Dieses Projekt wurde von NIH / NINDS finanziert (# 1R01-NS54252), NSF (# 0905041, # 0079874), das Texas Advanced Technology Program (# ATP-000.512-0146-2001, # ATP-000.512-0261-2001), Texas A &amp; M Research Foundation, Department of Computer Science and Engineering an der Texas A &amp; M University, und 3Scan. Wir möchten Bernard Mesa (Micro Star Technologies) für technische Beratung und Unterstützung für KESM Instrumentierung danken. Wesentliche Teile der Gestaltung und Umsetzung der KESM wurde von Bruce H. McCormick, der 2007 starb getan.

1. Probenvorbereitung: Golgi-Cox <ol><li> Dieses Protokoll lehnt sich eng an das Protokoll von Mayerich et al. 7. </li><li> Die C57BL/6J Maus ist tief narkotisiert mit Isofluran inhalant Anästhesie und dann enthauptet. </li><li> Das Gehirn wird entfernt und in einen Golgi-Cox Fixierung Lösung (1% Kaliumchromat, 1% Kaliumdichromat und 1% Quecksilberchlorid in VE-Wasser) gelegt. </li><li> Das Gehirn ist in der Golgi-Cox-Lösung im Dunkeln gelassen, bei Raumtemperatur für 10 bis 16 Wochen. </li><li> Die Gehirne werden in deionisiertem Wasser über Nacht im Dunkeln gespült. </li><li> Die gespült Gehirn ist in 5% Ammoniumhydroxidlösung in VE-Wasser für 7 bis 10 Tage im Dunkeln bei Raumtemperatur eingetaucht. Dieser langwierige Vorbereitungszeit war die Infiltration des gesamten Gehirns zu gewährleisten, so dass der schwarze Niederschlag eine Chance, vollständig in das Gewebe zu bilden hatte. </li><li> Das Gehirn ist wieder in deionisiertem Wasser bei Raumtemperatur gespült 4 Stunden und tHenne dehydriert durch eine abgestufte Reihe von Ethylalkohole: 50% und 70% in den Kühlschrank (4 ° C) und 85%, 95% (3 Änderungen), und 100% (4 bis 5 Änderungen) bei Raumtemperatur. Das Gehirn ist in jeder Lösung für 24 Stunden belassen. </li><li> Die entwässerten Gehirn ist dann in Aceton gelegt (3 bis 4 Änderungen, die jeweils für einen Tag), gefolgt von einem Araldit-Aceton-Gemisch mit Araldit-to-Aceton-Verhältnis von 1:2, 1:1, 2:1, und schließlich in 100% Araldit (3 ändert sich mit jedem über Nacht) im Kühlschrank (4 ° C). </li><li> Schließlich wird das behandelte Gehirn in 100% Araldit eingebettet und auf 60 ° C für 3 Tage (vgl. Einbettung Protokoll in Abbott und Sotelo 2). Wenn die Proben in LR White eingebettet sind dann die Proben aus den letzten 100% Ethanol-Lösung durch drei Änderungen des LR White-Lösung, die jeder im Kühlschrank über Nacht aufbewahrt, die eine Standard-Prozedur vor der Polymerisation übertragen wird. Drei Änderungen werden durchgeführt, um vollständige Infiltration gewährleisten. Die Probe wird tHenne auf frisches LR White, die in einem geschlossenen Behälter bei 60 polymerisiert ° C für 24 Stunden, die die erforderliche Menge von Zeit und Temperatur für die ordnungsgemäße Polymerisation wird. </li><li> Abb.. 1 zeigt ein Golgi-Cox gefärbten Gehirns in Araldite eingebettet. </li><li> Die ausgehärteten Probe Block wird dann auf dem Metall Probe Ring mit Epoxy angebracht, und die Seiten des Blocks abgeschnitten als notwendig (siehe Abb. 2).. </li></ol> 2. Probenvorbereitung: Nissl <ol><li> Die Maus ist tief narkotisierten mit Ketamin und Xylazin (1,7 mg Ketamin / 0,26 mg Xylazin pro 20 Gramm Körpergewicht) intraperitoneal und dann perfundierten transkardial mit 50 ml Raumtemperatur Phosphat-gepufferter Kochsalzlösung (pH 7,4), um 250 mL Platz, gefolgt Temperatur 10% neutral gepuffertem Formalin (pH 7,4). und schließlich mit 3,0 ccm unverdünnt Tusche. </li><li> Die Ganzkörper-Perfusion mit Kochsalzlösung und Fixativ ist notwendig, um das Blut aus dem Herz-Kreislauf-System klar und auf den ti fix<html> RAGEN. Perfusion mit Tusche ist notwendig, um vollständig zu füllen das Gefäßsystem des Herz-Kreislaufsystems. </li><li> Die daraus resultierende Gehirn wird dann durch eine Reihe von abgestuften Ethylalkohole (25% -100%) dehydriert und