Bu proje, NSF (# 0.905.041, # 0.079.874), Texas İleri Teknoloji Programı (ATP-000.512-0146-2001, # ATP-000.512-0261-2001), Texas (# 1R01-NS54252), Ulusal Sağlık Enstitüsü / NINDS tarafından finanse edildi A &amp; M Araştırma Vakfı, Texas A &amp; M Üniversitesi, 3Scan Bilgisayar Bilimi ve Mühendisliği Bölümü. Bernard Mesa (Micro Star Teknolojileri) Biz, teknik danışmanlık ve KESM enstrümantasyon destek için teşekkür etmek istiyorum. Bruce H. McCormick, 2007 yılında ölen KESM tasarım ve uygulama ana bölümden yapıldı.

1. Örnek hazırlama: Golgi-Cox <ol><li> Bu protokol Mayerich ark protokol 7 yakından takip etmektedir. </li><li> C57BL/6J fare izofluran uçucu anestezi ile derin anestezi ve daha sonra dekapite. </li><li> Beyin Golgi Cox, fiksasyon solüsyonu (% 1 potasyum kromat,% 1 potasyum dikromat, ve deiyonize su içinde% 1 civa klorür) kaldırılır ve yerleştirilir. </li><li> Beyin, 10 ila 16 hafta boyunca oda sıcaklığında, karanlıkta Golgi-Cox çözüm bırakılır. </li><li> Beyinleri, karanlık bir gecede, deiyonize su ile durulanır. </li><li> Durulanır beyin deiyonize su karanlıkta oda sıcaklığında 7 ila 10 gün için% 5 amonyum hidroksit çözeltisi batırılır. Bu uzun hazırlık süresi, siyah bir çökelti tamamen doku oluşturmak için bir şansı vardı, böylece bütün beynin infiltrasyonu sağlamak için. </li><li> Beyin, 4 saat ve t oda sıcaklığında, deiyonize su ile tekrar durulanıroda sıcaklığında% 50 ve buzdolabında (4 ° C)% 70 ve% 85,% 95 (3 değişikliklerin) ve 100 (% 4 ila 5 değişiklik): etil alkoller kademeli serisi ile kurutulmuş tavuk. Beyni 24 saat boyunca her çözüm bırakılır. </li><li> Susuz beyin sonra araldit-aseton oranı 1:2, 1:1, 2:1, ve nihayet bir araldit-aseton karışımı ile takip (3 ila 4 değişiklikleri, her bir gün için), aseton içinde alınır % 100 araldit buzdolabında (3 değişiklik, bir gecede her zaman), (4 ° C). </li><li> Son olarak, tedavi beyin% 100 araldit gömülü ve 60 ° C&#39;de 3 gün (bkz. Abbott ve Sotelo 2 gömme protokolü) kadar ısıtılır. Örnekleri LR Beyaz gömülü Eğer örnekleri önce polimerizasyon için standart bir prosedür her gece buzdolabında tutulur üç değişiklikler, LR Beyaz çözüm yoluyla, son% 100 etanol çözüm aktarılır. Üç tam infiltrasyonu sağlamak için yapılır. Örnek t60 yaşında, kapalı bir kap içinde polimerize taze LR Beyaz transfer tavuk ° C, 24 saat boyunca uygun polimerizasyon için gerekli zaman ve sıcaklık miktarıdır. </li><li> Şekil 1 Araldite gömülü bir Golgi Cox lekeli beyin gösterir. </li><li> Sonra tedavi örnek blok, epoksi kullanarak metal örnek halkası monte, ve blok tarafına (bkz. Şekil 2) gerektiği gibi kesilmiş. </li></ol> 2. Numune hazırlama: Nissl <ol><li> Ketamin ve ksilizin (1.7 mg ketamin / 20 gram vücut ağırlığı başına 0,26 mg ksilizin) intraperitoneal olarak enjekte kullanarak fare derin anestezi ve sonra oda sıcaklığında 50 ml transcardially kullanmadan perfüze fosfat tamponlu salin (pH, 7.4 &#39;) 250 ml oda tarafından takip sıcaklık,% 10 nötr formalin tamponlu (pH, 7.4 &#39;). ve son olarak 3.0 cc sulandırılmamış Hindistan mürekkep. </li><li> Fiksatif ve tuzlu su ile bütün vücudu kardiyovasküler sistem perfüzyon kan temizlemek ve ti düzeltmek için gerekli<html> ssues. Hindistan mürekkep ile Perfüzyon kardiyovasküler sistem damarsal tamamen doldurmak için gereklidir. </li><li> Sonucu beyin kademeli etil alkoller (25% -100%), bir dizi sonra kurutulmuş ve daha sonra yukarıda 1,8-1,9 protokol araldit plastik