Wir möchten Gaby Martins und die Organisatoren der EMBO2010 3D Imaging Entwicklungs Werkstatt, in der diese Arbeit begann und alle von den Beitragszahlern der Fidschi und ImageJ Projekte danken.

Ein. 4D Zeitraffer-Imaging Experimente Die für die Bildaufnahme verwendete Einstellungen in Abhängigkeit von der verwendeten Ausrüstung variieren. Die Fähigkeit der konfokalen Mikroskopie optisch Schnitt einer Probe hängt von einer Anzahl von Faktoren: die Wellenlänge des Anregungs, Lochblendengröße, numerische Apertur des Objektivs, den Brechungsindex der Probe und des Mediums, in dem die Probe eingebettet ist. Die Größe des konfokalen Lochblende ausgewählt wird, die Dicke der gesammelten optischen Abschnitt zu bestimmen. Eine kleinere Lochblende ein dünner optischer Schnitt Erhöhung der z-Achse Auflösung, sondern die Verringerung der Menge an Licht, eingefangen zu produzieren. Eine größere Lochblende wird die Dicke des optischen Schnitt erhöhen, wodurch z-Achse Auflösung, sondern die Erhöhung der Menge von Licht eingefangen. Zusätzliche Faktoren, die bei der Sammlung von 4D-Daten vor der Korrektur berücksichtigen: <ol><li> Scan-Geschwindigkeit zu optimieren, zu beseitigen oder zu begrenzen, treiben dährend die Erfassung von einem einzigen Zeitpunkt. Deshalb ist die Zeit für die Bildsammlung entnommen sollte ein kleiner Bruchteil des Intervalls zwischen den Zeitpunkten. </li><li> Perfekt wiederholenden Strukturen oder Gitter, sind nicht als Strukturen für die Registrierung geeignet, da es nicht möglich ist, um die erforderliche Korrektur zu bestimmen. Zufällig verteilt Perlen oder unebene Strukturen eindeutige Registrierung zu ermöglichen. </li><li> Wenn Drift wird erwartet, erhöhen Sie den Scanbereich und obere und untere Grenzen Fokus, um sicherzustellen, das Gebiet von Interesse innerhalb des Bildstapels bleibt. </li><li> Neben der Sicherstellung eine angemessene räumliche Auflösung, die Strukturen von Interesse zu beheben, legen Sie die Abtastrate auf zeitliche Auflösung für die dynamischen Ereignisse untersucht werden. Hinweis: Die Zeit zwischen Bildzeitpunkten sollte daher mindestens die Hälfte der Zeit-Intervall zwischen regelmäßigen wiederkehrende Ereignisse und senken für unregelmäßige Veranstaltungen. </li></ol> 2. Öffnen der konfokale Dataset Die Open-Source-Paket Fidschi ist eine Verteilung der ImageJ-Programm, das vorinstallierte Plug-ins für zahlreiche Prozesse auf Daten aus der Mikroskopie Experimente durchführen gesammelt enthält. Die Software bietet einfacher Plug-in-Architektur und Update enthält eine Kopie der richtigen 3D-Drift-Plug-in für dieses Protokoll verwendet. Die Software unterstützt den Import einer Vielzahl von proprietäre Bildformate Mikroskopie durch den Einsatz des Open-Mikroskopie Umwelt Bio-Formate Import-Plug-in. <ol><li> Installieren Sie den Fidschi-Programm (http://fiji.sc/Downloads). </li><li> Laden Sie die Datenmenge mit Hilfe der erworbenen LOCI Bio-Formate Importer-Plugin. </li><li> Wenn die Datenmenge ist größer als der Speicher für das Programm, wählen Sie die Option &quot;Verwenden virtuellen Stack&quot; in der Speicherverwaltung. </li></ol> 3. Drift Korrektur eines 3D-Objekt im Post Processing Im Verlauf eines längeren Zeitrafferexperimentieren, eine Probe zu bewegen, auch wenn eingebettet. Um eine Bewegung zu korrigieren und damit die Migration Ereignisse abgebildet analysiert werden soll, kann die Nachbearbeitung der Bilder durchgeführt werden. Alle Bildnachbearbeitung muss deutlich in der Methodik der Analysen aus dieser Arbeit abgeleitet beschrieben werden. <ol><li> Sobald der Datensatz geladen wurde, führen Sie die korrekte 3D-Drift-Plugin. </li><li> Wenn es mehrere Bildkanäle, wählen Sie die zu verwendenden Kanal, um die Bilder zu registrieren. Dies sollte idealerweise stellen eine statische Struktur innerhalb der Probe eher als alle Zugvögel oder mobile Elemente. Jedoch, wenn dies nicht möglich ist der Kanal mit der geringsten Bewegung gewählt werden. </li><li> Wenn der verfügbare RAM auf dem für diese Analyse verwendete System ist weniger als doppelt so groß wie die ursprüngliche Datenmenge, wählen Sie die Option Gebrauch virtuellen Stack. Dies wird die registrierte Hyperstack als Bildsequenz zu speichern, anstatt die Datei in den Arbeitsspeicher. <ol><li> Wählen Sie einen Ordner für das Plug-in die Ausgabe einzelner korrigiert images-Dateien. Eine separate Bilddatei wird für jeden Kanal an jeder z-Position erstellt werden. </li></ol></li><li> Das Plugin wird dann führen eine paarweise Phasenkorrelationsanalyse zwischen den einzelnen Zeitpunkt, um die erforderliche Korrektur, die dann auf den Datensatz angewandt bestimmen. </li></ol>

