Research in the Gunzer laboratory was supported by the German Federal Ministry of Education and Research (grant no. 0315590 A-D) and by the German Research Foundation (grant no. GU769/4-1, GU769/4-2). We further thank the IMCES for technical support and Sebastian Kubat for help with 3D cartoon modeling.

All animal experiments were conducted in accordance with EU guidelines and were approved by the relevant local authorities in Essen (AZ 84-02.04.2014.A036 - Landesamt f&#252;r Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Essen, Germany). The animals were used and housed under specific pathogen-free (SPF) conditions.
1. Induction of Myocarditis by Diphtheria Toxin (DTX)
<ol>
	<li>Prepare the DTX solution for the induction of myocarditis by diluting the DTX stock solution with phos...

<ol>
	<li>Siedentopf, H., Uber Zsigmondy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sichtbarmachung+und+Gr&#246;&#223;enbestimmung+ultramikoskopischer+Teilchen,+mit+besonderer+Anwendung+auf+Goldrubingl&#228;ser.">Sichtbarmachung und Gr&#246;&#223;enbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingl&#228;ser.</a> Annalen der Physik. 315 (1), 1-39 (1902).</li><li>Voie, A. H., Burns, D. H., Spelman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthogonal-plane+fluorescence+optical+sectioning:+three-dimensional+imaging+of+macroscopic+biological+specimens.">Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens.</a> J. Microsc. 170 (Pt 3), 229-236 (1993).</li><li>Epp, J. 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The authors have nothing to disclose.

In modern life science, the microscopic visualization of biological processes plays an increasingly important role. In this context, many developments have been achieved during the last two centuries that help to answer questions not addressable up to this point, . First, there has been a clear tendency to fundamentally increase the resolution ability of light microscopes. With structured illumination microscopy (SIM)21,22, stimulated emission-depletion (STED) mi...