gömülü. LR beyaz gömme ortamı ise, o zaman yukarıda 1.10 &#39;da protokol izledi. </li></ol> 4. Numune hazırlama: Genel türleri, genel organlar <ol><li> Genel durum için, fiksasyon ya da% 10&#39;luk nötral tamponlu formalin veya% 4 paraformaldehid ile yapılabilir. </li><li> Küçük bir doku birimler için, difüzyon yerine perfüzyon fiksasyon ve boyama için tavsiye edilir. Küçük organizmalar veya organlar durumunda, ayrıca boyama dehidratasyon işlemi sırasında yapılabilir. </li><li> Gömme protokolü çözüm infiltrasyon için organ veya tüm fare beyin çok daha küçük organizmalar için azaltılmış olmasına rağmen, yukarıdaki ile aynı. </li></ol>jove_title &quot;&gt; 5 KESM kurulum ve görüntüleme <ol><li> Pompa, sahnede açmak, kamera açmak, ışığını açın. </li><li> Bıçak ve amaç kaldırın. </li><li> Örnek yüzük takın. </li><li> Ölçü numune boyut ve KESM Sahne Controller2 uygulama için bir yapılandırma dosyası oluşturmak. </li><li> KESM Sahne Controller2 başlatın ve sahne başlatılamadı. </li><li> Alt bıçak / amaç, pompa açın. </li><li> Optik tren odaklanarak düğmeyi çevirerek ve bıçak kenarına göre kameranın görüş alanında ayarlayarak, objektif ve kamera odaklanın. </li><li> Görüntüleme başlatmak için [] tuşuna basın. </li><li> Bkz: Şekil. 3-8. 9, 7 teknik detaylar için bkz. </li></ol> 6. Görüntü işleme ve veri hazırlama <ol><li> KESM Yığın İşlemci çalıştırılabilir (00000, 00001, vb.) Yapılandırma dosya klasörü altında veri klasörünün içine kopyalayın. </li><li> Aydınlatma artifakı kaldırmak ve tüm arka plan yoğunluğu normale KESM Yığın İşlemci çalıştırıngörüntüler. Tüm verileri alt klasörler için tekrarlayın. </li><li> Veri ölçek fayans hazırlayın ayarlamak ve KESM Beyin Atlas web sunucu dosyalarını yükleyebilir ve kurmak. </li><li> Bkz: Şekil. 9 - </li></ol> 7. Veri analizi ve görselleştirme <ol><li> KESM Beyin Atlası kullanarak Görselleştirme. Git <a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> ve uygun atlas seçin. <ol><li> Google Maps gibi gidin. </li><li> Google Maps gibi Yakınlaştırma ve uzaklaştırma. </li><li> [-] Z derinliği arttırmak veya azaltmak için farklı bir derinlik gitmek için, ya da [+] veya üzerine tıklayın daha sonra açılan menüden her adımda ne kadar derin seçin ve. </li><li> Açılan menüyü kullanarak bindirme sayısını ayarlayın. </li><li> Açılan menü ile kaplamayı aralığı ayarlayın. </li><li> Bkz: Şekil. 11. </li></ol></li><li> MeVisLab kullanarak Görselleştirme. <ol><li> MeVisLab başlatın. </li><li> Yeni bir proje oluşturun. </li><li> Aşağıda, yeni modüller ty oluşturmaya olabilirping komutu kutusunda modül adı. </li><li> Oluştur modülü [Compose3Dfrom2DFiles], ve istediğiniz klasöre gidin. Dosya dize girin ve [GetFileList] tıklayın. Seçilen görüntüleri hafıza sığabilecek emin olun. [Create3D] birim oluşturmak için. </li><li> Oluşturun modülü [SetWorldMatrix] ve altında &quot;Ölçek&quot; uygun voksel boyutu (0.6, 0.7, 1.0 10X 0.45NA bir amaç için) x, y, z &quot;İlköğretim Dönüşümleri&quot;. Link [Compose3Dfrom2DFiles] [SetWorldMatrix]. </li><li> Matrix ayarlayın ve &quot;İleri&quot;, &quot;Yoksay&quot;, &quot;Yoksay&quot;, &quot;Matrix Kompozisyon&quot; altında Dönüşümler. </li><li> Oluştur modülü [Arithmetic1] &quot;(Max-Img) İnvert&quot; &quot;Fonksiyon&quot; ayarlayın. Link [SetWorldMatrix] [Arithmetic1]. </li><li> Oluştur modülü [View3D] ve [Arithmetic1] bağlayın. </li><li> View3D, sağ fare tuşu basılı iken, ayarlayabilirsiniz.