<ol>
	<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Abramoff, M. D., Magalhaes, P. J., Ram, 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=Image+Processing+with+ImageJ.">Image Processing with ImageJ.</a> Biophotonics International. 11 (7), 42-42 (2004).</li><li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+Microscopy.">ImageJ for Microscopy.</a> BioTechniques. 43 (S1), 25-30 (2007).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> The Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE transactions on image processing : a publication of the IEEE Signal Processing Society. 7 (1), 27-41 (1998).</li><li>Kuglin, C. D., Hines, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+correlation+image+alignment+method.">The phase correlation image alignment method.</a> Proceedings of the IEEE, International Conference on Cybernetics and Society. , 163-165 (1975).</li><li>Preibisch, S., Saalfeld, S., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Globally+optimal+stitching+of+tiled+3D+microscopic+image+acquisitions.">Globally optimal stitching of tiled 3D microscopic image acquisitions.</a> Bioinformatics. 25 (11), 1463-1465 (2009).</li><li>Kaufmann, A., Mickoleit, M., Weber, M., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+mounting+enables+long-term+imaging+of+zebrafish+development+in+a+light+sheet+microscope.">Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.</a> Development. 139 (17), 3242-3247 (2012).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+motility+and+actin+cytoskeleton+of+individual+neurons+and+neural+crest+cells+in+zebrafish+embryos.">Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.</a> J VIs. Exp. (36), (2010).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+cell+extrusion+from+the+epidermis+of+developing+zebrafish.">Live imaging of cell extrusion from the epidermis of developing zebrafish.</a> Journal of Visualized Experiments: JoVE. (52), (2011).</li><li>Benard, E. L., vander Sar, A. M., Ellett, F., Lieschke, G. J., Spaink, H. P., Meijer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments: JoVE. (61), (2012).</li><li>Nguyen-Chi, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+Cell+Fate+Determination+within+the+Adaxial+Cell+Equivalence+Group+of+the+Zebrafish+Myotome.">Morphogenesis and Cell Fate Determination within the Adaxial Cell Equivalence Group of the Zebrafish Myotome.</a> PLoS Genetics. 8 (10), (2012).</li><li>Moore, C. A., Parkin, C. A., Bidet, Y., Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+the+Myoblast+city+homologues+Dock1+and+Dock5+and+the+adaptor+proteins+Crk+and+Crk-like+in+zebrafish+myoblast+fusion.">A role for the Myoblast city homologues Dock1 and Dock5 and the adaptor proteins Crk and Crk-like in zebrafish myoblast fusion.</a> Development. 134 (17), 3145-3153 (2007).</li><li>Higashijima, S., Okamoto, H., Ueno, N., Hotta, Y., Eguchi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+generation+of+transgenic+zebrafish+which+reliably+express+GFP+in+whole+muscles+or+the+whole+body+by+using+promoters+of+zebrafish+origin.">High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.</a> Developmental Biology. 192 (2), 289-299 (1997).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Unsere Fähigkeit, Post-Processing-Software verwenden, um Probendrift von Datensätzen aus erweiterten Zeitraffer-Mikroskopie Experimente abgeleitet korrigieren wird durch eine Reihe von Faktoren eingeschränkt. Die Fähigkeit, gegenüber Drift Wanderungs einer Probe zu erkennen ist abhängig von den zellulären Marker verwendet. Cellular Marker, die entweder weit innerhalb einer Probe ausgedrückt sind oder während der Bildaufnahme nicht in Migrations Veranstaltungen beteiligt bieten die beste Quelle für Driftkorrekt...

