynağı </td><td> Hamamatsu </td><td> M9012 </td><td> H7422-PA ile kullanım için </td><td> 1 </td></tr><tr><td> HC PL APO CS Amaç </td><td> Leica </td><td> 11506284 </td><td> 10x/0.40 </td><td> 1 </td></tr><tr><td> Solios EA / XA Framegrabber Kart </td><td> Matrox </td><td> Solios EA / XA </td><td> MIL yazılım gereklidir; OEM tavsiye birbirine </td><td> 1 </td></tr><tr><td> 12V Güç Kaynağı </td><td&gt; MeanWell </td><td> LPV-100-12 </td><td> +12 V, 8.5A </td><td> 1 </td></tr><tr><td> 5x Mikroskop Objektif </td><td> Newport </td><td> M-5X </td><td> 0.10 NA, 25,4 mm Odak Uzunluğu </td><td> 1 </td></tr><tr><td> Tutarlı Görüntü fiber </td><td> Sumitomo </td><td> 8-30N </td><td></td><td> 1 </td></tr><tr><td> 1 / 4 &quot;-20 Kapak Vida ve Donanım Kiti </td><td> ThorLabs </td><td> HW-Takımı 2 </td><td></td><td> 1 </td></tr><tr><td> 100 mikron İğne Atlı </td><td> ThorLabs </td><td> P100S </td><td> Uzaysal filtreler oluşturmak için idealdir. </td><td> 1 </td></tr><tr><td> 30 mm Kafes Küp Kelepçe </td><td> ThorLabs </td><td> B6C </td><td></td><td> 1 </td></tr><tr><td> 30 mm Kafes Sistem Küp, 4-Way </td><td> ThorLabs </td><td> C4W </td><td></td><td> 1 </td></tr><tr><td> 406 nm, 5 mW, B Pin Kodu, SM Fiber Pigtailed Lazer Diyot, FC / PC </td><td> ThorLabs </td><td> LPS-406-FC </td><td> Eskimiş Ürün; LP405-SF10 tarafından yerine </td><td> 1 </td></tr><tr><td> 5-Minute Epoxy, 1 Ons </td><td> ThorLabs </td><td> G14250 </td><td></td><td> 1 </td></tr><tr><td> 6 Eksen Kinematik Optik Dağı </td><td> ThorLabs </td><td> K6X </td><td></td><td> 1 </td></tr><tr><td> 8-32 Kapak Vida ve Donanım Kiti </td><td> ThorLabs </td><td> HW-KIT1 </td><td></td><td> 1 </td></tr><tr><td> 8-32 ayar vidası ve Donanım Kiti </td><td> ThorLabs </td><td> HW-KIT3 </td><td></td><td> 1 </td></tr><tr><td> Dış RMS Konuları ve İç SM1 Konuları adaptör </td><td> ThorLabs </td><td> SM1A4 </td><td></td><td> 1 </td></tr><tr><td> Adj. FC / PC ve FC / APC Kolimatör, f = 2.0 mm, ARC: 400-600 nm </td><td> ThorLabs </td><td> CFC-2X-A </td><td> f = 2.0 mm </td><td> 1 </td></tr><tr><td> Ayarlanabilir Fiber Kolimatör Adaptörü, Dişli SM1 </td><td> ThorLabs </td><td> AD9.5F </td><td></td><td> 1 </td></tr><tr><td> Alüminyum Breadboard, 12 &quot;x 18&quot; x 1 / 2 &quot; </td><td> ThorLabs </td><td> MB1218 </td><td> 1 / 4 &quot;-20 Dişli </td><td> 2 </td></tr><tr><td> Benchtop Lazer Diyot / TEC Kontrolörü </td><td> ThorLabs </td><td> ITC4001 </td><td> 1 A/96 W </td><td> 1 </td></tr><tr><td> DMLP 425 nm Uzun-Pass Dikroik Ayna </td><td> ThorLabs </td><td> DMLP425 </td><td></td><td> 1 </td></tr><tr><td> Ø1 &quot;Optik Kinematik Dağı </td><td> ThorLabs </td><td> KM100 </td><td></td><td> 3 </td></tr><tr><td> LD / TEC Mount ThorLabs Pigtailed Fiber Lazer Diyot </td><td> ThorLabs </td><td> LM9LP </td><td></td><td> 1 </td></tr><tr><td> Ø18 mm Optik Lens Bayoneti </td><td> ThorLabs </td><td> LMR18 </td><td> Bir emniyet halkasıyla </td><td> 1 </td></tr><tr><td> 2 &quot;Optik Lens Mounts </td><td> ThorLabs </td><td> LMR2S </td><td> Hizmetkar halka dahil, iç ve dış iş parçacığı ile </td><td> 2 </td></tr><tr><td> Mini Serisi Cage Meclisi Rod, 6 &quot;Uzun, 4 mm çaplı, Miktar 1 </td><td> ThorLabs </td><td> SR6 </td><td></td><td> 4 </td></tr><tr><td> Ø1.0 &quot;Ayak Pillar Mesaj, 8-32 Musluklar, 1&quot; Uzun </td><td> ThorLabs </td><td> RS1P8E </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Pillar Mesaj Uzatma, Boy = 0.5 </td><td> ThorLabs </td><td> RS05 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Pillar Mesaj Uzatma, Boy = 0.75&quot; </td><td> ThorLabs </td><td> RS075 </td><td></td><td> 4 </td></tr><tr><td> Ø1 &quot;Korunan Gümüş Ayna, kalın 3,2 mm </td><td> ThorLabs </td><td> ME1-P01 </td><td></td><td> 1 </td></tr><tr><td> Ø1 &quot;SM1 Döner Ayarlanabilir Odak Element, L = 1&quot; </td><td> ThorLabs </td><td> SM1V10 </td&gt; <td></td><td> 1 </td></tr><tr><td> Ø2 &quot;Korunan Gümüş Ayna, kalın 3,2 mm </td><td> ThorLabs </td><td> ME2-P01 </td><td></td><td> 2 </td></tr><tr><td> P100S - Ø100 mm İğne Atlı </td><td> ThorLabs </td><td> P100S </td><td></td><td> 1 </td></tr><tr><td> Polaris Düşük Drift Ø1 &quot;Kinematik Ayna Dağı </td><td> ThorLabs </td><td> POLARIS-K1 </td><td> Düşük sürüklenme </td><td> 1 </td></tr><tr><td> SM1 Lens Tube, L = 1 &quot; </td><td> ThorLabs </td><td> SM1L-10 </td><td> Bir emniyet halkasıyla </td><td> 4 </td></tr><tr><td> SM1 Konu 30 mm Kafes Tabak, 0.35 &quot;Kalın </td><td> ThorLabs </td><td> CP02 </td><td></td><td> 2 </td></tr><tr><td> SM1 M25 Optik Bileşen Threading Adaptör </td><td> ThorLabs </td><td> SM1A24 </td><td> , Dış SM1 Konuları ve İç M25.5x0.5 Konuları </td><td> 1 </td></tr><tr><td> Küçük Işın Çapıaçmak mümkündür galvo Sistemi </td><td> ThorLabs </td><td> GVSM001 </td><td></td><td> 1 </td></tr><tr><td> Küçük Sıkma Çatal </td><td> ThorLabs </td><td> CF125 </td><td> 1 / 25 &quot;, evrensel yuvası counterbored </td><td> 15 </td></tr><tr><td> Mekansal Filtre Sistemi </td><td> ThorLabs </td><td> KT310 </td><td> İğne ayrı ayrı satılır </td><td> 1 </td></tr><tr><td> 5.6 ve 9 mm Lazerler TE-Soğutmalı Dağı </td><td> ThorLabs </td><td> TCLDM9 </td><td></td><td> 1 </td></tr><tr><td> Breadboards için Dikey Braketi </td><td> ThorLabs </td><td> VB01 </td><td> Her </td><td> 2 </td></tr><tr><td> Plan-Apochromat </td><td> Zeiss </td><td> 1101-957 </td><td> 20x/0.75 NA </td><td> 1 </td></tr></tbody></table>