Özellik etrafında fareyi hareket ettirin. Bölgeler, değişimin yönünü (sol fare tuşu basılı iken hareket fare), değişim aydınlatma (hacim renderin kırpabilirsinizg veya MIP), vb / arka plan kapatmak </li><li> Bkz: Şekil. 10. </li></ol></li></ol> 8. Temsilcisi Sonuçlar: Burada, bütün beyin veri ve detay sunuyoruz. İncir. 12-15 gösterisi Hindistan Mürekkep, Golgi, tüm beyin ve Nissl veri, 1, 4, 3 ayarlar . <img alt="Şekil 1" src="/files/ftp_upload/3248/3248fig1.jpg" /> Şekil 1, Sabit, lekeli, ve gömülü fare beyin <img alt="Şekil 2" src="/files/ftp_upload/3248/3248fig2.jpg" /> Şekil 2. Gömülü fare beyin örnek numune halkası üzerine monte edilmiş. <img alt="Şekil 3" src="/files/ftp_upload/3248/3248fig3.jpg" /> Şekil 3 Knife Edge Tarama Mikroskobu (KESM). Önemli bileşenleri işaretli bir fotoğrafı KESM gösterilir: (1) yüksek hızlı hat-tarama kamera, (2) mikroskop objektif, (3) elmas bıçak montaj ve ışık kolimatör, (4) SPEÇimen tankı (su altında kalacak görüntüleme için) (5) üç-eksenli hassas hava taşıyan sahne, (6) beyaz ışık mikroskobu aydınlatıcı, (7) kesitli dokusunun çıkarılması için su pompası (arka), (8) sahne kontrolü ve görüntü alımı, (9) granit taban, ve (10) granit köprü için PC sunucu. <img alt="Şekil 4" src="/files/ftp_upload/3248/3248fig4.jpg" /> Şekil 4 KESM Görüntüleme ilkeleri. KESM çalışma başlıca gösterilmiştir. Objektif ve bıçak örnek ise 20 nm ve 1-5 seyahat hızı çözünürlükte konumlandırma aşamasında hamle (düz çizgi ile ok) yapıştırılmış (5 mm genişliğinde, bir yerde tutulur ve elmas bıçakla karşı kazınarak alır 10X objektif), bıçak (düz çizgi ile ok) üzerinden akan ince bir bölümü oluşturuyor. Line-tarama görüntüleme bıçak ucuna yakın yapılır. <img alt="Şekil 5" src="/files/ftp_upload/3248/3248fig5.jpg" /> Şekil 5KESM kamera ve objektif odaklama kontrolleri. <img alt="Şekil 6" src="/files/ftp_upload/3248/3248fig6.jpg" /> Şekil 6 KESM bıçak montaj ve kontrolleri. <img alt="Şekil 7" src="/files/ftp_upload/3248/3248fig7.jpg" /> Şekil 7 gözlem bağlantı noktası üzerinden odaklama İlk. <img alt="Şekil 8" src="/files/ftp_upload/3248/3248fig8.jpg" /> Şekil 8 KESM Sahne Controller 2 uygulama (ekran görüntüsü). <img alt="Şekil 9" src="/files/ftp_upload/3248/3248fig9.jpg" /> Şekil 9 KESM yığın işlemci uygulamasını (ekran görüntüsü). <img alt="Şekil 10" src="/files/ftp_upload/3248/3248fig10.jpg" /> Şekil 10 MeVisLab uygulama (ekran görüntüsü). <img alt="Şekil 11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; Şekil 11 KESM Beyin Atlas: Web arayüzü (ekran görüntüsü). <img alt="Şekil 12" src="/files/ftp_upload/3248/3248fig12.jpg" /> Şekil 12 KESM bütün beyin Hindistan mürekkep veri. KESM veri yığınlarının Cilt görselleştirme vasküler veri seti için gösterilmiştir. (A) Yakın çekim vasküler veri. Genişlik ~ 100 um. (Bd) tüm fare beynin damarsal Üç standart görünümler (yüksek çözünürlüklü veri alt örneklendirilmiştir). Genişlik 10mm ~. <img alt="Şekil 13" src="/files/ftp_upload/3248/3248fig13.jpg" /> Şekil 13 KESM tüm beyin fare Golgi veri. <img alt="Şekil 14" src="/files/ftp_upload/3248/3248fig14.jpg" /> Şekil 14 KESM Golgi veri ayrıntılar. <img alt="Şekil 15" src="/files/ftp_upload/3248/3248fig15.jpg" /> Şekil 15 KESM tüm beyin Nissl veri. K (a) Yakın çekimissl veri. ~ 300 um 3. (Bd) tüm fare beyin Nissl veri Üç standart görünümler (yüksek çözünürlüklü veri alt örneklendirilmiştir). Kesit iç yapıları net bir şekilde görmek için gösterilmiştir.