Die konfokale Bildgebung ist weit verbreitet in Zell-und Entwicklungsbiologie, Zell Bewegungen und Veränderungen in der Morphologie zu folgen. Erfassen einer Reihe von optischen Schnitten in verschiedenen Brennebenen ermöglicht die Erzeugung einer dreidimensionalen (3D) Modells der Probe, die dann durch die Schaffung eines Zeitserien von 3D-Datensätzen in vier Dimensionen (4D) verlängert werden können. Die Generation von 4D-Datensätzen ermöglicht die detaillierte Messung von Zellbewegungen und Verhaltensweisen. In Langzeit-Zeitraffer-Experimente ist es üblich, Probenbewegung zu beobachten. Dies kann durch kleine Ungenauigkeiten in der Hardware-Steuerung Bühne und Fokuspositionen verursacht werden. Während in anderen Fällen ist ein Ergebnis der Driftbewegungen Probe Wachstum oder Flexibilität in der Probenmontage Medien induziert. Methoden existieren, um auszugleichen oder zu begrenzen, diese Bewegungen einschließlich der Verbesserung Hardware Fokussiersysteme und erhöhte Steifigkeit der Montage Medium. Jedoch sind diese Ansätze können in vielen Fällen aufgrund der Abbildungs ​​s angewendet werdenet up erforderlich, geeignete Bedingungen für die Proben Wartung und Wachstum. Open-Source-Software-Lösungen nicht für die Korrektur der Bewegung in 2D über die Zeit vorhanden ist, durch den Einsatz der STACKREG und TURBOREG (http://bigwww.epfl.ch/thevenaz/stackreg/) 5 Plugins in ImageJ oder Fidschi, aber diese kann nicht 4D Datensätze angewendet werden. Um für die Probe Drift zu korrigieren haben wir eine Plug-in (Richtiges 3D-Drift), um die Open-Source-Bildverarbeitungsplattform, Fidschi 1 nutzen entwickelt. Unser Plug-in ist in der Lage, Phasenkorrelation Registrierung durchzuführen, um eine Bewegung, die als Ergebnis der Probendrift im dreidimensionalen Zeitraffer-Experimente erfolgt korrigieren. Phase 6 ist eine Korrelation Rechen effiziente Methode zur Übersetzung zwischen den Bildern zu bestimmen. Die hier beschriebene Plug-in nutzt die Phasenkorrelation Bibliothek von Preibisch et al. 7 entwickelt. Der Mehrkanal-Experimenten die Steck nutzt einen Kanal zur Bestimmungne die erforderliche Korrektur. Diese Korrektur wird dann auf zusätzliche Kanäle, was zu der Eintragung der 4D-Datensatz angelegt. Im Zebrafisch-Modell-System ist es möglich, Zeitraffer-Bildgebung über einen Zeitraum von vielen Stunden oder sogar mehrere Tage 8. Eine gängige Methode für die Montage des Zebrafisch ist es, die betäubt Live-Embryo in niedrig schmelzender Agarose (0,8-1,5%) einbetten, die Beschränkung ihrer Bewegung 11.09. Während Bewegung eingeschränkt ist noch Wachstum der Probe auftritt, was in den Zellen innerhalb des Sichtfeldes Schaltposition. Um die Bewegung der Zellen innerhalb des Embryos zu folgen ist es notwendig, zuerst für die Bewegung der gesamten Probe zu korrigieren. Dieses Protokoll wurde mit Zebrafisch-Proben entwickelt und hat zu Bild 12 Somitenentwicklung verwendet worden kann aber zu jedem 4D konfokalen Datenmenge angewendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5040</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Low gelling temperature agarose</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9414-25G</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:216px;">Dumont #4 Forceps</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">0208-4-PO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable 3mL graduated</td>
			<td rowspan="2" style="width:71px;">Samco</td>
			<td rowspan="2" style="width:119px;">212</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Polyethylene transfer pipette</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">9cm bacterial grade Petri dishes</td>
			<td style="width:71px;">Greiner Bio One</td>
			<td style="width:119px;">632180</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fluorinated ethylene propylene (FEP) tubing</td>
			<td style="width:71px;">Bola</td>
			<td style="width:119px;">S1815-04</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Zeiss LSM-710 Confocal microscope</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">W Plan-Apochromat 20x/1.0 DIC Objective</td>
			<td style="width:71px;">Zeiss</td>
			<td style="width:119px;">421452-9600-000</td>
		</tr>
	</tbody>
</table>

