This study was funded by grants from the National Institutes of Health (NIH), Heart, Lung, and Blood Institute (NIHLB): K01-HL-121045, K08-HL-094703, 5T32HL007411-35, P20 HL101425, U01 HL100398. 
Imaging and tissue processing (after NaOH maceration) were performed through the use of the Vanderbilt University Medical Center (VUMC) Cell Imaging Shared Resource (CISR) (supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126). We are especially grateful to the VUMC CISR core directors (Dr. Sam Wells and Dr. W. Gray (Jay) Jerome) for valuable technical advice and also for providing core space and resources for the purposes of filming the technique highlighted in this paper. 
We would like to extend our deepest appreciation to Dr. Yan Ru Su and Ms. Kelsey Tomasek in the Cardiology Core Lab for Translational and Clinical Research at Vanderbilt University for providing technical expertise and for collecting human tissue samples used in this study.

Ethics Statement: The protocols for animal handling were approved by the Vanderbilt Institutional Animal Care and Use Committee (IACUC, protocols number M/10/117 (swine) and M/10/219 (mice) and conducted according to AAALAC-International standards. Usage of banked human cardiac tissues was approved by the Vanderbilt University Medical Center IRB (protocol number 100887).
1. Sample Collection and Storage
<ol>
	<li>Make fresh 4% glutaraldehyde in 0.1 M phosphate buffer (PB) solution. 
	CAUTION: Glutaraldehyde is toxic, wear gloves and work in a fume hood.
	<ol>
		<li>Make a 0.2 M stock solution of PB, pH 7.4 using 21.8 g Na2HPO4 and 6.4 g NaH2PO4. Quantum satis (Q.s.) to 1,000 ml dH2O.</li>
		<li>Add 400 &#181;l of 50% glutaraldehyde to 2.5 ml of PB stock solution and 2.1 ml dH2O.</li>
	</ol>
	</li>
	<li>Immerse a chunk (no larger than 2 cm2) of tissue into the 4% glutaraldehyde solution. Note: Smaller pieces can also be used but should be of sufficient size (no smaller than 5 mm2) to be easily visualized by eye in order to facilitate later steps of the protocol.</li>
	<li>Incubate at room temperature for 1 hr, then store indefinitely at 4 &#176;C.</li>
</ol>
2. Decellularization of Heart Tissue
<ol>
	<li>Make fresh 10% aqueous NaOH solution using 10 g NaOH pellets/100 ml dH2O. 
	CAUTION: NaOH solutions are corrosive and can cause alkali-burns, wear gloves.</li>
	<li>Rinse tissue in dH2O.</li>
	<li>Incubate in 10% NaOH solution at room temperature for 6 - 10 days (until tissue changes from reddish-brown to off-white or white).</li>
	<li>Rinse in dH2O until tissue becomes transparent.</li>
	<li>Immerse tissue in 1% tannic acid for 4 hr at room temperature. Use 1 ml 5% stock solution per 4 ml dH2O. 
	CAUTION: Tannic acid is a strong irritant, wear gloves.</li>
	<li>Rinse in dH2O overnight.</li>
</ol>
3. Osmication and Dehydration of Heart Tissue (in Fume Hood for Safety)
<ol>
	<li>Make a 0.2 M stock solution of sodium cacodylate buffer using 21.4 g sodium cacodylate, 10.0 g calcium chloride and 450 ml dH2O. Mix, then add hydrochloric acid as needed to adjust pH to 7.4. Q.s. to 500 ml with dH2O. 
	CAUTION: Sodium cacodylate and hydrochloric acid are toxic, wear gloves and work in a fume hood.</li>
	<li>Make 2% aqueous stock solution of osmium tetroxide in fume hood for safety by dissolving 1 g osmium tetroxide crystal in 50 ml dH2O. 
	CAUTION: Osmium tetroxide is a severe inhalation hazard; vapor fixation of mucus membranes or eyeballs is possible, hence handle only in the fume hood with gloves. A splash guard is recommended.</li>
	<li>Rinse tissue in 0.1 M sodium cacodylate buffer (mix stock solution 1:1 with dH2O) for 5 min on rotator (or gentle agitation).</li>
	<li>Repeat previous step twice, for a total of three buffer rinses.</li>
	<li>Immerse tissue in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (mix stock sodium cacodylate and stock osmium tetroxide 1:1) on rotator for 1 hr.</li>
	<li>Rinse tissue in 0.1 M sodium cacodylate buffer 3 times for 5 min each on rotator.</li>
	<li>Rinse tissue using increasing concentrations of ethanol (30%, 50%, 75%, 85%, 95%, and finally 100%) for 15 min each on rotator.</li>
</ol>
4. Cross Section Surface Preparation for SEM
<ol>
	<li>Transfer tissue in 100% ethanol to a shallow Petri dish also containing 100% ethanol.</li>