dann in Araldit Kunststoff nach dem Protokoll in 1,8-1,9 oben eingebettet. Wenn LR weiß ist das Einbettmedium dann das Protokoll in 1.10 oben folgt. </li></ol> 4. Probenvorbereitung: Generic Arten, generische Organe <ol><li> Für den allgemeinen Fall, kann Fixierung mit entweder 10% neutral gepuffertem Formalin oder 4% Paraformaldehyd durchgeführt werden. </li><li> Für kleine Gewebe Volumen, ist die Diffusion statt Perfusion für die Fixierung und Färbung empfohlen. Im Fall von kleinen Organismen oder Organe können Verfärbungen auch während der Entwässerung Prozess gemacht werden. </li><li> Die Einbettung Protokoll ist das gleiche wie oben, obwohl die Zeiten für die Lösung Infiltration von Organen oder Organismen, die viel kleiner sind als ganze Gehirn der Maus reduziert werden kann. </li></ol>jove_title &quot;&gt; 5. KESM Setup-und Imaging- <ol><li> Pumpe ausschalten, auf der Bühne drehen, drehen vor der Kamera, drehen Sie am Illuminator. </li><li> Raise Messer und objektiv. </li><li> Installieren Probe Ring. </li><li> Measure Probe Dimension und eine Konfigurationsdatei für die KESM Bühne Controller2 Anwendung. </li><li> Starten KESM Bühne Controller2 und initialisieren Bühne. </li><li> Untermesser / Ziel, an der Pumpe ab. </li><li> Fokus Ziel und Kamera durch Drehen der Fokussierknopfes auf der optischen Bahn und Einstellen der Kamera das Sichtfeld des in Bezug auf die Messerschneide. </li><li> Drücken Sie auf [Start], um Imaging zu initiieren. </li><li> Siehe Abb.. 3-8. Siehe 9, 7 für technische Details. </li></ol> 6. Bildverarbeitung und-aufbereitung <ol><li> Kopieren Sie die KESM Stack-Prozessor ausführbare Datei in den Ordner Data unter der Konfigurationsdatei Ordner (00000, 00001, etc.). </li><li> Führen Sie das KESM Stack-Prozessor für die Beleuchtung Artefakt zu entfernen und normalisieren die Hintergrund-Intensität in allenBilder. Wiederholen Sie dies für alle Daten Unterordnern. </li><li> Bereiten Multiskalen Fliesen aus dem Datensatz und Upload und Einrichten der Dateien in der KESM Brain Atlas Web-Server. </li><li> Siehe Abb.. 9. </li></ol> 7. Datenvisualisierung und-analyse <ol><li> Visualisierung mit dem KESM Brain Atlas. Zum <a href="http://kesm.cs.tamu.edu">http://kesm.cs.tamu.edu</a> und wählen Sie den entsprechenden Atlas. <ol><li> Navigieren Sie in Google maps. </li><li> Zoom in und aus, wie in Google Maps. </li><li> Um zu einer anderen Tiefe, wählen Sie, wie tief bei jedem Schritt aus dem Pull-Down-Menü gehen, und dann klicken Sie entweder auf [+] oder [-] zu erhöhen oder verringern z Tiefe. </li><li> Passt die Overlay-Nummer über das Pulldown-Menü. </li><li> Passt die Overlay-Intervall mit dem Pull-Down-Menü. </li><li> Siehe Abb.. 11. </li></ol></li><li> Visualisierung mit MeVisLab. <ol><li> Starten Sie MeVisLab. </li><li> Erstellen Sie ein neues Projekt. </li><li> Im Folgenden können neue Module, die von ty werden die Schaffungping in den Modul-Namen in der Befehlszeile ein. </li><li> Neues Modul [Compose3Dfrom2DFiles], und gehen Sie auf den gewünschten Ordner. Geben Sie den Dateinamen ein und klicken Sie in string [GetFileList]. Achten Sie darauf, die ausgewählten Bilder können in den Speicher passen. Klicken Sie auf [Create3D] zu erstellen Volumen. </li><li> Neues Modul [SetWorldMatrix], und stellen Sie die &quot;Scale&quot; unter &quot;Elementare Transformationen&quot; x, y, z die entsprechenden Voxelgröße (0,6, 0,7, 1,0 für eine 10X 0.45NA Ziel). Link [Compose3Dfrom2DFiles] bis [SetWorldMatrix]. </li><li> Set Matrix und Transformationen unter &quot;Matrix Composition&quot; auf &quot;Ignorieren&quot;, &quot;Ignorieren&quot;, &quot;Forward&quot;. </li><li> Neues Modul [Arithmetic1] und stellen Sie die &quot;Function&quot; auf &quot;Invert (Max-Img)&quot;. Link [SetWorldMatrix] bis [Arithmetic1]. </li><li> Neues Modul [View3D] und connect [Arithmetic1]. </li><li> In View3D, während die rechte Maustaste gedrückt wird, bewegen Sie die Maus, um die Schwelle einstellen. Sie können Bilder zuschneiden Regionen, die Ausrichtung ändern (bewegen Sie die Maus, während linke Maustaste gedrückt ist), ändern Beleuchtung (Volumen-zeichnungeng oder MIP), on / off Hintergrund drehen, etc. </li><li> Siehe Abb.. 10. </li></ol></li></ol> 8. Repräsentative Ergebnisse: Hier präsentieren wir Whole-Brain-Daten und Details. Feigen. 