During the evolution of light microscopy, many specialized forms appeared, all of them developed to minimize limitations in the visualization process for particular specimens. One such method is light-sheet fluorescence microscopy (LSFM). First developed in 1903 by Siedentopf and Zsigmondy1 and finding its first fundamental biological applications in the early 1990s2, LSFM has become the most powerful microscopic tool to date for the visualization of large specimens, such as intact mouse organs, with a fluorescence signal resolution down to the cellular level. Because of these benefits, in combination with its potential for live-cell imaging, Nature Methods named LSFM the &#34;Method of the Year 20143.&#34;
As the name suggests, the sample illumination in an LSFM is conducted by light sheets, which are orientated perpendicularly to the axis of the objective used for emission-light collection and subsequent image formation. The light sheet is usually generated either by focusing wide, collimated laser beams with a cylindrical lens or by the rapid sideways movement of narrow, focused laser beams in just one horizontal or vertical plane4,5. In this way, only the focal plane of the imaging optics is illuminated, typically with a thickness of 1-4 &#181;m. Consequently, for a fluorescent sample placed in the illumination plane, both the generation of scattered light and the effects of photobleaching from regions above or below the focal plane are eliminated or greatly suppressed6,7. As all out-of-focus planes are not illuminated, photobleaching effects are omitted in these areas. In contrast to standard confocal or multi-photon microscopy, the paths of illuminated and emitted light are separate from each other, so the final image quality does not depend on the perfect focusing of the excitation light beam through the objectives. Depending on the underlying question, it is therefore possible to use objectives with an enormous field of view (FOV) so that the largest possible area of the illuminated plane can be imaged without any component parts moving in the xy-direction.
In modern LSFM systems, a fluorescent image of the generated optical section is captured on a highly sensitive charge-coupled device (CCD) or complementary metal-oxide semiconductor (CMOS) cameras, which are able to acquire the entire field of view (FOV) in microseconds. Therefore, by moving the sample through the light sheet and by acquiring images at defined z-steps, it is possible to obtain the full 3D information of a specimen in a reasonable amount of time8,9, making this technique applicable for live-cell studies10,11,12.
Nevertheless, although LSFM offers a rapid, sensitive, and fluorescence-friendly method, light transmission through the specimen is still a major issue, especially when large biological samples are the target for a 3D analysis. Light transmission is critically modified by physical aspects of absorption and by light scattering at interfaces of structures with different refractive indices13. Therefore, when imaging samples several millimeters in size, LSFM is mostly combined with clearing protocols to render the samples optically transparent. These techniques are based on the idea of removing water from the respective biological tissue and exchanging it with water- or (organic) solvent-based immersion media, which are chosen to narrowly match the refractive indices of the particular target tissue components. As a result, lateral light scattering is minimized, and all wavelengths of light can much more efficiently pass through the tissue13. In many cases, biological specimens treated in this way appear macroscopically crystal-clear, which enables LSFM to be conducted, even on entire mouse organs, using long working-distance, low magnification objectives.
Here, we present a preparation protocol for large-sample imaging in a light-sheet microscope (see the table of materials), which we have established to investigate the cellular cardiac infiltrate in a murine model of myocarditis15. CD11c.DTR mice express the primate diphtheria toxin receptor (DTR) under the control of the CD11c promotor16. Consequently, cells in these mice, which express CD11c along with the DTR, are rendered sensitive to the exotoxin of Corynebacterium diphtheria (diphtheria toxin, DTX); the systemic treatment of these animals with DTX results in a depletion of all CD11c+ cells. CD11c is an integrin and, as a cell-surface receptor for a variety of different soluble factors, is involved in activation and maturation processes mainly in cells of the monocytic lineage17. Consequently, the CD11c.DTR mouse model has been intensively used to study the role of dendritic cells and macrophage subsets in the context of many different immunological questions. Over time, it has been reported that CD11c.DTR mice treated systemically with DTX can develop adverse side effects and can display strongly elevated mortality rates18,19. Recently, we were able to identify the underlying cause of death15, describing the development of fulminant myocarditis after intratracheal DTX application in these animals. The toxin challenge caused cellular destruction, including in central parts of the stimulus transmission system in the heart. This was accompanied by massive CD45+ leukocyte infiltrate, finally leading to lethal cardiac arrhythmias. In this case, not only was the appearance of the leukocyte population important, but also its spatial distribution inside the heart. This experimental question is a challenge for modern microscopic imaging, which we have solved by a light-sheet microscopy approach that is supported by intravital antibody staining and an organic solvent-based optical clearing protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>diphtheria toxin</td>
			<td>Sigma Aldrich</td>
			<td>D0564</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Biochrom</td>
			<td>L182-10</td>
			<td></td>
		</tr>
		<tr>
			<td>ketamine [50 mg/ml]</td>
			<td>Inresa</td>
			<td>PZN 4089014</td>
			<td></td>
		</tr>
		<tr>
			<td>xylazine [2 %]</td>
			<td>Ceva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>syringe</td>
			<td>Braun</td>
			<td>REF 9161502</td>
			<td>Braun Omincan F&#160;</td>
		</tr>
		<tr>
			<td>indwelt venous catheter&#160;</td>
			<td>Becton Dickinson</td>
			<td>REF 381923</td>
			<td>BD Insyte Autoguard</td>
		</tr>
		<tr>
			<td>small animal respirator</td>
			<td>Harvard Apparatus</td>
			<td>73-0044</td>
			<td>MiniVent</td>
		</tr>
		<tr>
			<td>anit-CD45 antibody (AF647)</td>
			<td>BioLegend</td>
			<td>103124</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate</td>
			<td>Carl Roth</td>
			<td>8043.2</td>
			<td></td>
		</tr>
		<tr>
			<td>catheter (21 G)</td>
			<td>BD Biosciences</td>
			<td>REF 387455</td>
			<td>BD valu-set</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PE tube (5 ml)</td>
			<td>Carl Roth</td>
			<td>EKY9.1</td>
			<td>Rotilabo</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Carl Roth</td>
			<td>9065.4</td>
			<td></td>
		</tr>
		<tr>
			<td>dibenzyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>108014</td>
			<td></td>
		</tr>
		<tr>
			<td>tube rotator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>MACSmix</td>
		</tr>
		<tr>
			<td>agarose (low gelling)</td>
			<td>Sigma Aldrich</td>
			<td>A4018</td>
			<td></td>
		</tr>
		<tr>
			<td>mold (15x15x5 mm)</td>
			<td>Tissue Tek</td>
			<td>4566</td>
			<td>Cryomold&#160;</td>
		</tr>
		<tr>
			<td>light sheet microscope system</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td>Ultramicroscope&#160;</td>
		</tr>
		<tr>
			<td>microscope body</td>
			<td>Olympus</td>
			<td></td>
			<td>MVX10&#160;</td>
		</tr>
		<tr>
			<td>objective</td>
			<td>Olympus</td>
			<td></td>
			<td>0.5&#160;NA MVPLAPO 2XC, WD 5.5&#160;mm&#160;</td>
		</tr>
		<tr>
			<td>sCMOS camera 5.5MP</td>
			<td>Andor Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>488 nm OPSL (50mW) laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>647 nm&#160; diode laser (50mW)</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3D image processing &#38; analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>IMARIS Ver. 8.3</td>
		</tr>
		<tr>
			<td>transgenic mouse strain</td>
			<td>Steffen Jung et al.</td>
			<td></td>
			<td>CD11c.DTR</td>
		</tr>
		<tr>
			<td>wt mouse strain</td>
			<td>Envigo</td>
			<td></td>
			<td>Balb/c Ola Hsd J</td>
		</tr>
		<tr>
			<td>Laser Module</td>
			<td>LaVision Biotec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MVPLAPO 2XC Objective Lens</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative visualization of leukocyte infiltrate in a murine model of fulminant myocarditis by light sheet microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

The presented LSFM approach analyzes the leukocyte distribution and amount in murine hearts upon induction of severe myocarditis. Figure 1A explains the pre-treatment protocol for the transgenic CD11c.DTR mice for myocarditis induction. This step represents the necessary trigger for the recruitment of leukocytes to the myocardium. After successful DTX application, the animals develop severe disease symptoms, such as general weakness, anorexia, and weight loss within the r...