	<li>Hold two very sharp razor blades such that the flats of the sides are in contact with one another and the cutting edges cross to form two sides of an equilateral triangle above the specimen. To accomplish this, use the left hand to place one blade on the far right side of the sample and cut leftward. At the same time, use the right hand to place the second blade on the far left of the sample and slice rightward. Thus, the blades will be slid against each other from opposite directions to make a single smooth cut.</li>
	<li>Slide the flat blade sides against each other to make a very clean cut of the specimen with minimal distortion or tearing force, preferably exposing as large a surface area as possible without damaging the specimen.</li>
	<li>Repeat for each specimen to expose the cleanest possible cross section surfaces for examination in the SEM.</li>
</ol>
5. Critical Point Drying (CPD) of Heart Tissue
<ol>
	<li>Use a spatula or tweezers to transfer tissue to the sample holder of the critical point dryer (CPD), ensuring that tissue remains in 100% ethanol at all times. Ensure that the holder is immersed in ethanol and that the transfer is achieved with the tissue exposed to air for no more than a few seconds.</li>
	<li>Operate CPD per user&#39;s manual to complete 3 purge-and-fill cycles to replace ethanol with liquid carbon dioxide.</li>
	<li>Operate CPD per user&#39;s manual to achieve critical point drying of the samples and return them to atmospheric pressure with controlled venting of CO2.</li>
</ol>
6. Mounting of Heart Tissue Specimens for SEM
<ol>
	<li>Prepare an SEM sample stub for each specimen, by adhering a carbon adhesive tab to the top surface of the aluminum stub.</li>
	<li>With the aid of a stereomicroscope, carefully adhere the specimen to the adhesive tab with the cross section surface of interest facing up (away from the tab, visible), and as close as possible to parallel with the plane of the sample stub surface. Do not probe or touch the surface of interest.</li>
	<li>Break a wooden applicator stick to achieve a tapered brush, ideal for paint application. Apply silver or carbon paint at the base and sides of the specimen, to improve adherence to the stub.</li>
	<li>Extend a very thin line of silver or carbon paint up to an edge of the surface of interest, to provide a charge path from the surface of interest to ground.</li>
	<li>Apply 2 or 3 small dabs of silver or carbon paint around the perimeter of the carbon tab, to provide a conductive path from the carbon tab surface to the metal stub and thus to ground.</li>
	<li>Allow conductive paint to dry for two hrs.</li>
	<li>Operate sputter coater per user&#39;s manual to apply a relatively heavy coating (target range of 30 - 40 nm) of gold-palladium alloy or gold. Pump specimen chamber to approximately 0.1 mbar; set Timer to 40 sec. Open Set valve to the 8:00 position (moderate Argon gas flow). Press Start to initiate sputter coating at 30 mA. Purple glow discharge should be visible in the specimen chamber.</li>
</ol>
7. SEM Examination of Heart Tissue Specimens
<ol>
	<li>Perform scanning electron microscopy at relatively low accelerating voltage to minimize imaging problems associated with poor charge dissipation in the sample (charging). Suggested initial imaging condition are: 5 kV accelerating voltage, 10 mm working distance.</li>
	<li>With assistance of an experienced operator, employ increased working distance to extend depth of field in imaging conditions requiring focus of fibers in multiple focal planes, or fibers extending for considerable length in the z dimension.
	<ol>
		<li>For the microscope used here (see Table of Materials), in the user interface access the Navigation tab at upper right.</li>
		<li>Access the Coordinates tab from the Stage menu. To increase working distance, enter a larger value in millimeters for the Z coordinate then click on the Go To tab to move the sample stage to the entered working distance.</li>
	</ol>
	</li>
	<li>With assistance of an experienced operator, employ specimen tilt and rotation to position the surface of interest orthogonal to the electron beam. Additional tilt of 10 to 30 degrees from this position may improve observation and documentation of the matrix structure.
	<ol>
