Name: a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy
Uploaded: 2015-10-27T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy at JoVE.com

This project was supported by Grant Numbers HL96593 from NIH and D56HP20783 from HRSA/ HHS. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH or HRSA / HHS.

Note: The acquisition process for obtaining capillary images has previously been published and is accomplished using a still digital camera with a corresponding image acquisition and analysis computer program. 1121 This lab utilizes still images for analysis, not videos, simplifying image acquisition for analysis. The following describes the new technique for quantitating the capillaries from the images.
1. Image Enhancement Process
<ol>
	<li>Obtain digital images with a monochrome digital camera. Calibrate the images to an object of known size by taking a picture of an object of known length such as a ruler with the camera. This process allows the computer program to measure and count capillaries accurately after processing. Ideally for the highest precision, a reticule (scientifically manufactured piece of glass with a ruler etched into it) should be used. Measure the number of pixels in a 1 mm square box using a computer program. 
	Note: The key to the reproducibility and standardization of this protocol depends largely upon proper placement of the 1 mm2 box that is counted.</li>
	<li>Use contrast enhancement tools to darken the capillaries and lighten the background, which will maximize visualization of the capillaries. Initial differentiation of capillaries from background is important for proper cropping of the image in later steps. Do this by adjusting the image using a &#8220;best fit histogram.&#8221; To do this click on: capture, intensity, image histogram, best fit.</li>
	<li>Crop a region of interest (ROI) for capillary counting as a new image (select, regions of interest). Use a 1 mm2 box, which was determined by calibrating the images at 530 pixels equaling 1 mm. check that the cropped image places the apex of the capillary loops at the very top of the image.</li>
	<li>Flatten the image so all future image adjustments will be evenly applied to the image. To do this click on: process tab, 2D filters, flatten, BG intensity of &#8220;bright,&#8221; object width to &#8220;75,&#8221; apply.</li>
	<li>Raise the contrast of the image so capillaries are maximally visualized. To do this click on: adjust tab, display, raise contrast to 75.</li>
	<li>Despeckle the image to smooth the edges of the capillaries by clicking on process tab, 2D filters, despeckle, kernel size 7 x 7, apply.</li>
	<li>Finalize the image contrast so capillaries are black and the background is white. Perform this step by adjusting the histogram to the &#8220;best fit&#8221; model. 
	Note: Please refer to Figure 2 in Representative Results for an example of what the processed image should look like following these steps.</li>
</ol>
2. Performing Capillary Counts / Quantitating Capillary Density
<ol>
	<li>On each image, manually select one part of a well-defined capillary using the &#8220;target object&#8221; feature to be recognized as objects to be counted by the program. Then, select a small part of background, using the &#8220;background&#8221; feature, as a reference to areas that need to be disregarded by the counting algorithm. 
	Note: The combination of these highlights causes all capillaries to be highlighted while disregarding the background noise. The quantitation (counting) protocol utilizes computer functions to differentiate parts of the image based on color and morphology.</li>
	<li>Use the count function to instantly count all capillaries in the image with the imaging equipment. Set the minimum diameter of counted objects to 5 pixels in order to avoid counting background noise as capillaries.</li>
	<li>For each individual, count the average of 3 -&#160;4 images in order to obtain a more reliable assessment. 
	Note: Please refer to Figure 3 in Representative Results for an example of what the image should look like during the counting procedure.</li>
</ol>
3. Creating and Using Macros to Automate Image Processing
Note: To save time, macros can be created in order to automatically perform a specific sequence of processes on one or many images. These sequences can be customized in order to make the image modifications quicker. In essence, these macros remember how the images are processed, and perform all the steps quickly and with no user input. Performing counts on 12 capillary images takes this lab between 20 to 30 min with the macros (2 to 3 min per picture), as opposed to about 8 min per picture without the macros. Therefore using the macros is 3 to 5 times more efficient than manually processing each individual image.
<ol>
	<li>In order to create a macro, select &#8220;record macro&#8221; and on one image perform the steps and processes desired, as described in steps 1 and 2 above. Name the macro based on which image processing steps were performed for future reference. When using the macro on future pictures, simply click &#8220;run macro&#8221; and the program will automatically apply the recorded enhancements to the image(s) desired. 
	Note: This lab uses a macro to perform all but one of the steps in Section 1 of the methods in a few seconds. The only step that requires user input is choosing where to crop the image, Step 1.2.</li>
</ol>

