This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

This study is in accordance with the National University of Singapore Institutional Animal Care and Use Committee (approved protocol no. R15-0225).
1. Surgical Preparation of the Animal Model
<ol>
	<li>Vessel cannulations
	<ol>
		<li>Anesthetize a male Sprague-Dawley rats (6 - 7 weeks old) weighing (203 &#177; 20) g with ketamine (37.5 mg/ml) and xylazine (5 mg/ml ) cocktail through intraperitoneal (i.p.) injection (2 ml/kg). Do not recap the needle or remove it from the syringe after the injection.</li>
		<li>Once the animal has been anesthetized (confirmed by toe pinching), place it on a heating pad to maintain its body temperature at 37 &#176;C. Gently shave the hair on the scapulae, anterior cervix, lower abdomen, medial hind leg and scrotal sac. Gently restrain the legs using adhesive paper tapes.</li>
		<li>Perform all surgical procedures using microdissection scissors and angled forceps while viewing through a stereomicroscope. Place all sharp surgical tools on a puncture-resistant tray to prevent injury during the surgery.</li>
		<li>Scrub all surgical sites 3 times with alternating iodine and 70% alcohol before performing the incision. Flush all catheters with 30 IU/ml heparin-saline solution.</li>
		<li>Make a 1 - 1.5 cm midline skin incision on the scapulae using a pair of surgical scissors over the right jugular vein. Separate the fascia by blunt dissection to expose the jugular vein and cannulate it with a polyethylene tube (PE-50) filled with heparin-saline using 5-0 silk sutures. Infuse supplemental anesthesia when necessary (1/3 to 1/2 of initial dosage, intravenous (i.v.)) throughout the course of surgery and experiment.</li>
		<li>Perform tracheostomy to maintain airway patency. Make a 1 - 1.5 cm incision in the anterior cervical area. Cannulate the trachea using a polyethylene tube (PE-205) with 2-0 silk sutures to secure the catheter in place.</li>
		<li>Monitor blood pressure through cannulation at the femoral artery. Make a 1 - 1.5 cm incision at the left medial surface of the hind leg. Separate the femoral artery by blunt dissection. Cannulate the femoral artery with a polyethylene tube (PE-10) filled with heparin-saline using 5-0 silk sutures.</li>
	</ol>
	</li>
	<li>Cremaster Muscle Preparation and Flow Visualization
	<ol>
		<li>Insert a 5-0 silk suture through the apex of the scrotal sac to extend it. Make an incision along the ventral surface of the scrotal sac. Regularly apply warm isotonic solution (37 &#176;C; pH 7.4) to the exposed muscle.</li>
		<li>Remove surrounding connective tissues carefully and thoroughly using a cotton-tipped applicator.</li>
		<li>Insert a 5-0 silk suture through the apex of the cremaster muscle. Cut the suture into two pieces of equal length and tie a knot on each side. Cut the muscle between the two knots and stretch it onto a customized transparent Plexiglas platform by gently pulling the suture. Fix the end of the suture onto the platform with blue tack. 
		NOTE: Thorough removal of surrounding connective tissues is crucial in obtaining optimum image contrast.</li>
		<li>Repeat step 1.2.3 until 5 to 6 fixations are made. Carefully remove the cremaster muscle from the epididymis using high temperature cautery. Superfuse warm isotonic solution to the exposed muscle to prevent dehydration of the tissue.