		<li>For the microscope used here (see Table of Materials), in the user interface, click and hold the right mouse button, then slide left or right to focus the specimen near the periphery of the prepared surface plane of interest, noting the focused working distance.</li>
		<li>Navigate with the Manual User Interface joystick to move near the opposing edge of the surface and repeat focus. If working distance is not approximately equal to the first position, tilt specimen to achieve approximate agreement at both locations using the Coordinates tab of the Stage menu (see 7.2) and enter a tilt value in the T coordinate.</li>
		<li>Rotate specimen ninety degrees (enter value into R Coordinate field) and repeat the process until all positions are focused at approximately the same working distance. 
		Note: Variable pressure SEM may be employed to improve charge dissipation if available. High vacuum is the standard operating mode for scanning electron microscopy.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Cross section surface preparation is the most critical step during the protocol. To preserve fine structure, dehydrated specimens must remain in 100% ethanol at all times until introduced to the critical point drying process. Therefore the slicing of the specimens to achieve surfaces for EM examination must be done while specimens are submersed in ethanol in a shallow dish. It is also critical that the exposed surface is not touched or probed during subsequent handling. No major modifications are anticipated for application of this technique to other tissue types for similar matrix observations, although the SEM portion of the protocol might require basic electron microscopy troubleshooting regardless of sample origin. Images collected from the fibrous samples are prone to artifacts introduced by poor charge dissipation (&#34;charging&#34;). Charging problems can usually be minimized by reducing accelerating voltage, increasing scan speed (also called dwell time) and reducing spot size of the electron beam. Integration of several scans collected quickly enough to avoid charge artifacts will produce an image of comparable signal to noise quality without the charge artifacts present in a slower, higher quality single scan image.
This technique is inherently qualitative and thus complements when considered alongside quantitative measurements (e.g., Masson&#39;s trichrome or picoserius red staining, measurement of hydroxyproline content, mass spectrometry and RNASeq) to ascertain how visualized structural differences might be related to various developmental or disease states. However, despite this limitation, the method is significant beyond cardiac fibrosis specifically, because the extracellular matrix is an essential component of almost every organ in the body. In the heart, the cardiac matrix provides critical mechanical support for continuous pumping characterized by complex stretching, twisting, and deformation, which confers optimal entry and efflux of oxygenated and deoxygenated blood 49 for the &#62;2.5 billion beats that occur over the average human lifespan. Given the extremely low regenerative capacity of cardiac tissue, a dynamic matrix that can be remodeled according to contextual needs makes logical sense. With only a slight stretch of the imagination, one might infer that there exist therapeutic targets for manipulating matrix remodeling to enhance the healing process, while limiting adverse fibrosis. Application of the demonstrated technique at a minimum showcases the sophistication and beauty of the cardiac matrix and in doing so further underscores its functional importance.
While the value of quantitative measures is a core principle for evaluation of practically all experimental studies, the technique highlighted here can be used to reveal qualitative ultrastructural variations that not only complement standard matrix measurements but might suggest alternate investigative paths to understand the fundamental biochemical alterations that underscore the qualitative changes. Anticipated future applications of this technique are its use in cardiac disease models and human tissues as a complementary tool for assessing matrix changes, as well as broadened usage to study other organs for which matrix changes are a component of the disease process.