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+skin+capillary+recruitment+in+essential+hypertension+is+caused+by+both+functional+and+structural+capillary+rarefaction.">Impaired skin capillary recruitment in essential hypertension is caused by both functional and structural capillary rarefaction.</a> Hypertension. 38, 238-242 (2001).</li><li>Shore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillaroscopy+and+the+measurement+of+capillary+pressure.">Capillaroscopy and the measurement of capillary pressure.</a> Br J Clin Pharmacol. 50, 501-513 (2000).</li><li>Rieder, M. J., O'Drobinak, D. M., Greene, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computerized+method+for+determination+of+microvascular+density.">A computerized method for determination of microvascular density.</a> Microvasc Res. 49, 180-189 (1995).</li><li>Vermeulen, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+angiogenesis+in+solid+human+tumours:+an+international+consensus+on+the+methodology+and+criteria+of+evaluation.">Quantification of angiogenesis in solid human tumours: an international consensus on the methodology and criteria of evaluation.</a> Eur J Cancer. 32A, 2474-2484 (1996).</li><li>Cheng, C., Daskalakis, C., Falkner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+Assessment+of+Microvascular+and+Endothelial+Function.">Non-invasive Assessment of Microvascular and Endothelial Function.</a> Journal of Visualized Experiments. , (2012).</li><li>Antonios, T. 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The authors have no conflicts of interest.

Nailfold capillaroscopy shows promise as a clinically useful tool in the future for various oncology, cardiovascular, and rheumatologic disease applications. The image acquisition process is fairly consistent among researchers, yet there are currently multiple methods for image processing and analysis. Methods currently include computerized and manual capillary counts. Manual counts are problematic as they are time-consuming, and subject to user subjectivity and fatigue. Current computer based methods require a high level of user input, both in the image enhancement process and the quantification process. The new method described here requires relatively little user training or involvement, as the image enhancement steps are entirely automated. User input is only required for the counting process initially to distinguish capillary from background in the processed images. Using the automated macros as described here is three to five times more efficient than manually processing each individual image.
Reliable standardized computer based algorithms to quantitate capillaroscopy data are lacking.
Reliable standardized computer based methods are needed for capillary quantitation in order to reduce subjectivity and promote efficiency. The technique described here demonstrates excellent reliability with good results for both intra and inter-observer reproducibility with intraclass correlations of 0.93 to 0.98. We have previously reported the correlation of results obtained via computerized capillary density assessment, compared to the gold standard of manual counting.21 Pearson correlations between baseline, post-ischemic, and venous congestion counts done with the software and corresponding manual counts in 10 subjects were 0.78, 0.78, and 0.71 respectively (all p &#60;0.05), indicating reasonable agreement between the two methods.
This lab&#8217;s image manipulation steps utilize a number of computer processing tools. Step 1.3, image &#8220;flattening,&#8221; removes the various &#8220;layers&#8221; that are present in each image. This must be done first so all future image processing procedures will be applied evenly to all parts of the image. Contrast adjustment both darkens the capillaries and pales out the background, therefore making the capillaries more visible. The &#8220;despeckle&#8221; process smooths the edges of the capillaries while maintaining their size and shape. Although there appear to be no differences in a despeckled image to the naked eye, this is an important process to help ensure multiple capillaries do not blend together during the counting process. Finalizing the image using a &#8220;best fit histogram&#8221; excludes all pixels at either extreme of the histogram. This helps define the borders of the capillaries while further enhancing the contrast between the capillaries and the background. Overall there are three contrast enhancement steps, and all three are necessary to maximize the clarity of the final image for capillary counting.
Occasionally, the program will count too many or too few capillaries. The first step to fix this problem is to undo the highlighting and simply try the highlighting process again. If the capillaries are being highlighted incorrectly, adjusting the minimum capillary diameter may be necessary. The authors recommend a default minimum diameter of 5 pixels. If the program is counting too many capillaries or counting one capillary as multiple capillaries the user should increase the minimum diameter by one or two pixels. On the other hand, if the program is not counting dark pixel groups that are capillaries, the user can reduce the minimum diameter by one pixel.
There is also a need to standardize the position for these counts within the nailbed. As seen in the Table, counts in the same individual are highly position dependent, varying greatly based on which part of the nailbed is being counted.
Critical steps of the protocol include proper and optimal visualization of the capillaries. The steps allowing optimal visualization of the capillaries in this protocol are fully automated, allowing for rapid and accurate image manipulation. While these methods represent a major advancement in the reliability and ease of processing and counting capillaroscopic image, the major limitation of the technique is the semi-automated counting process. Ideally, a fully automated process will be created in the near future. Researchers should feel encouraged to build upon the methodology described in this paper in order to develop a fully automated clinically useful technology that allows rapid quantification of a patient&#8217;s nailfold capillary density.