		<ol>
			<li>Surround the cremaster muscle with folded pieces of gauze. Cover the exposed muscle with a polyvinyl film. The gauze pieces with the film form a shallow basin to hold warm isotonic solution for the water-immersion microscope objective (Figure 1A).</li>
		</ol>
		</li>
		<li>Transfer the animal onto the animal stage of an intravital microscope (Figure 1C). Connect the arterial cannulation to a physiological data-acquisition system for continuous pressure monitoring (Figure 1E).</li>
		<li>Maintain the muscle temperature at 35 &#176;C with a heating element attached beneath the animal platform (Figure 1B). Place a temperature probe beside the muscle to provide negative feedback to the power controller of the heating element (Figure 1D).</li>
		<li>Leave the animal on the stage for 15 min to equilibrate with the environment.</li>
		<li>Visualize blood flow under an intravital microscope with a 40X water-immersion objective and a long working condenser.</li>
		<li>Choose an unbranched arteriole (&#60; 60 &#181;m) based on a clear image focus and contrast between the RBC core, CFL and vessel walls, in order to focus the microscope on the diametric plane of the blood vessel. Rotate the camera mounted onto the microscope to align the vessel wall vertically.</li>
		<li>Record the blood flow using a high-speed video camera at a frame rate of 3,000/sec for 1 sec. Save the recorded video as uncompressed 8-bit grayscale AVI format to preserve the image quality. 
		NOTE: A minimum recording frame rate of 3,000 frames/sec is recommended to ensure that the CFL measurement can be performed at least once per RBC under physiological arteriolar flow conditions.</li>
		<li>Use a blue filter with peak transmission at a wavelength of 394 nm and spectral bandpass at 310 - 510 nm to enhance the contrast between the RBCs and plasma. 
		NOTE: Ensure that the light spectrum passing through the blue filter from the microscopic light source (100 W halogen lamp) is of low light intensity to prevent any potential tissue damage.</li>
		<li>At the end of the experiment, euthanize the animal with an overdose of pentobarbital sodium.</li>
	</ol>
	</li>
</ol>
2. Image Analysis
<ol>
	<li>Preprocessing for the CFL width measurement
	<ol>
		<li>Open MATLAB and run the &#39;CFL_pre.m&#39; file. (This and other MATLAB files can be found in the <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/CFL_MATLAB_script.zip">Supplemental MATLAB Archive</a>.)</li>
		<li>Click &#39;Open file&#39; to select the video file to analyze.</li>
		<li>Adjust the &#39;Rotation&#39; slider to align the vessel walls vertically. 
		NOTE: Users can display the assisting grid lines for the vessel alignment by selecting the &#39;Grid On&#39; radio button, and adjust the zoom level of the image by sliding the &#39;Zoom&#39; slider.</li>
		<li>Click the &#39;Confirm Editing&#39; button to confirm the vessel alignment.</li>
		<li>Click the &#39;Set ROI to crop&#39; button to define the region of interest (ROI). The aligned image will be displayed in a pop-up window. Adjust the rectangular objective on the image, and double click to confirm the ROI. Skip this step if the cropping of image is not required. 