Fibrosis disrupts the normal myocardial collagen network, which is critical for normal mechanistic functions of cardiomyocytes 1,2 as well as for inter-cellular communication, intracellular signaling, and cell survival 3. The development of fibrosis is a major determinant of cardiac function, and fibrotic remodeling of the cardiac interstitium arising from a variety of etiologies leads to increased left ventricular stiffness and diastolic dysfunction 4. Myocardial fibrosis may also lead to arrhythmias, and whether the progression of fibrotic remodeling is a general or local phenomenon, it is highly associated with a poor prognosis in patients with ischemic and non-ischemic cardiomyopathy 5. Likewise, the absence of myocardial fibrosis is a strong predictor of ventricular functional recovery and long-term survival 6. 
The hallmark of fibrosis is the deposition of excess collagen, which has the tensile strength of steel 7 and can adversely affect cardiomyocyte function on multiple levels. Mechanical forces resulting from an excessively collagenous matrix can lead to cardiomyocyte atrophy 8,9, passive tissue stiffness 10, tonic contraction-induced myocardial stiffness 11-13, and reduced delivery of oxygen to the remaining population of cardiomyocytes. Gap junction coupling of cardiomyocytes and myoFbs can also compromise the heart's electrical characteristics, creating a greater risk for the development of arrhythmias 14-16. Perivascular fibrosis alters vasomotor reactivity of intramural coronary arteries and arterioles 17 and contributes to luminal narrowing that reduces the supply of oxygen and thus the survival of cardiomyocytes 17-22. Pathogenic fibrotic and electrical remodeling, emanating from an initial site of ischemic injury or energy imbalance, inevitably progresses to heart failure. 
Cardiomyocyte necrosis initiates the fibrotic response, and subsequent adverse fibrotic remodeling can occur irrespective of etiology. Finding a way to control cardiac fibrosis would be clinically beneficial for the treatment of ischemic and idiopathic cardiomyopathies, hypertensive heart disease, hypertrophic cardiomyopathy, valvular heart disease and dystrophinopathies 23-42. Regardless of how the fibrotic disease process begins, soluble, profibrotic factors can cross the interstitial space and provoke activation of interstitial and adventitial fibroblasts at sites remote to the initial fibrotic scar, creating a cascade effect that ultimately leads to heart failure. The optimum scenario would be to exploit the fibrillogenic process using a targeted therapeutic that can be applied during the compensative hypertrophic stage of cardiomyopathy before it progresses to systolic pump failure, diastolic heart failure, or other end-stage outcomes. The ultimate goal would be to reverse fibrosis so that dead cardiomyocytes can be replaced and heart function restored completely. 
The importance of the matrix is widely understood, yet methods to study the matrix are limited mainly to quantitative measurements of major structural components, particularly collagen, and relative levels of different matrix and matricellular proteins. This protocol highlights a rarely used technique that is useful for assessing qualitative differences in the cardiac matrix. This technique has been recently used to compare and contrast fundamental differences in heart matrices from different etiologies of heart disease (in human explants), to examine hearts from post-infarcted pigs treated with the glial growth factor (GGF) isoform of neuregulin-1&#946;, relative to untreated animals 43, and to probe for differences in the matrices of cardiac tissues from mdx mice (a commonly used animal model of Duchenne Muscular Dystrophy) at different ages and compared to wild-type controls. This technique was first introduced by Drs. Caulfield and Borg in 1979 44, but few studies have since employed this powerful technique 45-47, re-introduced here with only slight modification. This methodology is a valuable research tool, because it provides qualitative information about extracellular matrix ultrastructure that might otherwise be overlooked when simply measuring matrix component content and/or level of fibrosis.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Calcium Chloride</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12340</td>
			<td style="width:167px;">100g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Carbon Adhesive</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12664</td>
			<td>30g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Carbon Adhesive Tabs</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">77825</td>
			<td>order to fit stubs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Double edge razor blades stainless steel</td>
			<td style="width:207px;">Ted Pella, Inc</td>
			<td style="width:119px;">121-6</td>
			<td>250/pkg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ethanol</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15055</td>
			<td>450mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gluteraldehyde, 50% solution</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">16310</td>
			<td>EM grade, distillation purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Hydrochloric Acid</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">16760 or 16770</td>
			<td>100mL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Monosodium phosphate NaH2PO4</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9251-250G</td>
			<td>250g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Osmium Tetroxide</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">19100</td>
			<td>1g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Silver Conductive Adhesive</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12686-15</td>
			<td>15g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium hydroxide (NaOH)</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8045-1KG</td>
			<td>1KG</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">S3264-500G</td>
			<td>500g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Tannic acid, 5% aqueous&#160;</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">21702-5</td>
			<td>500mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trihydrate Sodium Cacodylate</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">12300</td>
			<td>100g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Gold-palladium Alloy or Gold</td>
			<td style="width:207px;">Refining Systems, Inc.&#160;</td>
			<td style="width:119px;">varies</td>
			<td>&#160;specific to the sputter coater make and model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Critical Point Dryer</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plain Wooden Applicators</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Quanta 250 Environmental SEM</td>
			<td style="width:207px;">FEI</td>
			<td style="width:119px;">Q250 SEM</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sputter coater</td>
			<td style="width:207px;">Cressington Scientific Instruments Ltd.</td>
			<td style="width:119px;">Model 108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Alluminum SEM Sample Stubs</td>
			<td style="width:207px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">75220-12</td>
			<td>specific to the miscroscope</td>
		</tr>
	</tbody>
</table>