Microvascular imaging is a rapidly growing field with many potential clinical applications.2 For example, oncologists are using microvessel imaging to determine the extent of tumor angiogenesis, yielding valuable information concerning the state of the tumor and insight into possible treatment options. 34 However, nailfold capillaroscopy is perhaps the most cost efficient and widely applicable form of microvascular imaging. Researchers are using video nailfold capillaroscopy to study blood flow rates and investigate capillary morphology.5 6 Both video and still-picture nailfold capillaroscopy are adjuncts to care for diagnosing and treating Raynaud&#8217;s phenomenon and various connective tissue diseases such as systemic sclerosis.2
Nailfold capillaroscopy has various potential cardiovascular applications as well. Current research using nailfold capillaroscopy suggests that diabetes mellitus Type 1 and Type 2 patients exhibit a high prevalence of abnormal capillary morphology, yet have unchanged capillary densities compared to non-diabetic individuals.7-8 Capillaroscopy has also been studied experimentally in hypertension. Structural capillary rarefaction leading to a reduced capillary density has been demonstrated in hypertensive individuals compared to non-hypertensive individuals.9-10 In contrast to these older hypertensive patients (mean age 40 and above) who exhibit structural rarefaction, more recent research has demonstrated that younger hypertensive patients (mean age under 40 years old) have functional rarefaction without structural rarefaction.11 This suggests that functional rarefaction occurs before and may progress over time to structural rarefaction.
Interestingly, hypertensive patients treated with specific antihypertensive drugs such as Perindopril/Indapamide displayed normal capillary density and endothelial function after treatment, while those treated with ACE (angiotensin-converting-enzyme) inhibitors or diuretics maintained a low capillary density despite comparable blood pressure control.12 This suggests that some antihypertensive medications may normalize capillary density by reversing the capillary rarefaction caused by hypertension. In addition, other researchers have shown that a reduction in salt intake leads to reversal of both functional and structural capillary rarefaction in hypertensive individuals.13
Despite the various potential clinical applications of this technology, there is little standardization in technique for quantitating capillary density images.2 To date, researchers have found that capillary density results are reproducible from both an intra-observer and inter-observer perspective only if the exact same area is being counted each time. 1,14 15 Of note, previous researchers have largely performed capillary counts manually using the naked eye, 9 16 17 18 which is a slow and subjective process.
Standardized, computer based algorithms for quantitation of capillary images theoretically provide more efficient and reproducible data analysis with less subjectivity, facilitating clinical applications of capillaroscopy. Some researchers have indeed used computer-based programs to quantitate the data from nailfold capillaroscopic pictures. 1,6 19 20 However, only one publication to date describes reliability of a complex software program available for quantitating capillaroscopy data,1 and this program is complicated as previously noted above by the requirement to count the exact same visual field. Here, we present a simpler, reliable protocol for quantitating capillaries using a standardized algorithm which allows for the use of multiple visual fields. The use of multiple visual fields not only simplifies the procedure, but also permits the assessment of normal biological variation in capillary count.
The aim of this study is to describe a reproducible and efficient computer based algorithm which standardizes the capillary quantitation process. While these methods are not fully automated they require very little user input, and provide rapid and reliable quantitation of the pictures.