sample drift correction following 4d confocal time-lapse imaging

Die Erzeugung des vierdimensionalen (4D) konfokale Datensätze; bestehend aus 3D-Bildsequenzen über die Zeit; bietet eine hervorragende Methode, um Zellverhalten in Entwicklungsprozessen beteiligt zu erfassen. Die Fähigkeit zu verfolgen und folgen Zellbewegungen wird durch Probenbewegungen, die durch die Probe während der Bildaufnahme treiben oder, in einigen Fällen das Wachstum auftreten beschränkt. Tracking-Zellen in Datensätzen durch Drift und / oder Wachstums betroffen, diese Bewegungen in jede Analyse von Zell-Position zu übernehmen. Dieser kann aus der scheinbaren Bewegung des statischen Strukturen innerhalb der Probe führen. Daher vor der Zellverfolgung sollte jede Probe Drift korrigiert werden. Mit dem Open-Source-Distribution Fidschi 1 von ImageJ 2,3 und die einge LOCI Werkzeuge 4 des richtigen 3D-Drift-Plug-in, entwickelten wir zu fehlerhaften Probenbewegung in der konfokalen Datensätze zu entfernen. Dieses Protokoll effektiv kompensiert Probeübersetzung oder Änderungen in der Brenn position durch die Verwendung Phasenkorrelation zu jedem Zeitpunkt von einer vier-dimensionalen Datensätzen konfokalen registrieren und gleichzeitig die Fähigkeit, über längere Zeitraffer-Experimente visualisieren und messen Zellbewegungen.

In der sich entwickelnden Zebrafisch, verschmelzen schnellen Muskelkernige Zellen in Fasern von 19 Stunden nach der Befruchtung (20 - Somitenstadium) 13. Um die Bewegung der Kerne und Fusion von Muskelzellen führten wir 4D konfokalen Zeitraffer-Bildgebung unter Verwendung eines transgenen Stamm, der grün fluoreszierendes Protein (GFP) unter der Kontrolle des α-Skelett-Aktin-Promotors exprimiert, um alle Muskeln kenn visualisieren Zellen 14 und injizierten RNA die rote fluoreszierende Protein mCh...

Watch this Scientific Journal Video about Sample Drift Correction Following 4D Confocal Time-lapse Imaging at JoVE.com

Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Zeitraffer-Mikroskopie ermöglicht die Visualisierung von Entwicklungsprozessen. Wachstum oder Drift der Proben während der Bildaufnahme reduziert die Fähigkeit, genau zu verfolgen und zu messen Zellbewegungen während der Entwicklung. Wir beschreiben die Verwendung von Open-Source-Bildbearbeitungssoftware für dreidimensionale Probe Drift über die Zeit zu korrigieren.

Beispiel Drift Korrektur Nach 4D Konfokale Zeitraffer-Imaging

The generation of four-dimensional (4D) confocal datasets; consisting of 3D image sequences over time; provides an excellent methodology to capture ...

sample-drift-correction-following-4d-confocal-time-lapse-imaging

Research

JoVE Journal

Bioengineering

16.3K Aufrufe.  Monash University.  Zeitraffer-Mikroskopie ermöglicht die Visualisierung von Entwicklungsprozessen. Wachstum oder Drift der Proben während der Bildaufnahme reduziert die Fähigkeit, genau zu verfolgen und zu messen Zellbewegungen während der Entwicklung. Wir beschreiben die Verwendung von Open-Source-Bildbearbeitungssoftware für dreidimensionale Probe Drift über die Zeit zu korrigieren. 

Serie: Sample Drift Correction Following 4D Confocal Time-lapse Imaging

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Biolumineszenz-Bildgebung für die Beurteilung von Immunantworten nach der Implantation von Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Forschung

Biotechnik

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 ...

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 ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Zeitraffer-Fluoreszenz-Imaging von Arabidopsis Wurzelwachstum mit Rapid Manipulation der Root-Umgebung mit dem RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Probenvorbereitung Strategien für Massenspektrometrie Imaging von 3D-Zellkulturmodelle

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

Eines mikrofluidischen Chips für ICPMS Probenaufgabe

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Selektive Zellelimination aus Mixed 3D Culture Mit einem Nah-Infrarot-Technik Photoimmunotherapy

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

Säugetier-Zellteilung in 3D Matrizen über Quantitative konfokale Reflexion Mikroskopie

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

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Erweiterte 3D-Lebermodelle für In-vitro-Genotoxizitätstests nach langfristiger Nanomaterial-Exposition

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...