scanning electron microscopy of macerated tissue to visualize the extracellular matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

The highlighted technique was applied to cardiac tissues from an unused human heart transplant donor (Figure 1), explanted tissues from transplant recipients, hearts from wild-type and dystrophic mice (Figure 3), and in post-myocardial infarction heart samples from a swine model of heart injury (Figure 2). As shown in Figure 1, the human cardiac matrix is an intricate weave of cross-linked proteins that display a honeycomb-like pattern when viewed in cross section. Each &#39;honeycomb&#39; structure is approximately 40 &#181;m wide, normally circumventing a single myocyte, when considering the planar view in Figure 1. When connected by intercalated discs, several cardiomyocytes can be envisioned as a rod running lengthwise through the &#39;tunnel&#39; when the metaphor is extended to three-dimensionality. Figure 1 also highlights the importance of the cutting procedure, with greater precision yielding more revelatory topographical data (Figure 1, top left) than sections that are &#34;smashed&#34; during the cutting process (Figure 1, top right).
Removal of resident cardiac cells, blood vessels, and circulatory cells prior to SEM processing uncovers additional ultra-structural details that would be less appreciable by SEM of whole heart tissue blocks. Each individual collagenous &#34;strut&#34;, for example (Figure 1, bottom panels) is apparently aligned at regular intervals and perpendicular to myofibril sarcomeres. This arrangement is suitable for helping maintain heart structure by counter-acting deformations exerted during contraction and relaxation cycles, similar to the &#39;warp and weft&#39; of textiles that helps to maintain fabric body and form against stretching.
<img alt="Figure 1" src="/files/ftp_upload/54005/54005fig1.jpg" /> 
Figure 1: Representative Scanning Electron Micrographs of the Three-dimensional Arrangement of the Extracellular Matrix in Decellularized Left Ventricular Tissue Obtained from an Unused Human Donor Heart. The top two panels show the matrix in cross section at low magnification (bars = 500 &#181;m), providing an aerial view of the architecture of normal human heart tissue. At higher magnification, one can better observe the typical honeycomb-like structure of supportive fibers that provide mechanical support for regularly spaced myofibers (middle left and right panel bars = 100 &#181;m and 50 &#181;m, respectively). Upon closer inspection, each &#39;honeycomb&#39; is composed of fibers that are organized in parallel with one another while perpendicular to resident cardiomyocytes (bottom left and right panel bars = 10 &#181;m and 5 &#181;m, respectively). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54005/54005fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In addition to providing structural information, SEM of decellularized tissues can allow meaningful, qualitative assessment of extracellular matrix changes in response to injury or non-injurious forms of cardiomyopathy. For example, this technique was recently used to examine the extracellular matrices of post-infarcted swine cardiac tissues 43. During the course of this large-animal experiment, designed specifically to assess the efficacy of the GGF2 isoform of NRG-1&#946; as a potential therapeutic for heart failure, post-infarcted swine which received intravenous NRG-1&#946; administration exhibited striking alterations in the cardiac matrix, compared to untreated cardiac tissues of post-infarcted animals. These results were subsequently published 43, and the technique has remained a valuable tool for building upon this initial, serendipitous discovery. Figure 2 includes example micrographs, produced during the course of that study, which highlight drastic matrix differences between untreated and NRG-1&#946;-treated matrices.