<table><tbody><tr><td>Image-Pro Premier</td><td>Media Cybernetics, Inc</td><td>9.1</td><td>Image processing software</td></tr></tbody></table>

a reproducible computerized method for quantitation of capillary density using nailfold capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The capillaroscopy technique can provide insight into a patient&#8217;s microvascular health, leading to a variety of potentially valuable dermatologic, ophthalmologic, rheumatologic and cardiovascular clinical applications. In addition, tumor growth may be dependent on angiogenesis, which can be quantitated by measuring microvessel density within the tumor. However, there is currently little to no standardization of techniques, and only one publication to date reports the reliability of a currently available, complex computer based algorithms for quantitating capillaroscopy data.1 This paper describes a new, simpler, reliable, standardized capillary counting algorithm for quantitating nailfold capillaroscopy data. A simple, reproducible computerized capillaroscopy algorithm such as this would facilitate more widespread use of the technique among researchers and clinicians. Many researchers currently analyze capillaroscopy images by hand, promoting user fatigue and subjectivity of the results. This paper describes a novel, easy-to-use automated image processing algorithm in addition to a reproducible, semi-automated counting algorithm. This algorithm enables analysis of images in minutes while reducing subjectivity; only a minimal amount of training time (in our experience, less than 1 hr) is needed to learn the technique.