video-rate scanning confocal microscopy and microendoscopy

Mikroskopisi, hızlı, yüksek hassasiyet ve karmaşık sistemlerin yüksek çözünürlüklü optik kesitler sağlayarak, biyoloji ve biyomedikal bilimler paha biçilmez bir araç haline gelmiştir. Mikroskopisi, canlı hücreleri 2-4 spesifik hücresel hedefler 1, monitör dinamikleri rutin çalışma ve üç boyutlu tüm organizmalar 5,6 evrim görselleştirmek, örneğin kullanılır . Konfokal mikroendoskopların gibi konfokal görüntüleme sistemleri, Uzantıları, in vivo 7 yüksek çözünürlüklü görüntüleme için izin verir ve henüz klinik ortamlarda 8,9 hastalık görüntüleme ve teşhis uygulanmaktadır. Mikroskopisi basit geometrik optik kullanarak sözde &quot;optik bölümler&quot; oluşturarak üç boyutlu çözünürlük sağlar. Standart bir geniş alan mikroskobu, floresan bir örneklem üzerinden üretilen bir objektif lens ile toplanır ve bir dedektör doğrudan iletti. Kabul ederkengörüntüleme ince örnekleri için mümkün, kalın örnekleri objektif odak düzlem üstünde ve altında üretilen floresan bulanık olur. Buna karşılık, konfokal mikroskopi reddederek, sanal örnek, optik kesit sağlar out-of-focus, örneklerin yüksek çözünürlüklü üç boyutlu temsilleri inşa ışık. Konfokal mikroskoplar algılama ışın yolu bir konfokal diyafram kullanarak bu feat ulaşmak. Nesnel bir örnek alınan floresan tarama aynalar aracılığıyla ve geri birincil dikroik ayna, uzun, Stokes-kaymıştır floresans emisyon geçerken, lazer uyarma ışını gibi kısa dalga boylarında yansıtmak için dikkatlice seçilen bir ayna aracılığıyla iletti. Bu uzun dalga boyunda floresans sinyalinin ardından, objektif lens odak düzlemli ile tam olarak konjuge bir düzlemde konumlandırılmış bir iğne deliği iki tarafında lenslerin bir çift geçti. Nesne odak hacmi toplanan Fotonlar collimatedobjektif lens ve iğne deliğinden konfokal lensler odaklanmıştır. Bu nedenle odak düzlem üzerinde veya altında üretilen Floresans düzgün collimated olmayabilir, ve mikroskop odak sadece ışık görünür olduğu bir optik kesit yaratırken, konfokal iğne deliği 1 ile geçmek olmaz . (Şekil 1). Böylece iğne deliği etkili, sadece tek bir sınırlı mekansal konumu tespit edilen emisyon hapsetmesi odak düzlemli bir sanal diyafram gibi davranır. Modern ticari konfokal mikroskoplar, nispeten basit ve erişilebilir eski karmaşık görüntüleme yöntemleri, kullanıcıların tam otomatik çalışma imkanı sunar. Bu sistemlerin esneklik ve güç olmasına rağmen, ticari konfokal mikroskoplar birçok in vivo görüntüleme uygulamaları gibi tüm konfokal görüntüleme görevleri için uygun değildir. Onların ihtiyaçlarını karşılamak için özelleştirilmiş görüntüleme sistemleri yaratma yeteneği olmadan, tepkiler, önemli deneyler dışında kalanpek çok bilim adamı saat. Bu makalede, biz temel bileşenlerinden özel bir video-rate konfokal görüntüleme sistemi yapımı için adım adım bir yöntem sağlar. Dik mikroskop, standart bir hız rezonans galvanometric ayna yavaş eksen taraması, hızlı tarama ekseni sağlamak için bir rezonans galvanometric ayna kullanılarak inşa edilecek. Objektif lens odak kesin bir taranan ışını oluşturmak için, bu aynalar dört röle lensler kullanarak sözde telesentrik uçakları konumda olacaktır. Konfokal algılama standart, off-the-raf photomultiplier tüp (PMT) kullanılarak gerçekleştirilebilir ve yakalanan görüntülerin ve Matrox Framegrabber kartı kartı ile birlikte gelen yazılımı kullanarak görünecektir.