<img alt="Figure 2" src="/files/ftp_upload/54005/54005fig2.jpg" /> 
Figure 2: Representative Scanning Electron Micrographs of Left Ventricular Extracellular Matrix in Untreated and NRG-1&#946;-treated Pigs, after NaOH Maceration. The matrix in cross section highlights the regular spatial arrangement of fibers in untreated post-myocardial infarction (post-MI) pigs (top left), compared to post-MI NRG-1&#946;-treated animals (top right). When viewed in longitude, the matrix in untreated swine exhibits a thick, matte-like appearance (bottom left), whereas the matrix of NRG-1&#946;-treated pigs displays regular spatial arrangement of fibers (bottom right). White bar = 40 &#181;m (all four panels). More detailed results and figures are included in the associated manuscript 43. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54005/54005fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Exploration of matrix changes that occur in dystrophic hearts has also yielded qualitative insights into the progression and development of an animal model of Duchenne muscular dystrophy cardiomyopathy (DMD). In mdx mice, a commonly used mouse model of DMD, there are significant and age-dependent differences among wild-type and mdx hearts when viewed by SEM after fixation and NaOH treatment. As shown in Figure 3, matrix components were relatively normal in 6 week-old mdx hearts lacking functional dystrophin relative to normal mice. More interesting, extracellular matrix organization was clearly disrupted or perhaps degraded in older dystrophin-deficient mice compared to younger mdx mice, illustrating the progressive nature of DMD in the heart. Such profound differences were not expected, because mdx mice are poorly representative of human DMD cardiomyopathy due to the fact that they exhibit a much milder cardiomyopathy phenotype and slower mortality rate than humans with DMD 48. This suggests that even small changes in heart function can be captured using the visualization technique presented in this manuscript. This methodology should also be readily adaptable to the extracellular matrices of other organs, for which there are likewise no currently available fibrosis-targeting therapies.
<img alt="Figure 3" src="/files/ftp_upload/54005/54005fig3.jpg" /> 
Figure 3: Representative Scanning Electron Micrographs of Left Ventricular Extracellular Matrix in Wild-type versus mdx Mice. The left ventricular cardiac matrix in wild-type mice (top panel) is similar to that observed in other species. The matrix in mdx mice at 6 weeks of age looks relatively normal, although slightly &#34;fluffy&#34; in appearance (middle panel). Conversely, the cardiac matrix of older mdx mice appears severely degenerated (bottom panel), indicating that the dystrophic process can be qualitatively captured using SEM in fixed, NaOH-macerated tissues. White bar = 10 &#181;m (all three panels). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54005/54005fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

