Tim Daley und Andrew Smith lieferte viele nützliche Rechen Diskussionen in den frühen Tagen; Tim Daley schrieb das Programm rotate_translate_cylindrical notwendig für das Protokoll 5. Computational Ressourcen, die von der High Performance Computing Cluster an der University of Southern California zur Verfügung gestellt wurden. Diese Arbeit wurde von NIH Zuschusses # GM098536 (MDD) unterstützt.

Alle Verfahren und Personal wurden von der University of Southern California Institute for Animal Care und Use Committee (IACUC), Protokoll # 11394 zugelassen. 1. Baculum Dissection und Vorbereitung <ol><li> Euthanize eine geschlechtsreife männliche Maus über Kohlendioxid Überbelichtung, nach Protokollen, die von der jeweiligen Institutional Animal Care und Use Committee (IACUC) dargelegt. </li><li> Legen Sie das Tier in Rückenlage, und verschleppen den Penis durch Druck mit den Daumen auf die Präputialöffnung lateral. </li><li> Sobald der Penis langwierig ist, das Gewebe durch die Vorhaut erstrecken so weit wie möglich. </li><li> Mit Schere, schneiden Sie den Penis Körper proximal zu der Eichel, wo die baculum befindet. </li><li> Übertragen Sie die seziert Penis zu einem 1,7 ml-Röhrchen und mit 200 &amp; mgr; l Leitungswasser. Stellen Sie sicher, dass der Penis vollständig in die Flüssigkeit eingetaucht ist. </li><li> Inkubieren des Gewebes in Wasser bei ~ 50 ° C für 3-5 Tage. </li><li&gt; Nach der ordnungsgemäßen Inkubation entfernen baculum das umgebende Gewebe, Pinzette unter einem Präpariermikroskop mit. Sanft spritzen 70% Ethanol verbleibende Gewebe abzustoßen und den Knochen zu reinigen. </li><li> Platzieren Sie den sezierten baculum in ein neues Reaktionsgefäß mit der Kappe geöffnet. Lassen Kappe offen O / N Knochen austrocknen. </li></ol> 2. MicroCT Scanning <ol><li> Drücken Sie eine microCT Scan zylindrischen Halter in einen Ziegel von Floristen Schaum einen Zylinder von Floristen Schaum zu erzeugen. </li><li> Extrahieren Sie den Zylinder von Floristen Schaum Scheiben schneiden und ~ 2-5 cm dick. </li><li> Push getrocknet bacula in den Floristen Schaum, um den Umfang eines einzelnen Slice Störungen zu minimieren während des Scannens. Die genaue Ausrichtung der Knochen sollte 4 in Protokoll für eine angemessene Identifizierung einzelner Proben bemerkt werden. </li><li> Sanft legen Sie die Scheibe mit den eingebetteten Knochen in den microCT Halter. </li><li> Erwerben Sie microCT Scans. Im Fall der Maus 14 bacula </sup&gt; Verwendeten wir ein uCT50 Scanner (Scanco Medical AG, Brüttisellen, der Schweiz) an der USC Molecular Imaging Center unter den folgenden Einstellungen: 90 kVp, 155 &amp; mgr; A, 0,5 mm Al-Filter, 750 Projektionen pro 180 (360 Deckung), Belichtungszeit 500 ms und Voxelgröße von 15,5 mm. </li></ol> 3. MicroCT Verarbeitung: eine .dcm Stapel zu einem einheitlichen .xyz Datei konvertieren HINWEIS: Jeder microCT Scan einen Stapel von .dcm erzeugt, oder &quot;dicom&quot;, Dateien, die Bildscheiben durch das Objekt genommen repräsentieren. Alle nachgelagerten algorithmischen Geometrie erfordert flache .xyz Dateien, die einfach eine Textdatei, die