Watch this Scientific Journal Video about Video-rate Scanning Confocal Microscopy and Microendoscopy at JoVE.com

Video-rate Scanning Confocal Microscopy and Microendoscopy

Özel, gerçek zamanlı bir konfokal tarama görüntüleme sistemi komple inşaat açıklanmıştır. Video hızı mikroskopi ve mikroendoskopi için kolayca kullanılabilen bu sistem, maliyetinin bir kısmını, standart ticari konfokal sistemlerini kullanarak erişilebilir değil görüntüleme geometrileri ve uygulamaları bir dizi sağlar.

Video-rate Tarama Konfokal Mikroskop ve Mikroendoskopi

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

video-rate-scanning-confocal-microscopy-and-microendoscopy

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JoVE Journal

Bioengineering

27.7K Görüntüleme.  Harvard University. Özel, gerçek zamanlı bir konfokal tarama görüntüleme sistemi komple inşaat açıklanmıştır. Video hızı mikroskopi ve mikroendoskopi için kolayca kullanılabilen bu sistem, maliyetinin bir kısmını, standart ticari konfokal sistemlerini kullanarak erişilebilir değil görüntüleme geometrileri ve uygulamaları bir dizi sağlar.

Video: Video-rate Scanning Confocal Microscopy and Microendoscopy

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy. Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user.
We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences. The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ.

High-resolution Fiber-optic Microendoscopy for in situ Cellular Imaging

In many biological and clinical situations it is advantageous to study cellular processes as they evolve in their native microenvironment. Here we describe the assembly and use of a low-cost fiber-optic microscope which can provide real time imaging in cell culture, animal studies, and clinical patient studies.

Yüksek çözünürlüklü Fiber-optik Mikroendoskopi In situ Hücresel Görüntüleme

Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution.  ...

Biyomühendislik

Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are
 preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of
 data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative 
 cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the 
 assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This 
 cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM 
 studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols 
 for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a 
 reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

We demonstrate the fabrication of a low-cost cryogenic stage designed to fit most reflected light microscopes. This lab-built cryogenic stage enables efficient and reliable correlative imaging between cryo-light and cryo-electron microscopy.

Düşük Maliyetli Cryo-Işık Mikroskopi Sahne İmalatı İlişkili Işık / Elektron Mikroskopi

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular 
dynamics ...

Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at submicrometer resolution. One of the first such instruments producing whole-brain-scale data is the Knife-Edge Scanning Microscope (KESM)7, 5, 9, developed and hosted in the authors' lab. KESM has been used to section and image whole mouse brains at submicrometer resolution, revealing the intricate details of the neuronal networks (Golgi)1, 4, 8, vascular networks (India ink)1, 4, and cell body distribution (Nissl)3. The use of KESM is not restricted to the mouse nor the brain. We have successfully imaged the octopus brain6, mouse lung, and rat brain. We are currently working on whole zebra fish embryos. Data like these can greatly contribute to connectomics research10; to microcirculation and hemodynamic research; and to stereology research by providing an exact ground-truth. 
In this article, we will describe the pipeline, including specimen preparation (fixing, staining, and embedding), KESM configuration and setup, sectioning and imaging with the KESM, image processing, data preparation, and data visualization and analysis. The emphasis will be on specimen preparation and visualization/analysis of obtained KESM data. We expect the detailed protocol presented in this article to help broaden the access to KESM and increase its utilization.