The goal of this image processing procedure is to differentiate the capillaries from the background image so they can be accurately quantified. Both incomplete image processing and excessive image processing are detrimental to the program's ability to quantify the capillaries. As seen in Figure 3, incomplete image processing makes the capillaries difficult to distinguish from the background. It is critical that the user be able to readily distinguish the border of a capillary since the counting method described above depends on the user's ability to accurately highlight a few capillaries. On the other hand, as seen in Figure 3, the application of unnecessary image processing steps can lead to blurring of the capillaries and therefore can also be detrimental to the quantification process.
An optimally processed image can be counted within 30 secs and clearly distinguishes capillaries from background as well as individual capillaries from each other. An example of a processed image is seen in Figure 2 part D, with the counted image shown in Figure 3.
Capillary density differs depending on the location of the nailbed being counted. Table 1 shows that capillary density increases with distance from the top row of capillaries at the nailbed. Standardization of the ROI placement is crucial to count reproducibility. Figure 1 shows how images can be altered with different ROI placements.
<table>
	<tbody>
		<tr>
			<td>ID</td>
			<td>T1</td>
			<td>T2</td>
			<td>T3</td>
			<td>T4</td>
			<td>Mean Top</td>
			<td>M1</td>
			<td>M2</td>
			<td>M3</td>
			<td>M4</td>
			<td>Mean Middle</td>
			<td>L1</td>
			<td>L2</td>
			<td>L3</td>
			<td>L4</td>
			<td>Lowest</td>
		</tr>
		<tr>
			<td>Patient A</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>46</td>
			<td>45</td>
			<td>44</td>
			<td>46</td>
			<td>45.25</td>
			<td>64</td>
			<td>62</td>
			<td>62</td>
			<td>62</td>
			<td>62.5</td>
			<td>66</td>
			<td>67</td>
			<td>66</td>
			<td>66</td>
			<td>66.25</td>
		</tr>
		<tr>
			<td>Venous Occlusion</td>
			<td>51</td>
			<td>53</td>
			<td>49</td>
			<td>59</td>
			<td>53</td>
			<td>59</td>
			<td>61</td>
			<td>64</td>
			<td>69</td>
			<td>63.25</td>
			<td>70</td>
			<td>70</td>
			<td>75</td>
			<td>72</td>
			<td>71.75</td>
		</tr>
		<tr>
			<td>Patient B</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>47</td>
			<td>51</td>
			<td>48</td>
			<td>51</td>
			<td>49.25</td>
			<td>73</td>
			<td>74</td>
			<td>75</td>
			<td>81</td>
			<td>75.75</td>
			<td>76</td>
			<td>85</td>
			<td>81</td>
			<td>80</td>
			<td>80.5</td>
		</tr>
		<tr>
			<td>Venous Occlusion</td>
			<td>68</td>
			<td>57</td>
			<td>65</td>
			<td>64</td>
			<td>63.5</td>
			<td>75</td>
			<td>78</td>
			<td>76</td>
			<td>72</td>
			<td>75.25</td>
			<td>91</td>
			<td>89</td>
			<td>93</td>
			<td>83</td>
			<td>89</td>
		</tr>
		<tr>
			<td>Patient C</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Baseline</td>
			<td>51</td>
			<td>54</td>
			<td>51</td>
			<td>56</td>
			<td>53</td>
			<td>66</td>
			<td>59</td>
			<td>58</td>
			<td>60</td>
			<td>60.75</td>
			<td>60</td>
			<td>61</td>
			<td>62</td>
			<td>69</td>
			<td>63</td>
		</tr>
		<tr>
			<td>Venous Occlusion</td>
			<td>62</td>
			<td>66</td>
			<td>57</td>
			<td>59</td>
			<td>61</td>
			<td>63</td>
			<td>63</td>
			<td>73</td>
			<td>65</td>
			<td>66</td>
			<td>83</td>
			<td>74</td>
			<td>81</td>
			<td>77</td>
			<td>78.75</td>
		</tr>
	</tbody>
</table>
Table 1: Variation in Capillary Counts with Differential Positioning in the Fingernail Bed. This table shows the counts obtained for three different patients (A, B, C) when the ROI box is variably positioned at the top (counts T1 - T4), middle (M1 - M4), and lower regions (L1 - L4) of the fingernail bed. The mean counts increase from the top to lower regions, demonstrating the need for standardization of ROI box placement to compare counts obtained from different laboratories.
Performing counts in the area described in step 1.2, baseline counts should range from 30 to 60 capillaries/mm2 while venous occlusion counts can range anywhere from 50 to 100. As seen in Table 1, these densities differ from other literature. Capillary density counts obtained in the authors' laboratory are most likely lower because this lab begins counts at the first row of capillaries, where the density is lowest. As seen in Table 1, performing counts in lower regions of the nailbed increase the counts toward values obtained previously by Antonios et al9 and Debbabi et al.16 This discrepancy illustrates the need for standardization in quantification of nailbed capillaroscopy by counting the first (most proximal) row of capillaries. Counting at the first row of capillaries is also optimal because the capillaries are most clearly and completely visualized in the first row and progressively become less visible with subsequent rows.
Blinded reproducibility studies using N = 10 subjects and two independent observers were conducted. Reliability results refer to the average A, B, and C counts, obtained by averaging results across 4 images for each. The A, B, and C counts represent different physiologic states within the same individual that are used to assess microvascular health, briefly summarized here. Details have been previously published 21. Capillary Density is defined as the number of capillaries per square millimeter (mm2) of finger nailfold skin. Stage A is a resting baseline stage where the capillaries are continuously perfused 16. Stage B occurs during postocclusive reactive hyperemia. These counts represent the sum of continuously perfused and intermittently perfused (functional reserve) capillaries. This stage is used as a measure of capillary function 16.
Stage C occurs during venous occlusion, therefore showing maximal capillary density including both perfused (with active red blood cell (RBC) motion) and nonperfused (filled with stagnant, non-moving RBCs) capillaries.22
For intra-rater reliability, the intraclass correlations (ICC) were 0.93 for mean A counts, 0.93 for mean B counts, and 0.94 for mean C counts. For inter-rater reliability, the ICCs were 0.94 for mean A counts, 0.98 for mean B counts, and 0.94 for mean C counts. Accordingly, the technique described here demonstrates excellent reliability with good results for both intra and inter-observer reproducibility.