vier Spalten enthält - die x-, y- und z-Koordinaten jedes Pixels und die Intensität des Pixels, von -5.000 (schwarz) bis 5000 (Weiß). Ein Pixelschwelle über 3000 funktioniert im Allgemeinen gut als Schwelle für die Definition von Knochen. <ol><li> Installieren Sie Python (www.python.org) und die Python-Module BEFEHLE, DICOM, PYLAB, SYS und numpy. </li><li> Open &quot;01_process_dicom.py &quot;{Figshare} mit einem beliebigen Texteditor. Unter dem Abschnitt Variablen, ändern Weg, Pixelschwellen und Verzeichnisnamen wie nötig. </li><li> Run &quot;Python 01_process_dicom.py&quot;. Der Fortschritt wird auf Bildschirm gedruckt werden. Innerhalb jedes Verzeichnis in Schritt namens 3.2, wird eine neue Datei gemacht benannt; zum Beispiel directory_name.PT3000.xyz, wo PT3000 die Pixelschwelle anzeigt, in Schritt 3.2 angegeben. </li></ol> 4. MicroCT Verarbeitung: Segmentieren-out Einzel Specimen .xyz Dateien <ol><li> Installieren Sie R (https://www.r-project.org/) mit der Bibliothek RGL. </li><li> Öffnen Sie die Datei &#39;02_segment_dicoms.r&#39; {Figshare} mit einem beliebigen Texteditor. Unter dem Abschnitt Variablen, ändern Sie den Pfad zur Datei .xyz in Protokoll 3 oben erstellt zu zeigen. </li><li> Innerhalb R, führen Sie den Befehl &quot;Quelle ( &#39;02_segment_dicoms.r&#39;)&quot; (ohne Anführungszeichen). </li><li> Nach dem dreidimensionalen Bild der .xyz Datei in Protokoll 3 erscheint erstellt, geben Sie das number von Proben in der Gesamt .xyz Datei. Dann beschriften und die Punkte aus jeder Probe wählen Sie die Scroll- und Zoom-Funktionen. HINWEIS: Im Hintergrund getrennt .xyz Dateien werden für jede Probe durchgeführt werden. Diese erscheinen in einem Verzeichnis mit dem Namen, zum Beispiel XYZ_FILES_PT3000, wo PT3000 gibt die Pixelschwelle verwendet. </li></ol> 5. &quot;Ausrichten&quot; Specimen .xyz Dateien auf gemeinsamen Koordinaten. <ol><li> Öffnen Sie das Python-Skript &quot;03_transform.py&quot; {Figshare}, die das Zusatzmodul mattdean_modules.py {Figshare} erfordert, sowie zwei selbststehende Anwendungen: &quot;rotate_translate_cylindrical&quot; (https://github.com/timydaley/dean_cylindrical_tranform) und &quot;qconvex&quot; (www.qhull.org/html/qconvex.htm), die von diesem Skript verwendet werden. </li><li> Unter dem Abschnitt Variablen identifizieren die vollständigen Pfadnamen zu mattdean_modules.py, rotate_path und qconvex_dir. Darüber hinaus erkennen Sie den vollständigen Pfad zu dem Verzeichnis, das individuelle .xy enthältz-Dateien in Schritt 4 erstellt haben. </li><li> Führen Sie 03_transform.py, die pro Probe mit dem Suffix .TRANSFORMED.xyz eine neue Datei erstellt. </li></ol> 6. &quot;Slicing&quot; Aligned Specimen .xyz Dateien Halb Sehenswürdigkeiten zu identifizieren. <ol><li> Öffnen Sie und führen Sie das Python-Skript &quot;04_identify_landmarks.py&quot; {Figshare}. Im Abschnitt Variablen identifizieren vollständigen Pfadnamen in das Verzeichnis der .TRANSFORMED.xyz Dateien enthält. Dieses Skript identifiziert 802 semi-Wahrzeichen, die verwendet werden können, Größe und Form der Struktur zu quantifizieren. </li></ol>