12-15 zeigen, Whole-Brain Tusche, Golgi und Nissl Datensätze 1, 4, 3. <img alt="Abbildung 1" src="/files/ftp_upload/3248/3248fig1.jpg" /> Abbildung 1. Fixiert, gefärbt und eingebettete Gehirn der Maus <img alt="Abbildung 2" src="/files/ftp_upload/3248/3248fig2.jpg" /> Abbildung 2. Embedded Maushirn Probe montiert auf die Probe Ring. <img alt="Abbildung 3" src="/files/ftp_upload/3248/3248fig3.jpg" /> Abbildung 3. Der Knife Edge Scanning Mikroskop (KESM). Ein Foto des KESM ist mit seinen wesentlichen Komponenten markiert dargestellt: (1) High-Speed-Zeilenkamera, (2) Mikroskop-Objektiv, (3) Diamantmesser Montage-und Licht-Kollimator, (4) speCimen Tank (für Wasserlagerung imaging), (5) Drei-Achsen-Präzisions-Luftlagerung Bühne, (6) weiß-Lichtmikroskop-Hilfslicht, (7) Wasserpumpe (im Hintergrund) für die Entfernung von Gewebe geschnitten, (8) PC-Server für Bühne und Bildaufnahme, (9) Granitsockel, und (10) Granit-Brücke. <img alt="Abbildung 4" src="/files/ftp_upload/3248/3248fig4.jpg" /> Abbildung 4. Imaging Prinzipien der KESM. Das Prinzip der Betrieb von KESM dargestellt. Das Ziel und das Messer ist an ihrem Platz gehalten, während die Probe auf die Positionierung der Bühne bewegt (Pfeil mit durchgezogener Linie) mit der Auflösung von 20 nm und Fahrgeschwindigkeit von 1-5 angebracht und wird auf der Diamant-Messer abgeschabt (5 mm breit für 10X-Objektiv) und erzeugt einen dünnen Abschnitt fließt über das Messer (Pfeil mit durchgezogener Linie). Line-Scan-Bildgebung ist in der Nähe der Spitze des Messers gemacht. <img alt="Abbildung 5" src="/files/ftp_upload/3248/3248fig5.jpg" /> Abbildung 5. KESM Kamera und Objektiv Fokussierung steuert. <img alt="Abbildung 6" src="/files/ftp_upload/3248/3248fig6.jpg" /> Abbildung 6. KESM Messeranordnung und Kontrollen. <img alt="Abbildung 7" src="/files/ftp_upload/3248/3248fig7.jpg" /> Abbildung 7. Initial Fokussierung durch die Beobachtung Port. <img alt="Abbildung 8" src="/files/ftp_upload/3248/3248fig8.jpg" /> Abbildung 8. KESM Bühne Controller 2-Applikation (Screenshot). <img alt="Abbildung 9" src="/files/ftp_upload/3248/3248fig9.jpg" /> Abbildung 9. KESM Stack-Prozessor-Anwendung (Screenshot). <img alt="Abbildung 10" src="/files/ftp_upload/3248/3248fig10.jpg" /> Abbildung 10. MeVisLab Anwendung (Screenshot). <img alt="Abbildung 11" src="/files/ftp_upload/3248/3248fig11.jpg" /><br/&gt; Abbildung 11 KESM Brain Atlas:. Web-Schnittstelle (Screenshot). <img alt="Abbildung 12" src="/files/ftp_upload/3248/3248fig12.jpg" /> Abbildung 12. KESM Whole-Brain Tusche Daten. Volume Visualisierungen KESM Daten Stacks sind für die vaskuläre Datensatz angezeigt. (A) Close-up des vaskulären Daten. Breite ~ 100 um. (Bd) Drei Standard-Ansichten des ganzen Gehirn der Maus Gefäßsystem (subsampled von Daten mit hoher Auflösung). Breite ~ 10mm. <img alt="Abbildung 13" src="/files/ftp_upload/3248/3248fig13.jpg" /> Abbildung 13. KESM Whole-Brain-Maus Golgi-Daten. <img alt="Abbildung 14" src="/files/ftp_upload/3248/3248fig14.jpg" /> Abbildung 14. KESM Golgi Daten Details. <img alt="Abbildung 15" src="/files/ftp_upload/3248/3248fig15.jpg" /> Abbildung 15. KESM Whole-Brain Nissl Daten. (A) Close-up der NISSL Daten. ~ 300 UM 3. (Bd) Drei Standard-Ansichten des ganzen Gehirn der Maus Nissl Daten (subsampled von Daten mit hoher Auflösung). Querschnitt für eine klare Sicht auf die internen Strukturen gezeigt.