<img alt="Figure 1" src="/files/ftp_upload/53088/53088fig1.jpg" /> 
Figure 1. Standardizing the Crop Location. This figure illustrates how variable placement of the ROI box visibly alters the cropped image. On the left, the box is placed too low, cutting off the first row of capillaries. The middle box is placed too high, causing a blank space above the first row of capillaries. The box on the right is optimally placed. Its cropped image shows the first row of capillaries at the very top of the image. <a href="https://www.jove.com/files/ftp_upload/53088/53088fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53088/53088fig2.jpg" /> 
Figure 2. Stages of Image Processing.(A) Stage A shows the initial image taken from the subject's nailbed with a monochrome camera; (B) Stage B shows the original image after the first contrast enhancement. The green box shows a 530 x 530 pixel box, which is equivalent to 1 x 1 mm box for the camera; (C) Stage C represents the 1 mm box cropped from image B; (D) Stage D shows the enhanced image after applying the enhancements discussed above. <a href="https://www.jove.com/files/ftp_upload/53088/53088fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53088/53088fig3.jpg" /> 
Figure 3. Final Counted Image. The enhanced, counted image. The total count determined for this image was 54 capillaries/mm2. <a href="https://www.jove.com/files/ftp_upload/53088/53088fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53088/53088fig4.jpg" /> 
Figure 4. Improper Image Processing. <a href="https://www.jove.com/files/ftp_upload/53088/53088fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The photo on the left shows a photo that is not processed enough. The capillaries are difficult to distinguish from the background and the quantitation process will be negatively affected. The photo on the right shows the same image after incorrect image processing. Individual capillaries are difficult to distinguish from their neighbors and thus the quantitation process will be negatively affected.

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A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm.

Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy to learn methodology for directly visualizing the microcirculation. The ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

8.9K Views.  Thomas Jefferson University. Capillaroscopy is a non-invasive, efficient, relatively inexpensive and easy-to-learn methodology for directly visualizing capillaries in the microcirculation. However, only one publication to date describes the reliability of a complex software program available for quantitating capillaroscopy data. Here, we present a simple, reliable protocol for quantitating capillaries using a standardized algorithm. 

Video: A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC)&#160;to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using &#8220;off-the-shelf&#8221; microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) &#956;m in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.

We describe the use of a standard optical microscope to perform quantitative measurements of cellular mass, volume, and density through a combination of bright field and differential interference contrast imagery.

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a ...

Bioengineering

Non-invasive Assessment of Microvascular and Endothelial Function

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of cardiovascular disease. Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. Percent capillary recruitment is assessed by dividing the increase in capillary density induced by postocclusive reactive hyperemia (postocclusive reactive hyperemia capillary density minus baseline capillary density), by the maximal capillary density (observed during passive venous occlusion). Percent perfused capillaries represents the proportion of all capillaries present that are perfused (functionally active), and is calculated by dividing postocclusive reactive hyperemia capillary density by the maximal capillary density. Both percent capillary recruitment and percent perfused capillaries reflect the number of functional capillaries. The forearm blood flow (FBF) technique provides accepted non-invasive measures of endothelial function: The ratio FBFmax/FBFbase is computed as an estimate of vasodilation, by dividing the mean of the four FBFmax values by the mean of the four FBFbase values. Forearm vascular resistance at maximal vasodilation (FVRmax) is calculated as the mean arterial pressure (MAP) divided by FBFmax. Both the capillaroscopy and forearm techniques are readily acceptable to patients and can be learned quickly.
The microvascular and endothelial function measures obtained using the methodologies described in this paper may have future utility in clinical patient cardiovascular risk-reduction strategies. As we have published reports demonstrating that microvascular and endothelial dysfunction are found in initial stages of hypertension including prehypertension, microvascular and endothelial function measures may eventually aid in early identification, risk-stratification and prevention of end-stage vascular pathology, with its potentially fatal consequences.

Capillaroscopy is a non-invasive, relatively inexpensive methodology for directly visualizing the microcirculation. The forearm blood flow technique provides accepted non-invasive measures of endothelial function.