<ol>
	<li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geometrics+morphometrics.">Geometrics morphometrics.</a> Annu. Rev. Anthropol. 36, 261-281 (2007).</li><li>Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. 6, (2005).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2012).</li><li>Bookstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Morphometric tools for landmark data: geometry and biology. , (1991).</li><li>Rohlf, F. J., Marcus, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Revolution+in+Morphometrics.">A Revolution in Morphometrics.</a> Trends. Ecol. Evol. 8 (4), 129-132 (1993).</li><li>Zelditch, M. L., Swiderski, D. L., Sheets, H. D., Fink, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Geometric morphometrics for biologists: a primer. , (2004).</li><li>Rohlf, F. J., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensions+of+the+Procrustes+method+for+the+optimal+superimposition+of+landmarks.">Extensions of the Procrustes method for the optimal superimposition of landmarks.</a> Syst. Zool. 39 (1), 40-59 (1990).</li><li>Gunz, P., Mitteroecker, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semilandmarks:+a+method+for+quantifying+curves+and+surfaces.">Semilandmarks: a method for quantifying curves and surfaces.</a> Hystrix. 24 (1), 103-109 (2013).</li><li>Gunz, P., Ramsier, M., Kuhrig, M., Hublin, J. J., Spoor, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+bony+labyrinth+reconsidered,+introducing+a+comprehensive+geometric+morphometric+approach.">The mammalian bony labyrinth reconsidered, introducing a comprehensive geometric morphometric approach.</a> J. Anat. 220 (6), 529-543 (2012).</li><li>Mitteroecker, P., Gunz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+geometric+morphometrics.">Advances in geometric morphometrics.</a> Evol. Biol. 36 (2), 235-247 (2009).</li><li>Bookstein, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Landmark+methods+for+forms+without+landmarks:+morphometrics+of+group+differences+in+outline+shape.">Landmark methods for forms without landmarks: morphometrics of group differences in outline shape.</a> Med. Im. Anal. 1 (3), 225-243 (1997).</li><li>Gunz, P., Mitteroecker, P., Bookstein, F., Slice, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Modern morphometrics in physical anthropology. , 73-98 (2005).</li><li>Oxnard, C., O'Higgins, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+Clearly+Needs+Morphometrics.+Does+Morphometrics+Need+Biology?.">Biology Clearly Needs Morphometrics. Does Morphometrics Need Biology?.</a> Biological Theory. 4 (1), 84-97 (2009).</li><li>Schultz, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+baculum+size+and+shape+variation+in+mice.">The genetic basis of baculum size and shape variation in mice.</a> G3. 6 (5), 1141-1151 (2016).</li><li>Schultz, N. G., Lough-Stevens, M., Abreu, E., Orr, T. J., Dean, M. 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+baculum+was+gained+and+lost+multiple+times+during+mammalian+evolution.">The baculum was gained and lost multiple times during mammalian evolution.</a> Integr Comp Biol. 56 (4), 644-656 (2016).</li><li>Dines, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+selection+targets+cetacean+pelvic+bones.">Sexual selection targets cetacean pelvic bones.</a> Evolution. 68 (11), 3296-3306 (2014).</li><li>Adams, D. C., Ot&#225;rola-Castillo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=geomorph:+an+R+package+for+the+collection+and+analysis+of+geometric+morphometric+shape+data.">geomorph: an R package for the collection and analysis of geometric morphometric shape data.</a> Methods Ecol. Evol. 4 (4), 393-399 (2013).</li></ol>