<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 ist mit 3Scan, ein kommerzielles Unternehmen, produziert und vermarktet das KESM Technology (US-Patent # 6.744.572).

Die KESM ermöglicht eine Sub-Mikrometer-Ebene Umfrage großer Mengen biologischer Proben (~ 1 cm 3). Diese Art von Volumen reicht aus, um ganze kleine tierische Organe, wie Hirn, Lunge, Herz, Nieren halten, Scanned etc. Bilder von solcher Organe können beispiellose quantitative Informationen über die strukturelle Organisation dieser Organe vorsehen und ermöglichen rechnerische Modellierung der verschiedenen Funktionsbereiche Aspekte dieser Organe, einschließlich Schaltung und Netzwerk-Dynamik, elektrische Eigenschaften, Flüssigkeit und Luft Strömungsdynamik und muskulös Dynamik. Die Probenvorbereitung Protokoll und post-Analyse-Protokolls in diesem Artikel beschrieben wird erwartet, dass außerhalb Arbeitsgruppen helfen, einen leichteren Zugang zu den KESM und die daraus resultierenden Daten zu haben. Die KESM Betrieb Protokoll wird dazu beitragen, diese externen Forschern, die Vorteile und Grenzen dieser einzigartigen Bildgebungsverfahren zu schätzen wissen.