The authors have utilized capillaroscopy and forearm blood flow techniques to investigate the role of microvascular dysfunction in pathogenesis of ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence of calcification inhibitors, that concomitantly lead to a loss of vessel elasticity and function. Vascular calcification is an important contributor to morbidity and mortality in many pathologies, including chronic kidney disease, diabetes mellitus, and atherosclerosis. Current research models to study vascular calcification are limited and are only viable at the late stages of calcification development in vivo. In vitro tools for studying vascular calcification use end-point measurements, increasing the demands on biological material and risking the introduction of variability to research studies. We demonstrate the application of a novel fluorescently labeled probe that binds to in vitro calcification development on human vascular smooth muscle cells and determines the real-time development of in vitro calcification. In this protocol, we describe the application of our newly developed calcification assay, a novel tool in disease modeling that has potential translational applications. We envisage this assay to be relevant in a broader spectrum of mineral deposition research, including applications in bone, cartilage, or dental research.

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Cardiovascular disease is the leading cause of death worldwide. Vascular calcification contributes substantially to the burden of cardiovascular morbidity and mortality. This protocol describes a simple method to quantify vascular smooth muscle cell-mediated calcium precipitation in vitro by fluorescent imaging.

Vascular calcification involves a series of degenerative pathologies, including inflammation, changes to cellular phenotype, cell death, and the absence ...

Biochemistry

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Biology

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill should occur within 2 s after releasing 5 s of firm pressure (e.g., by the physician&#39;s finger) in the normal healthy supine patient. A slower refill time indicates poor skin perfusion, which can be caused by conditions including sepsis, blood loss, hypoperfusion, and hypothermia. Since its introduction, the clinical usefulness of the test has been debated. Advocates point out its feasibility and simplicity and claim that it can indicate changes in vascular status earlier than changes in vital signs such as heart rate. Critics, on the other hand, stress that the lack of standardization in how the test is performed and the highly subjective nature of the naked eye assessment, as well as the test&#39;s susceptibility to ambient factors, markedly lowers the clinical value. The aim of the present work is to describe in detail the course of the refill event and to suggest potentially more objective and exact endpoint values for the capillary refill test using diffuse polarization spectroscopy.

This protocol describes how the use of diffuse polarization spectroscopy can improve the clinical usefulness of the capillary refill test. We suggest a more detailed analysis of the course of the capillary refill in healthy volunteers using diffuse reflectance spectroscopy videos and new informatic endpoints.

The capillary refill test was introduced in 1947 to help estimate circulatory status in critically ill patients. Guidelines commonly state that refill ...

Neuroscience

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis algorithms which can be used effectively to count various biological particles. When counting large numbers of cell samples, the hemocytometer presents a bottleneck with regards to time. Likewise, counting membranes from migration/invasion assays with the ImageJ plugin Cell Counter, although accurate, is exceptionally labor intensive, subjective, and infamous for causing wrist pain. To address this need, we developed two plugins within ImageJ for the sole task of automated hemocytometer (or known volume) and migration/invasion cell counting. Both plugins rely on the ability to acquire high quality micrographs with minimal background. They are easy to use and optimized for quick counting and analysis of large sample sizes with built-in analysis tools to help calibration of counts. By combining the core principles of Cell Counter with an automated counting algorithm and post-counting analysis, this greatly increases the ease with which migration assays can be processed without any loss of accuracy.

This paper describes the quantification of hemocytometer and migration/invasion micrographs through two new open-source ImageJ plugins Cell Concentration Calculator and migration assay Counter. Furthermore, it describes image acquisition and calibration protocols as well as discusses in detail the input requirements of the plugins.

The National Institute of Health&#39;s ImageJ is a powerful, freely available image processing software suite. ImageJ has comprehensive particle analysis ...

A Reproducible Computerized Method for Quantitation of Capillary Density using Nailfold Capillaroscopy

Summary

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Chapters in this video

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

Non-invasive Assessment of Microvascular and Endothelial Function

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

A Semi-Automated and Reproducible Biological-Based Method to Quantify Calcium Deposition In Vitro

Visual Detection of Multiple Nucleic Acids in a Capillary Array

NanoDrop Microvolume Quantitation of Nucleic Acids

Diffuse Reflectance Spectroscopy: Getting the Capillary Refill Test Under One's Thumb

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Automated Quantification and Analysis of Cell Counting Procedures Using ImageJ Plugins