The authors declare that they have no competing financial interests.

Die kritischen Schritte in dem obigen Protokoll sind 1) sezieren die Bacula, 2) die microCT Bilder sammeln, 3) Umwandeln der microCT Ausgabe in eine flache Datei von XYZ-Koordinaten, 4) Segmentieren aus jeder Punkt Cloud Probe, 5) die Umwandlung jeder Probe zu ein standardisiertes System, und 6), die halb Sehenswürdigkeiten koordinieren. Diese Schritte werden leicht modifiziert, um verschiedene Objekte aufzunehmen. Diese Verfahren können wahrscheinlich auf jedes Objekt angewendet werden, d...

Das Feld der morphometrics umfasst eine Vielfalt von Methoden , um die Größe und Form der biologischen Form, einen entscheidenden Schritt in der wissenschaftlichen Forschung 1, 2, 3, 4, 5, 6 , zu quantifizieren. Herkömmlicherweise beginnt die statistische Analyse der Größe und Form von Landmarken auf einer biologischen Struktur, und dann Messen linearen Abstände, Winkel und Verhältnisse zu identifizieren, die in einer multivariaten Rahmen analysiert werden konnte. Landmark-basierte Geometrische Morphometrie ist ein Ansatz, der die räumliche Lage von Sehenswürdigkeiten behält, geometrische Informationen von der Datenerfassung durch die Analyse zu erhalten und Visualisierung 5. Generalized Prokrustes-Analyse (GPA) kann Variation in Position, Skalierung und Drehung von Sehenswürdigkeiten entfernt werden angewendet, um eine Ausrichtung zwischen Proben zu erzeugen, die minimizes ihre quadrierten Differenzen - was bleibt Form Unähnlichkeit 7. Ein wichtiges Konzept jeder morphometrische Analyse ist Homologie, oder die Idee, dass man Ziele zuverlässig identifizieren können biologisch sinnvolle und diskrete Merkmale darstellt, die zwischen Proben oder Strukturen entsprechen. Zum Beispiel haben menschliche Schädel homologe Prozesse, Foramen, Nähte und Kanäle, die morphometrischen Analysen ermöglichen können. Leider ist die Identifizierung der entsprechenden Ziele schwierig in vielen biologischen Strukturen, insbesondere solche mit glatten Oberflächen oder Kurven 8, 9, 10. Wir nähern uns, dieses Problem unter Verwendung von algorithmischen Geometrie. Der allgemeine Arbeitsablauf ist eine dreidimensionale Abtastung des Objekts zu erzeugen, die als Punktwolke dargestellt werden kann, und dann drehen und dass die Punktwolke verwandeln, so dass alle specimens sind auf einem gemeinsamen Koordinatensystem orientiert. Dann definieren wir mathematisch semi-Grenzsteine ​​aus bestimmten Regionen des Objekts. Diskrete Halb Sehenswürdigkeiten auf solche Regionen platziert sind biologisch willkürlich 11. Durchführung von GPA und die anschließende statistische Analysen können unerwünschte Artefakte 8, 12 produzieren , weil willkürlich platziert Sehenswürdigkeiten biologisch nicht homolog sein kann. Deshalb lassen wir diese Halb Sehenswürdigkeiten mathematisch &quot;gleiten&quot;. Dieses Verfahren minimiert die Potentialdifferenz zwischen den Strukturen. Wie an anderer Stelle der Schiebe Algorithmus ist hier geeignet , verwendet argumentiert ähnliche anatomische Bereiche zu quantifizieren ohne Anhaltspunkte leicht identifiziert entsprechende 3, 6, 8, 10, 11, 12. Diese Verfahren haben ihre limitations 13, sollte aber auf Gegenstände unterschiedlicher Größe und Form anpassungsfähig sein. Hier veranschaulichen wir , wie diese Methode in einer neueren Studie der Maus baculum 14 angelegt wurde, ein Knochen in den Penis, der gewonnen wurde und verlor mehrere unabhängige Zeiten während Säuger evolution 15. Wir diskutieren die Präparation und Herstellung einer bestimmten Knochen, die baculum (Protokoll 1), die Erzeugung von microCT Bilder (Protokoll 2), und die Umwandlung dieser Bilder in ein Format, das alle nachgelagerten algorithmischen Geometrie (Protokolle 3 und 4) ermöglicht. Nach diesen Schritten wird jede Probe dargestellt durch ~ 100K xyz-Koordinaten. Wir gehen dann durch eine Reihe von Transformationen, die alle Proben in eine gemeinsame Ausrichtung (Protokoll 5) effektiv ausrichten, dann definieren halb Sehenswürdigkeiten aus ausgerichteten Proben (Protokoll 6). Protokolle 1-4 sollte unabhängig davon, wobei das Objekt analysiert ähnlich sein. Protokoll 5 und das Protokoll 6 sind speziell für einen baculum entworfen, aber es ist unsere Hoffnung, dass diese Schritte durch Detaillierung, können die Ermittler Änderungen vorstellen, die für ihr Objekt von Interesse relevant wäre. So wurden beispielsweise Modifikationen dieser Methoden Beckenknochen und Rippenknochen 16 zu studieren Wal angewendet.

<table border="0" cellpadding="0" cellspacing="0" width="743">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting scissors</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">470106-338</td>
			<td style="width:167px;">Most sizes should work</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Dissecting Forceps, Fine Tip, Curved</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">82027-406</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:211px;">1.7 mL microcentrifuge tube</td>
			<td style="width:287px;">VWR</td>
			<td style="width:79px;">87003-294</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Absolute Ethanol</td>
			<td style="width:287px;">Fisher Scientific</td>
			<td style="width:79px;">CAS 64-17-5</td>
			<td>To be diluted to 70% for dissections</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:211px;">Floral Foam</td>
			<td style="width:287px;">Wholesale Floral</td>
			<td style="width:79px;">6002-48-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">uCT50 scanner&#160;</td>
			<td colspan="2">Scanco Medical AG, Bruttisellen, Switzerland</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection, microct scanning and morphometric analyses of the baculum

Moderne morphometrics bietet leistungsfähige Methoden Größe und Formvariation zu quantifizieren. Eine Grundvoraussetzung ist eine Liste von Koordinaten, die Sehenswürdigkeiten zu definieren; aber solche Koordinaten müssen homologe Strukturen in Proben repräsentieren. Während viele biologische Objekte leicht identifiziert Sehenswürdigkeiten bestehen die Annahme der Homologie zu erfüllen, fehlen viele solcher Strukturen. Eine mögliche Lösung ist mathematisch Platz halb Sehenswürdigkeiten auf ein Objekt, das den gleichen morphologischen Region über Proben repräsentieren. Hier zeigen wir eine kürzlich entwickelte Pipeline mathematisch halb Sehenswürdigkeiten aus der Maus baculum (Penisknochen) definieren. Unsere Methoden sollten auf eine breite Palette von Objekten anwendbar sein.