Watch this Scientific Journal Video about Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy at JoVE.com

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The overall goal of this protocol is to chemically clear the heart of a mouse, with an induced cardiac arrhythmia for a whole organ visualization of the leukocytic cardiac infiltrate by Fluorescence Light Sheet Microscopy. This method can help answer key questions in the field of whole organ research about prosthesis and structures at the cellular resolution level. The main advantage of this technique is that it allows both the quantification and the localisation of inflammation in whole organs.Giving detailed insides into neurological prosthesis. We first had the idea for this method when we were in urgent need of a visualization tool to identify cellular explanations for cardiac arrhythmia in the depletion module. After exposition of the heart from an eight to 10 week old mouse, use a 21 gauge catheter and penetrate the right ventricle.Incise the aorta to allow blood drainage and then perfuse with five milliliters of PBS plus EDTA, with a slow constant pressure. Follow the PBS EDTA with five milliliters of 4%paraformaldehyde. At the end of the perfusion, use serrated forceps to gently grasp the fixed heart, and pull the organ slightly upwards to cut all of the tissue connections.Including the incoming and outgoing arteries and veins. Then, store the heart for four hours in fresh 4%PFA for further fixation. To dehydrate the tissue, incubate the heart in an ascending ethanol series, in black five milliliter polypropylene reaction tubes in the dark, with constant slow rotation on a tube rotator, to prevent air bubble formation.Then, clear the tissue with a 98%dibenzyl ether incubation with constant rotation overnight. To conduct Light Sheet Fluorescence Microscopy, first place dehydrated agarose blocks into the standard sample holder, to hold the sample in place. Then, transfer the sample onto the sample holder.Next, transfer the sample holder into an appropriately sized dibenzyl ether filled cuvette. Use the appropriate excitation and emission filter settings, to detect the fluorescence signals of interest. So correct positioning and fixation of the sample is fundamental to minimize shadowing effects during imaging.Further, a loose specimen can cause moving artifacts, which are hard to illuminate during post-processing. After requiring all of the images, open the appropriate 3D and 4D image processing analysis software, and select surpass. Choose file and open, and select the folder containing the data from the first acquired channel to open the image stacks.Select edit and add channels, to add the appropriate acquired fluorescent channel data. Next, click edit and select image properties, to correct the x, y and z voxel dimensions. Adjusting the parameters in the newly opened window.To adjust the fluorescent signals, first open the edit menu and select, show display adjustment. A new window very display all of the open channels. To change the autofluorescence channel to greyscale, select the autofluorescence channel, choose the white display style and click okay.Then, adjust the view by removing the grid. Then, zoom in and deselect one channel. Then, adjust the black level value to exclude any unwanted background signals that do not represent the sample structures, the signal intensity and the contrast.Adjust the antibody standing channel in relation to the revised autofluorescence channel signal. Setting the channel mode, fire to set the visualization of the antibody signal to a heat map color lookup table. Then, select the blend mode, to visualize the tissue structures in detail.To virtually cut open the organ, before the 3D scene export, select the clipping plane icon in the object list. A yellow frame and a white spindle manipulator will appear in the image view. Use the mouse to rotate the spindle at the thinner end, to change the orientation of the clipping plane, and move the thicker middle part of the spindle to select the desired depth of clipping.Then, uncheck the respective check boxes to hide the frame and manipulator and create the desired snapshots. The acquisition of an entire image stack, composed of two fluorescent channel signals, elicits an artificial 3D rendering, in which the leukocyte distribution is displayed in a heat map view. The strongly inflamed areas of hearts from a Diphtheria toxin treated animal can be perceived by their reddish, whitish appearances.Particularly in the regions of the cardiac conduction system, such as the atrial ventricular bundle and the Purkinje fibers. In contrast, the hearts of controlled PBS treated animals, do not demonstrate this pattern of inflammation. Once mastered, this technique allows the digital ready analysis of a target organ, from its harvest to its computer assisted rendification in four to five days.After its development, this technique paved the way for researchers in the field of emmenology, to explore cellular distribution patterns in whole mouse organs. This may age in the analysis and the treatment of a variety of pathophysiological disease prosthesis. After watching this video, you should have a good understanding of how to prepare whole mouse organs for Fluorescence Light Sheet Microscopy and how to generate and process 3D data stacks.

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Research

JoVE Journal

Medicine

7.6K visualizações.  University of Duisburg/Essen. The overall goal of this protocol is to chemically clear the heart of a mouse, with an induced cardiac arrhythmia for a whole organ visualization of the leukocytic cardiac infiltrate by Fluorescence Light Sheet Microscopy. This method can help answer key questions in the field of whole organ research about prosthesis and structures at the cellular resolution level. The main advantage of this technique is that it allows both the quantification and the localisation of inflammation in whole orga...