		NOTE: Only include a single vessel in the ROI to analyze the CFL width from the vessel. Click the &#39;Reset Image&#39; button to restore the image to its original form, if necessary.</li>
		<li>Click the &#39;Extract Images&#39; button to extract all edited video frames into consecutive bit map images (8-bit grayscale &#39;bmp&#39; format). The extracted images can be found in the folder with the same name as the selected video file.</li>
	</ol>
	</li>
	<li>Measurement of CFL width
	<ol>
		<li>Open MATLAB and run the &#39;CFL_measure.m&#39; file.</li>
		<li>Click &#39;Select Folder&#39; to select the folder containing the extracted images.</li>
		<li>Click on the folder containing the images and click &#39;Select Folder&#39;. The first image frame in the folder will be loaded and shown in the &#39;Grayscale image&#39; panel, along with its gray intensity histogram in the &#39;Image Histogram&#39; panel.</li>
		<li>Select the desired image frame from the list box to perform the analysis, otherwise the first image frame will be selected.</li>
		<li>Click &#39;Find Vessel Walls&#39; to identify the inner vessel wall in the image, which is determined at the location where the light intensity profile peak transits from dark to light over two pixels.</li>
		<li>Check &#39;Median Filter&#39; to apply a median filter to the image to reduce the &#39;salt and pepper&#39; noise.</li>
		<li>Check &#39;Auto Contrast&#39; to adjust the image intensities digitally to enhance the image contrast.</li>
		<li>Select a thresholding algorithm in the list box which automatically determines a thresholding value (&#964;) that divides the gray levels into two classes &#8212; white pixels with gray levels above &#964; (CFL), and black pixels with gray levels below &#964; (RBC core). 
		NOTE: As an alternative method, use manual thresholding if none of automated-thresholding algorithm provides an appropriate image thresholding. Click on the &#39;Manual&#39; radio button and adjust the slider to define the manual thresholding value.</li>
		<li>To measure the spatial variation of the CFL widths, enter the pixel resolution in the &#39;Pixel Resolution&#39; box (the resolution with this experimental setup was 0.42 &#181;m/pixel).</li>
		<li>Click the &#39;Calculate&#39; button to obtain the spatial variation of the CFL widths. Click &#39;Export .csv&#39; to export the CFL width data in a tabulated format.</li>
		<li>To measure the temporal variation of the CFL widths at a specific analysis line along the vessel, click on the &#39;Temporal Variation&#39; radio button and enter the frame rate information (the frame rate used in this experimental setup was 3,000 frames/sec).</li>
		<li>Enter the first frame and last frame of the images for the analysis in the &#39;Start Frame&#39; and &#39;Last Frame&#39; boxes, respectively.</li>
		<li>Select the position of the analysis line along the vessel by sliding the &#39;Analysis Line&#39; slide bar. Confirm the position of the analysis line, which is illustrated on both the &#39;Grayscale image&#39; and &#39;Binary image&#39;.</li>
		<li>Click &#39;Calculate&#39; to obtain the temporal variation of the CFL widths. Click &#39;Export .csv&#39; to export the CFL width data in a tabulated format.</li>
	</ol>
	</li>
</ol>