The full process from brain specimen preparation to serial sectioning imaging using the Knife-Edge Scanning Microscope, to data visualization and analysis is described. This technique is currently used to acquire mouse brain data, but it is applicable to other organs, other species.

Bıçak kenar Tarama Mikroskopi için Numune Hazırlama, Görüntüleme ve Analiz Protokolleri

Major advances in high-throughput, high-resolution, 3D microscopy techniques have enabled the acquisition of large volumes of neuroanatomical data at ...

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

Kuantum Nokta Nanopartiküller kullanma Korelatif Işık- ve Elektron Mikroskobu

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of spectroscopy techniques. Combining imaging and spectroscopy techniques into a single optical probe may provide a more complete analysis of tissue health. In this article, two dissimilar modalities are combined, high-resolution fluorescence microendoscopy imaging and diffuse reflectance spectroscopy, into a single optical probe. High-resolution fluorescence microendoscopy imaging is a technique used to visualize apical tissue micro-architecture and, although mostly a qualitative technique, has demonstrated effective real-time differentiation between neoplastic and non-neoplastic tissue. Diffuse reflectance spectroscopy is a technique which can extract tissue physiological parameters including local hemoglobin concentration, melanin concentration, and oxygen saturation. This article describes the specifications required to construct the fiber-optic probe, how to build the instrumentation, and then demonstrates the technique on in vivo human skin. This work revealed that tissue micro-architecture, specifically apical skin keratinocytes, can be co-registered with its associated physiological parameters. The instrumentation and fiber-bundle probe presented here can be optimized as either a handheld or endoscopically-compatible device for use in a variety of organ systems. Additional clinical research is needed to test the viability of this technique for different epithelial disease states.

Multimodal Imaging and Spectroscopy Fiber-bundle Microendoscopy Platform for Non-invasive, In Vivo Tissue Analysis

The assembly and use of a multimodal microendoscope is described which can co-register superficial tissue image data with tissue physiological parameters including hemoglobin concentration, melanin concentration, and oxygen saturation. This technique can be useful for evaluating tissue structure and perfusion, and can be optimized for individual needs of the investigator.

Multimodal Görüntüleme ve Non-invaziv için Spektroskopisi Fiber paket Mikroendoskopi Platformu,<em&gt; İn Vivo</em&gt; Doku Analizi

Recent fiber-bundle microendoscopy techniques enable non-invasive analysis of in vivo tissue using either imaging techniques or a combination of ...

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; however such coordinates must represent homologous structures across specimens. While many biological objects consist of easily identified landmarks to satisfy the assumption of homology, many lack such structures. One potential solution is to mathematically place semi-landmarks on an object that represent the same morphological region across specimens. Here, we illustrate a recently developed pipeline to mathematically define semi-landmarks from the mouse baculum (penis bone). Our methods should be applicable to a wide range of objects.

Many biological structures lack easily definable landmarks, making it difficult to apply modern morphometric methods. Here we illustrate methods to study the mouse baculum (a bone in the penis), including dissection and microCT scanning, followed by computational methods to define semi-landmarks that are used to quantify size and shape variation.

Baculum diseksiyonu, mikroBT Tarama ve Morfometrik Analizleri

Modern morphometrics provides powerful methods to quantify size and shape variation. A basic requirement is a list of coordinates that define landmarks; ...

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.

Memeli hücre bölünmesi yılında niceliksel Confocal yansıma mikroskobu ile 3D matrisler

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

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

Eğik tarama Lazer Ophthalmoscopy (oSLO) ve optik koherens tomografi (OCT) tarafından Multimodal hacimsel Retina Görüntüleme

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

Hücreleri tarafından seçilmiş adezyon oluşumunda görselleştirme gelişmiş Disk-toplam iç yansıma floresans mikroskobu iplik

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Canlı Hücrelerde Eşzamanlı Lazer Tarama Konfokal ve Atomik Kuvvet Mikroskobu (Conpokal) Yakınında

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Methods Article

Video-rate Tarama Konfokal Mikroskop ve Mikroendoskopi