Die XYZ - Koordinaten der Halb Sehenswürdigkeiten in Protokoll erzeugt 6 kann direkt in jeder Grenzstein-basierte Analyse geometrische Morphometrie 17 importiert werden. Die rechnerische Pipeline oben angewendet wurde 16 Maus bacula 14 sowie Wal Becken- und Rippenknochen zu studieren. Weitere Einzelheiten über die rechnerische Definition von Halb Sehenswürdigkeiten werden hier vorgestellt, in einem Versuch, die Fo...

Watch this Scientific Journal Video about Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum at JoVE.com

Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

Viele biologische Strukturen fehlen leicht definierbaren Sehenswürdigkeiten, was es schwierig macht moderne morphometrische Methoden anzuwenden. Hier zeigen wir Methoden, um die Maus baculum (ein Knochen im Penis) zu studieren, einschließlich Dissektion und microCT Scannen, gefolgt von Rechenmethoden halb Sehenswürdigkeiten zu definieren, die verwendet werden, eine Größe zu quantifizieren und Variation zu gestalten.

Sezierung, MicroCT Scanning und morphometrische Analysen der Baculum

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

dissection-microct-scanning-and-morphometric-analyses-of-the-baculum

Research

JoVE Journal

Bioengineering

7.5K Aufrufe.  University of Southern California (USC).  Viele biologische Strukturen fehlen leicht definierbaren Sehenswürdigkeiten, was es schwierig macht moderne morphometrische Methoden anzuwenden. Hier zeigen wir Methoden, um die Maus baculum (ein Knochen im Penis) zu studieren, einschließlich Dissektion und microCT Scannen, gefolgt von Rechenmethoden halb Sehenswürdigkeiten zu definieren, die verwendet werden, eine Größe zu quantifizieren und Variation zu gestalten. 

Serie: Dissection, MicroCT Scanning and Morphometric Analyses of the Baculum

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.

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 ...

Forschung

Biotechnik

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 ...

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and expels it with a precisely orchestrated contraction. A number of congenital and acquired urological anomalies including posterior urethral valves, benign prostatic hyperplasia, and neurogenic bladder secondary to spina bifida/spinal cord injury can result in pathologic tissue remodeling leading to impaired compliance and reduced capacity1. Functional or anatomical obstruction of the urinary tract is frequently associated with these conditions, and can lead to urinary incontinence and kidney damage from increased storage and voiding pressures2. Surgical implantation of gastrointestinal segments to expand organ capacity and reduce intravesical pressures represents the primary surgical treatment option for these disorders when medical management fails3. However, this approach is hampered by the limitation of available donor tissue, and is associated with significant complications including chronic urinary tract infection, metabolic perturbation, urinary stone formation, and secondary malignancy4,5.
Current research in bladder tissue engineering is heavily focused on identifying biomaterial configurations which can support regeneration of tissues at defect sites. Conventional 3-D scaffolds derived from natural and synthetic polymers such as small intestinal submucosa and poly-glycolic acid have shown some short-term success in supporting urothelial and smooth muscle regeneration as well as facilitating increased organ storage capacity in both animal models and in the clinic6,7. However, deficiencies in scaffold mechanical integrity and biocompatibility often result in deleterious fibrosis8, graft contracture9, and calcification10, thus increasing the risk of implant failure and need for secondary surgical procedures. In addition, restoration of normal voiding characteristics utilizing standard biomaterial constructs for augmentation cystoplasty has yet to be achieved, and therefore research and development of novel matrices which can fulfill this role is needed.
In order to successfully develop and evaluate optimal biomaterials for clinical bladder augmentation, efficacy research must first be performed in standardized animal models using detailed surgical methods and functional outcome assessments. We have previously reported the use of a bladder augmentation model in mice to determine the potential of silk fibroin-based scaffolds to mediate tissue regeneration and functional voiding characteristics.11,12 Cystometric analyses of this model have shown that variations in structural and mechanical implant properties can influence the resulting urodynamic features of the tissue engineered bladders11,12. Positive correlations between the degree of matrix-mediated tissue regeneration determined histologically and functional compliance and capacity evaluated by cystometry were demonstrated in this model11,12. These results therefore suggest that functional evaluations of biomaterial configurations in rodent bladder augmentation systems may be a useful format for assessing scaffold properties and establishing in vivo feasibility prior to large animal studies and clinical deployment. In the current study, we will present various surgical stages of bladder augmentation in both mice and rats using silk scaffolds and demonstrate techniques for awake and anesthetized cystometry.