<ol>
	<li>Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+imaging+of+whole+small+animal+brains+with+the+knife-edge+scanning+microscope.">High-throughput imaging of whole small animal brains with the knife-edge scanning microscope.</a> Neuroscience Meeting Planner. Program No. 504.2, (2008).</li><li>Abbott, L. C., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+analysis+of+catecholaminergic+innervation+in+weaver+and+normal+mouse+cerebellar+cortices.">Ultrastructural analysis of catecholaminergic innervation in weaver and normal mouse cerebellar cortices.</a> Journal of Comparative Neurology. 426, 316-329 (2000).</li><li>Choe, Y., Abbott, L. C., Miller, D. E., Han, D., Yang, H. -. F., Chung, J. R., Sung, C., Mayerich, D., Kwon, J., Micheva, K., Smith, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+imaging,+analysis,+and+integration+of+mouse+brain+networks.">Multiscale imaging, analysis, and integration of mouse brain networks.</a> Neuroscience Meeting Planner. Program No. 516.3, (2010).</li><li>Choe, Y., Han, D., Huang, P. -. S., Keyser, J., Kwon, J., Mayerich, D., Abbott, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+submicrometer+scans+of+mouse+brain+microstructure:+Neurons+and+vasculatures.">Complete submicrometer scans of mouse brain microstructure: Neurons and vasculatures.</a> Neuroscience Meeting Planner. Program No. 389.10, (2009).</li><li>Choe, Y., Abbott, L. C., Han, D., Huang, P. S., Keyser, J., Kwon, J., Mayerich, D., Melek, Z., McCormick, B. H., Ravi Rao, A., Cecchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy:+High-throughput+imaging+and+analysis+of+massive+volumes+of+biological+microstructures.">Knife-edge scanning microscopy: High-throughput imaging and analysis of massive volumes of biological microstructures.</a> High-Throughput Image Reconstruction and Analysis: Intelligent Microscopy Applications. , 11-37 (2008).</li><li>Choe, Y., Abbott, L. C., Ponte, G., Keyser, J., Kwon, J., Mayerich, D., Miller, D., Han, D., Grimaldi, A. M., Fiorito, F., Edelman, D. B., McKinstry, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charting+out+the+octopus+connectome+at+submicron+resolution+using+the+knife-edge+scanning+microscope.">Charting out the octopus connectome at submicron resolution using the knife-edge scanning microscope.</a> BMC Neuroscience. 11, 136-136 (2010).</li><li>Mayerich, D., Abbott, L. C., McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knife-edge+scanning+microscopy+for+imaging+and+reconstruction+of+three-dimensional+anatomical+structures+of+the+mouse+brain.">Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain.</a> Journal of Microscopy. 231, 134-143 (2008).</li><li>Mayerich, D., Kwon, J., Sung, C., Abbott, L. C., Keyser, J., Choe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+macro-scale+Transmission+Imaging+of+Microvascular+Networks+Using+KESM.">Fast macro-scale Transmission Imaging of Microvascular Networks Using KESM.</a> Biomedical Optics Express. 2, 2888-2896 (2008).</li><li>McCormick, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Brain+Tissue+Scanner.">Development of the Brain Tissue Scanner.</a> Technical Report. , (2002).</li><li>Sporns, O., Tononi, G., K&#246;tter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+connectome:+A+structural+description+of+the+human+brain.">The human connectome: A structural description of the human brain.</a> PLoS Computational Biology. 1, 42-42 (2005).</li></ol>