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W., Calvard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+Thresholding+Using+an+Iterative+Selection+Method.">Picture Thresholding Using an Iterative Selection Method.</a> IEEE Trans. Syst., Man, Cybern. 8 (8), 630-632 (1978).</li><li>Shanbhag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Information+Measure+as+a+Means+of+Image+Thresholding.">Utilization of Information Measure as a Means of Image Thresholding.</a> Cvgip-Graph Model Im. 56 (5), 414-419 (1994).</li><li>Bishop, J. J., Popel, A. S., Intaglietta, M., Johnson, P. 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The authors declare no competing financial interests.

The measurement of CFL width is essential for a better understanding of the hemodynamics in the microcirculation. In particular, the measurement of CFL widths has been performed in mesenteric6, spinotrapezius24 and cerebral25 microcirculations. Conventional measurement of in vivo CFL widths was restricted to estimations by manual inspection of the recorded video frames. The manual measurements required the averaging of several successive video frames before visually identifying the boundaries of the RBC core and vessel walls15,16. In another study, fluorescein isothiocyanate (FITC)-labelled RBCs and rhodamine-B isothiocynate (RITC) labelled plasma were used to determine the mean CFL widths in cat cerebral microvessels25. These previous measurement methods are very time consuming and require additional steps for the fluorescent labelling, which limits the spatial and temporal resolution of the CFL width measurement. In contrast, by coupling high-speed camera recordings to an effective image segmentation and analysis, the technique demonstrated here permits the quantification of spatiotemporal variations of the CFL with a spatial resolution (0.42 &#181;m) of an order smaller than the size of a RBC and a temporal resolution of 1/3,000 sec.
Proper surgical preparation of the cremaster muscle is critical in determining the accuracy of the CFL width measurements. In particular, thorough removal of adjacent connective tissues is essential to ensure a good focus of the arterioles in the cremaster muscle. In addition, the temporal and spatial resolution of the measurement is dependent on the microscope and camera specifications. While a higher magnification objective may enhance the spatial resolution, it reduces the field of view, which in turn limits the obtainable vessel length for quantifying the spatial variation of the CFL width. Therefore, the microscopic configurations can be modified according to the specific application of the technique.
Image segmentation is another important factor for the accuracy of the CFL width measurement. Amongst the various techniques developed, image thresholding based on gray level histogram provides a simple and effective approach for image segmentation and analysis. Accordingly, foreground objects are extracted from the background based on the difference in their gray levels. In the ideal case, the image histogram will be bimodal and a threshold value at the bottom of the valley is trivial. However, in vivo experimental images do not always exhibit such grayscale level profiles. Our results have shown how the image quality and contrast can influence the image segmentation process. The use of an optical blue filter significantly enhanced the contrast between the RBCs and the plasma in an arteriole (Figure 3), and it seems to be essential when applying the histogram-based thresholding for the CFL width measurement regardless of the algorithms (Figure 4). This results in a distinct bimodal image histogram, which allows one to identify the threshold value effectively. However, it should be noted that even with a bimodal histogram obtained from the in vivo images, an extremely unequal variance of two peaks (local maxima) and a wide valley (local minimum) of the histogram can still influence the threshold selection (Table 1). Therefore, the selection of an appropriate thresholding algorithm needs to be examined based on the image quality and users have to consider the limitations of each thresholding algorithm for the best suitability in quantifying CFL widths.
As the CFL widths are largely dependent on the flow conditions, continuous arterial pressure measurement throughout the course of the experiment is essential. In order to determine the local flow conditions, the pseudoshear rate of the blood flow can be computed by measuring the mean flow velocity in the blood vessel5.
In summary, the protocols for the surgical preparation of a rat cremaster muscle and quantitative image analysis described here have been utilized to acquire quantitative information on the dynamic variation of the CFL widths in vivo. The primary challenges in ensuring the accuracy of the CFL width measurements include proper surgical preparation of the muscle and image segmentation, both of which have been addressed above. This technique can be readily adapted to other microcirculatory studies to investigate the hemorheological and hemodynamic aberrations in various physiological and pathological conditions. Consequently, these findings contribute to the future development of microvascular therapeutic approaches and clinical intervention.

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Intravital microscope</td>
			<td style="width:167px;">Olympus</td>
			<td style="width:119px;">BX51WI</td>
			<td style="width:253px;">Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">High speed camera</td>
			<td style="width:167px;">Photron</td>
			<td style="width:119px;">1024PCI</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blue filter</td>
			<td style="width:167px;">HOYA</td>
			<td style="width:119px;">B390</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pressure sensor &#38;biopac system</td>
			<td style="width:167px;">Biopac system</td>
			<td style="width:119px;">TSD104A, MP100</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Temperature controller</td>
			<td style="width:167px;">Shimaden</td>
			<td style="width:119px;">SR 1</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Plasma Lyte A</td>
			<td style="width:167px;">Baxter</td>
			<td style="width:119px;">NDC:0338-0221</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Saline 0.9%</td>
			<td style="width:167px;">Braun</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Heparin (5000 IU/ml)</td>
			<td style="width:167px;">LEO</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-10 polyethylene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427400</td>
			<td>.024&#34; OD X .011&#34; ID&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-50 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427411</td>
			<td>.038&#34; OD X .023&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">113-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">106-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water circulating heating pad</td>
			<td style="width:167px;">Gaymar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">Isotemp 205</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Cotton-tipped applicators</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14225</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel clip</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14120</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
		</tr>
	</tbody>
</table>

visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

The visualization of the CFL in vivo is largely dependent on the surgical preparations of the animal. Excessive blood loss or extended surgery duration may subject the animal to shock and blood flow aberrations. Maintenance of tissue temperature using a heating pad as well as a customized platform during the surgery and experiment is also crucial for maintaining the physiological conditions of the rat. By using a 100 W halogen lamp in the microscope system, no discernible tissue damage was observed even at the end of the experiment.
Figure 2A shows a typical RBC flow through an unbranched arteriole in the rat cremaster muscle, where the CFL can be observed between the RBC core and the inner vessel wall (Figure 2C). A good contrast between these components during the experiment is critical for ensuring the accuracy of CFL width measurements. The initial phase of the image analysis involves the detection of the inner vessel wall. By acquiring the light intensity profile along the analysis line perpendicular to the vessel, the location is approximated at the peak that transits from dark to light over two pixels (Figure 2B).
As RBCs and CFL possess different light transmittance, the difference in gray levels can be subdivided into two classes (binary image). However, the identification of an accurate threshold value between the two peaks in the image histogram may be restricted by poor image quality and contrast (Figure 3A). To improve the contrast between the RBCs and CFL, a blue filter can be used (Figure 3B). This is also evident in Figure 4, in which the boundaries of the RBC core can be more accurately identified with the use of a blue filter. Furthermore, the selection of thresholding algorithm20-23 can also influence the measurement of the CFL width (Figure 4). It is apparent in Figure 4A that different thresholding algorithms resulted in different RBC core boundary identified, which could in turn lead to erroneous CFL measurements. To better illustrate the influence of the thresholding algorithm on the CFL width measurement in Figure 4B, the spatial profiles for the CFL widths obtained using different thresholding algorithms are shown in Figure 5 and summarized in Table 1.
<img alt="Figure 1" src="/files/ftp_upload/54550/54550fig1.jpg" /> 
Figure 1: Intravital Microscopic System and Cremaster Muscle Preparation. A: Surgically exteriorized rat cremaster muscle. B: Customized platform with heating elements for placing the cremaster muscle and maintaining its temperature at 35 &#176;C. C: Microscopic system with customized animal stage and high-speed camera for the visualization of microcirculatory blood flows in the cremaster muscle. D: Negative feedback temperature controller and power supply. E: Physiological data-acquisition system for continuous pressure monitoring. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54550/54550fig2.jpg" /> 
Figure 2: Image Processing for Determination of Vessel Wall Position and CFL Width. A: Typical grayscale image of RBC flow in an arteriole (vessel diameter = 52 &#181;m). B: Light intensity profile along the analysis line (solid line in panel A). C: Representative result of CFL measurement along the vessel. The solid and dashed arrows indicate the inner vessel wall and outer edge of the RBC core, respectively. (LWB &#38; RWB: left and right vessel wall boundary, LCB &#38; RCB: left and right RBC core boundary) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54550/54550fig3.jpg" /> 
Figure 3: Image Contrast Enhancement with an Optical Blue Filter. Image histogram of the grayscale images obtained without (A) and with blue filter (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54550/54550fig4.jpg" /> 
Figure 4: RBC Core Width Determined using Five Different Thresholding Algorithms. Boundaries of RBC core and vessel wall superimposed on grayscale images in Figure 3. (Top row (A): without blue filter, bottom row (B): with blue filter) using (from left to right) the Otsu&#39;s method, minimum method, intermode method, iterative selection method (Isodata) and fuzzy entropic thresholding (Shanbhag). The solid and dashed lines indicate the inner vessel wall and outer edge of the RBC core, respectively. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54550/54550fig5.jpg" /> 
Figure 5: Spatial Variation of CFL Width. CFL widths corresponding to Figure 4B along the left (A) and right (B) vessel walls, respectively. (D: distance in vessel diameter) <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54550/54550fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54550/54550table1.jpg" /> 
Table 1: Threshold Values and CFL Width Data in Figure 5. * p &#60; 0.001: significant difference from Otsu&#39;s method. &#8224; p &#60; 0.001: significant difference from left. Statistical analyses were performed using two-tailed unpaired t-tests.

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Nanyang Technological University

Research

JoVE Journal

Biology

6.3K Views.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Video: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...