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

Große Fortschritte in hohen Durchsatz, hohe Auflösung, 3D-Mikroskopie-Techniken haben den Erwerb großer Mengen an neuroanatomischen Daten an Sub-Mikrometer-Auflösung aktiviert. Einer der ersten dieser Instrumente produziert Whole-Brain-Scale Data ist das Knife-Edge-Scanning-Mikroskop (KESM) 7, 5, 9, entwickelt und gehostet in der Autoren-Labor. KESM hat Abschnitt und Bild ganze Gehirn von Mäusen auf Submikrometer Auflösung verwendet wurde, enthüllt die komplizierten Details der neuronalen Netzwerke (Golgi) 1, 4, 8, Gefäßnetze (Tusche) 1, 4 und Zellkörper Verteilung (Nissl) 3 . Die Verwendung von KESM ist nicht auf die Maus noch das Gehirn beschränkt. Wir haben erfolgreich die Krake Gehirn 6, Maus Lunge und Gehirn der Ratte abgebildet. Wir arbeiten derzeit an insgesamt Zebrafisch-Embryonen. Daten wie diese können viel zur connectomics Forschung 10 beitragen; die Mikrozirkulation und Hämodynamik Forschung; und Stereologie Forschung durch Rückstellungending eine exakte ground-truth. In diesem Artikel beschreiben wir die Pipeline, darunter Probenvorbereitung (Fixierung, Färbung und Einbettung), KESM Konfiguration und Einrichtung, Schneiden und Bildgebung mit der KESM, Bildbearbeitung, Datenaufbereitung und Visualisierung von Daten und Analysen. Der Schwerpunkt wird auf die Probenvorbereitung und Visualisierung / Auswertung der gewonnenen Daten KESM werden. Wir erwarten, dass die detaillierten Protokoll in diesem Artikel vorgestellt werden, um zu erweitern den Zugang zu KESM und erhöhen ihre Auslastung.

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

Der gesamte Prozess vom Gehirn Probenvorbereitung auf serielle Schnitte Bildgebung mit dem Knife-Edge-Scanning-Mikroskop, um Daten-Visualisierung und Analyse beschrieben. Diese Technik wird derzeit verwendet, um Maus-Hirn-Daten zu erfassen, aber es ist auf andere Organe, andere Arten.

Probenvorbereitung, Imaging-und Analyse-Protokolle für Knife-edge-Scanning-Mikroskopie

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

specimen-preparation-imaging-analysis-protocols-for-knife-edge

Research

JoVE Journal

Bioengineering

17.5K Aufrufe.  Texas A&M University. Der gesamte Prozess vom Gehirn Probenvorbereitung auf serielle Schnitte Bildgebung mit dem Knife-Edge-Scanning-Mikroskop, um Daten-Visualisierung und Analyse beschrieben. Diese Technik wird derzeit verwendet, um Maus-Hirn-Daten zu erfassen, aber es ist auf andere Organe, andere Arten.

Serie: 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.

Dreidimensionale Optische Auflösung photoakustische Mikroskopie

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

Forschung

Biotechnik

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).

Bakterielle Immobilisierung für Imaging von 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 ...

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 konfokalen Mikroskopie und Mikroendoskopiesysteme

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.

Erstellen Adhesive und löslichen Farbverläufe for Imaging Cell Migration mit Fluoreszenz-Mikroskopie

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.

Hochauflösende Räumlich-zeitliche Analyse der Rezeptordynamik von Einzelmolekül-Fluoreszenzmikroskopie

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.

Herstellung von Mica Unterstützte Lipiddoppelschichten für Hochauflösende Optische Mikroskopie Imaging

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.

Hybrid μCT-FMT-Bildgebung und Bildanalyse

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.

Rasterkraftmikroskopie und Imaging Kraftspektroskopie von unterstützten Lipiddoppelschichten

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.

Korrelative Licht Elektronenmikroskopie (CLEM) für die Verfolgung und Imaging virales Protein verbundenen Strukturen in den Cryo-immobilisierten Zellen

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.

Visualisierung von Adhäsion Bildung in den Zellen durch erweiterte Spinning-Disk-Total Internal Reflection-Fluoreszenz-Mikroskopie

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