Todd Huffman 3Scan KESM Teknolojisi (US Patent # 6.744.572) üreten ve pazarlayan bir ticari şirket.

KESM biyolojik numune (~ 1 cm 3) geniş hacimli bir submicrometer düzeyinde araştırma için izin verir. Bu tür hacim, beyin, akciğer, kalp, böbrekler gibi tüm küçük hayvan organları, tutmak için yeterli, vb benzeri görülmemiş bu organların yapısal organizasyon hakkında niceliksel bilgi sağlamak ve çeşitli fonksiyonel sayısal modelleme etkinleştirmek gibi organların görüntüleri Taranmış devre ve ağ dinamiği, elektriksel özellikleri, sıvı ve hava akış dinamikleri, ve kas dinamikleri de dahil olmak üzere bu organların yönleri. Bu makalede ayrıntılı olarak numune hazırlama protokolü ve analiz sonrası protokol KESM kolay erişim ve ortaya çıkan veriler araştırma grupları dışında yardımcı olması beklenmektedir. KESM çalışma protokolü, bu benzersiz bir görüntüleme yönteminin yararları ve sınırlamaları takdir bu dış araştırmacılar yardımcı olacaktır.

A correction was made to <a href="http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

A correction was made to <a href="http://http://www.jove.com/video/3248">Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy</a>. Important modifications were made to the Nissl staining protocol.

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

specimen preparation, imaging, and analysis protocols for knife-edge scanning microscopy

Submicrometer çözünürlükte nöroanatomik veri geniş hacimli satın alma, yüksek verimlilik, yüksek çözünürlüklü, 3 boyutlu mikroskopi teknikleri büyük gelişmeler sağlamıştır. Tüm-beyin ölçekli veri üreten ilk bu tür araçların biri Knife-Edge Tarama Mikroskobu (KESM) 7, 5, 9, gelişmiş ve yazarların laboratuvar barındırılan . KESM, nöron ağları karmaşık ayrıntıları (Golgi) 1, 4, 8, damar ağları (Hindistan mürekkep) 1, 4, ve hücre gövdesi dağılımı (Nissl) 3 ifşa, bölüm ve tüm fare beyinleri submicrometer çözünürlükte görüntü için kullanılır olmuştur . KESM kullanımı, fare, ne de beyin sınırlı değildir. Biz başarıyla ahtapot beyin 6, fare akciğer ve sıçan beyin görüntülü var. Şu anda tüm zebra balık embriyoları üzerinde çalışıyoruz. Mikrodolaşım ve hemodinamik araştırma; bu gibi veriler connectomics araştırma 10 büyük katkıda bulunabilir ve yazarlara stereology araştırmading bir zemin gerçeği tam. Bu makalede, numune hazırlama (sabitleme, boyama ve gömme) dahil olmak üzere bir boru hattı, KESM konfigürasyon ve kurulum, kesit ve görüntüleme KESM ile, görüntü işleme, veri hazırlama ve veri analizi ve görselleştirme anlatacağız. Vurgu, numune hazırlama ve elde edilen KESM veri görselleştirme / analiz olacaktır. Biz bu yazıda ayrıntılı protokol KESM erişim ve kullanımını artırmak için genişletmeye yardımcı olmak için bekliyoruz.

Watch this Scientific Journal Video about Specimen Preparation, Imaging, and Analysis Protocols for Knife-edge Scanning Microscopy at JoVE.com

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

Beyin numune hazırlama, veri analizi ve görselleştirme, Bıçak-Kenar Tarama Mikroskobu kullanılarak seri kesit görüntüleme için tam bir süreç tarif edilir. Bu teknik şu anda fare beyni verileri elde etmek için kullanılan, ancak diğer organları, diğer türler için geçerli.

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 ...

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Bioengineering

17.5K Görüntüleme.  Texas A&M University. Beyin numune hazırlama, veri analizi ve görselleştirme, Bıçak-Kenar Tarama Mikroskobu kullanılarak seri kesit görüntüleme için tam bir süreç tarif edilir. Bu teknik şu anda fare beyni verileri elde etmek için kullanılan, ancak diğer organları, diğer türler için geçerli.