Surgical stages of bladder augmentation are described using 3-D scaffolds in murine and rat models. To test the efficacy of biomaterial configurations for use in bladder augmentation, techniques for both awake and anesthetized cystometry are presented.

Bewertung von Biomaterialien für Blasenvergrößerung mit Zystometrische Analysen in verschiedenen Tiermodellen

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Aplysia Ganglien-Vorbereitung für elektrophysiologische und molekulare Analysen einzelner Neuronen

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

Großflächige Scan Sonde Nanolithografie erleichtert durch automatische Ausrichtung und seine Anwendung auf Substrat Herstellung für Zell-Kultur-Studien

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Automated Slide Scanning and Segmentation in Fluorescently-labeled Tissues Using a Widefield High-content Analysis System

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. Unfortunately, many researchers spend large amounts of time and resources developing and optimizing workflows that are only relevant to their own experiments. In this article, we describe a protocol that can be used by those with access to a widefield high-content analysis system (WHCAS) to image any slide-mounted tissue, with options for customization within pre-built modules found in the associated software. Not originally intended for slide scanning, the steps detailed in this article make it possible to acquire slide scanning images in the WHCAS which can be imported into the associated software. In this example, the automated segmentation of brain tumor slides is demonstrated, but the automated segmentation of any fluorescently-labeled nuclear or cytoplasmic marker is possible. Furthermore, there are a variety of other quantitative software modules including assays for protein localization/translocation, cellular proliferation/viability/apoptosis, and angiogenesis that can be run. This technique will save researchers time and effort and create an automated protocol for slide analysis.

Here we describe a protocol for the automated segmentation of fluorescently labeled tissues on slides using a widefield high-content analysis system (WHCAS). This protocol has wide-ranging applications in any field which involves the quantitation of fluorescent markers in biological tissues including the biological sciences, medical engineering, and health sciences.

Automatisierte Dia scannen und Segmentierung in Gewebekulturen beschriftet Gewebe mit einem Weitfeld High-Content Analysesystem

Automated slide scanning and segmentation of fluorescently-labeled tissues is the most efficient way to analyze whole slides or large tissue sections. ...

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.

Multimodale Volumetrische Retinal Imaging von schrägen Scanning Laser Ophthalmoskopie (oSLO) und optische Kohärenztomografie (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 ...

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

Morphometrische Protokoll für die objektive Beurteilung der Blastozyste Verhalten während der Verglasung und wärmenden Schritte

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

Auswertung photosynthetischer Verhaltensweisen durch gleichzeitige Messungen von Blattreflexion simultanen und Chlorophyllfluoreszenzanalysen

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically been studied in vivo in animal models showing characteristic patterns of cell migration. However, in vivo models have several limitations, including intercellular mediators that are difficult to access and analyze, as well as the inability to directly analyze human neutrophils. Because of these limitations, there is a need for an in vitro platform that studies swarming with human neutrophils and provides easy access to the molecular signals generated during swarming. Here, a multistep microstamping process is used to generate a bioparticle microarray that stimulates swarming by mimicking an in vivo infection. The bioparticle microarray induces neutrophils to swarm in a controlled and stable manner. On the microarray, neutrophils increase in speed and form stable swarms around bioparticle clusters. Additionally, supernatant generated by the neutrophils was analyzed and 16 proteins were discovered to have been differentially expressed over the course of swarming. This in vitro swarming platform facilitates direct analysis of neutrophil migration and protein release in a reproducible, spatially controlled manner.

This protocol generates bioparticle microarrays that provide spatially controlled neutrophil swarming. It provides easy access to the mediators that neutrophils release during migration and allows for quantitative imaging analysis.

Biopartikel-Mikroarrays für chemotaktische und molekulare Analysen des menschlichen Neutrophilen-Schwärmens in vitro

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...