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9.8K Views.  Vanderbilt University Medical Center. Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

Video: Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Studying the Supramolecular Organization of Photosynthetic Membranes within Freeze-fractured Leaf Tissues by Cryo-scanning Electron Microscopy

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we describe a technique for studying the supramolecular organization of photosynthetic (thylakoid) membranes within leaf samples. This is achieved by high-pressure freezing of leaf tissues, freeze-fracturing, double-layer coating and finally cryo-SEM imaging. Use of the double-layer coating method allows acquiring high magnification (&#62;100,000X) images with minimal beam damage to the frozen-hydrated samples as well as minimal charging effects. Using the described procedures we investigated the alterations in supramolecular distribution of photosystem and light-harvesting antenna protein complexes that take place during dehydration of the resurrection plant Craterostigma pumilum, in situ.

Here we describe a procedure for studying freeze-fractured plant tissues. High-pressure frozen leaf samples are freeze-fractured and double-layer coated, yielding well preserved frozen-hydrated samples that are imaged using the cryo-scanning electron microscope at high magnifications with minimal beam damage.

Cryo-scanning electron microscopy (SEM) of freeze-fractured samples allows investigation of biological structures at near native conditions. Here, we ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the imaging modality in a focused ion beam scanning electron microscope (FIB-SEM), which is used to obtain stacks of sequential images for 3D reconstruction and modelling. High-pressure freezing (HPF) allows close to native structural preservation, but the subsequent freeze substitution often does not provide sufficient contrast, especially for a bigger specimen, which is needed for high-quality imaging in the SEM required for 3D reconstruction. Therefore, in this protocol, after the freeze substitution, additional contrasting steps are carried out at room temperature. Although these steps are performed in a microwave, it is also possible to follow traditional bench processing, which requires longer incubation times.The subsequent embedding in minimal amounts of resin allows for faster and more precise targeting and preparation inside the FIB-SEM. This protocol is especially useful for samples that require preparation by high-pressure freezing for a reliable ultrastructural preservation but do not gain enough contrast during the freeze substitution for volume imaging using FIB-SEM. In combination with the minimal resin embedding, this protocol provides an efficient workflow for the acquisition of high-quality volume data.

Here we present a protocol for combining two sample processing techniques, high-pressure freezing and microwave-assisted sample processing, followed by minimal resin embedding for acquiring data with a focused ion beam scanning electron microscope (FIB-SEM). This is demonstrated using a mouse tibial nerve sample and Caenorhabditis elegans.

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Array Tomography Workflow for the Targeted Acquisition of Volume Information using Scanning Electron Microscopy

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission Electron Microscopy (TEM) provided insight into cell ultrastructure, but in the recent decade, the development of modern Scanning Electron Microscopes (SEM) has changed the way of looking inside the cells. Even though the resolution of TEM is superior when protein-level structural details are needed, SEM-resolution is sufficient for the majority of the organelle-level cell biology-related questions. The advancement in technology enabled automatic volume acquisition solutions such as Serial block-face imaging (SBF-SEM) and Focused ion beam SEM (FIB-SEM). Nevertheless, to this day, these methods remain inefficient when the identification and navigation to areas of interest are crucial. Without the means for precise localization of target areas before imaging, operators need to acquire much more data than they need (in SBF-SEM), or, even worse, prepare many grids and image them all (in TEM). We propose the strategy of "lateral screening" using Array Tomography in SEM, which facilitates the localization of areas of interest, followed by automated imaging of the relevant fraction of the total sample volume. Array tomography samples are conserved during imaging, and they can be arranged into section libraries ready for repeated imaging. Several examples are shown in which lateral screening enables us to analyze structural details that are incredibly challenging to access with any other method.

We describe the preparation of ribbons of serial sections and their collection on large transfer support for use as Array Tomography samples, along with automated imaging procedures in a scanning electron microscope. The protocol allows screening, retrieval, and targeted imaging of local, rare events, and the acquisition of large data volumes.

Electron microscopy is applied in biology and medicine for imaging of cellular and structural details at nanometer resolution. Historically, Transmission ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Cryo-Electron Microscopic Grid Preparation for Time-Resolved Studies using a Novel Robotic System, Spotiton

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental challenge of great interest to the field of cryo-electron microscopy (cryo-EM). A few methodological strategies have been developed that support these &#34;time-resolved&#34; studies, one of which, Spotiton-a novel robotic system-combines the dispensing of picoliter-sized sample droplets with precise temporal and spatial control. The time-resolved Spotiton workflow offers a uniquely efficient approach to interrogate early structural rearrangements from minimal sample volume. Fired from independently controlled piezoelectric dispensers, two samples land and rapidly mix on a nanowire EM grid as it plunges toward the cryogen. Potentially hundreds of grids can be prepared in rapid succession from only a few microliters of a sample. Here, a detailed step-by-step protocol of the operation of the Spotiton system is presented with a focus on troubleshooting specific problems that arise during grid preparation.

The protocol presented here describes the use of Spotiton, a novel robotic system, to deliver two samples of interest onto a self-wicking, nanowire grid that mix for a minimum of 90 ms prior to vitrification in liquid cryogen.

The capture of short-lived molecular states triggered by the early encounter of two or more interacting particles continues to be an experimental ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...