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

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Üç boyutlu optik çözünürlük foto akustik Mikroskopi

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Biyomühendislik

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.

Live Gram-negative and Gram-positive bacteria can be immobilized on gelatin-coated mica and imaged in liquid using Atomic Force Microscopy (AFM).

Atomik Kuvvet Mikroskopi Görüntüleme Bakteriyel İmmobilizasyon

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface.  It has the ability to image cells and biomolecules, in a liquid ...

Video-rate Scanning Confocal Microscopy and Microendoscopy

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

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

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 ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Floresan Mikroskobu ile Görüntüleme Hücre Göç Yapıştırıcı ve Çözünür Degradeler oluşturma

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

Tek molekül Floresan Mikroskopi Reseptör Dinamiği Yüksek çözünürlüklü Spatiotemporal Analizi

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

Preparation of Mica Supported Lipid Bilayers for High Resolution Optical Microscopy Imaging

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction with other compounds, such as drugs or peptides. However SLB characteristics differ depending on the support used. 
Commonly used techniques for SLB imaging and measurements are single molecule fluorescence microscopy, FCS and atomic force microscopy (AFM). Because most optical imaging studies are carried out on a glass support, while AFM requires an extremely flat surface (generally mica), results from these techniques cannot be compared directly, since the charge and smoothness properties of these materials strongly influence diffusion. Unfortunately, the high level of manual dexterity required for the cutting and gluing thin slices of mica to the glass slide presents a hurdle to routine use of mica for SLB preparation. Although this would be the method of choice, such prepared mica surfaces often end up being uneven (wavy) and difficult to image, especially with small working distance, high numerical aperture lenses. Here we present a simple and reproducible method for preparing thin, flat mica surfaces for lipid vesicle deposition and SLB preparation. Additionally, our custom made chamber requires only very small volumes of vesicles for SLB formation. The overall procedure results in the efficient, simple and inexpensive production of high quality lipid bilayer surfaces that are directly comparable to those used in AFM studies.

We present a method of preparing mica supported lipid bilayers for high resolution microscopy. Mica is transparent and flat on an atomic scale, but rarely used in imaging because of handling difficulties; our preparation results in even deposition of the mica sheet, and reduces the material used in bilayer preparation.

Mika hazırlanması Yüksek Çözünürlüklü Optik Mikroskopi Görüntüleme için lipit tabakalarının Desteklenen

Supported lipid bilayers (SLBs) are widely used as a model for studying membrane properties (phase separation, clustering, dynamics) and its interaction ...

Hybrid &#181;CT-FMT imaging and image analysis

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to assess the biodistribution of novel probes and to assess disease progression using established molecular probes or reporter genes. The combination with an anatomical modality, e.g., micro computed tomography (&#181;CT), is beneficial for image analysis and for fluorescence reconstruction. We describe a protocol for multimodal &#181;CT-FMT imaging including the image processing steps necessary to extract quantitative measurements. After preparing the mice and performing the imaging, the multimodal data sets are registered. Subsequently, an improved fluorescence reconstruction is performed, which takes into account the shape of the mouse. For quantitative analysis, organ segmentations are generated based on the anatomical data using our interactive segmentation tool. Finally, the biodistribution curves are generated using a batch-processing feature. We show the applicability of the method by assessing the biodistribution of a well-known probe that binds to bones and joints.

Hybrid  &amp;#181;CT-FMT imaging and image analysis

We describe a protocol for hybrid imaging, combining fluorescence-mediated tomography (FMT) with micro computed tomography (&#181;CT). After fusion and reconstruction, we perform interactive organ segmentation to extract quantitative measurements of the fluorescence distribution.

Hibrid uCT-FMT görüntüleme ve görüntü analizi

Fluorescence-mediated tomography (FMT) enables longitudinal and quantitative determination of the fluorescence distribution in vivo and can be used to ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomik Kuvvet Mikroskobu Görüntüleme ve Desteklenen Lipid Bilayers Kuvvet Spektroskopisi

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Cryo-immobilize hücreler yapılarda bağdaşık ışık elektron izleme ve Viral Protein Imaging mikroskobu (CLEM) ilişkili

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

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 ...