Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The overall goal of this methodology is to provide a standardized procedure for obtaining images of live human resistance arteries for analysis of the relationship between the microarchitecture of the extracellular matrix and the mechanics of the arterial wall. This method can help answer key questions in vascular biology and mechanics such as how pressure induces changes in the overall geometry as well as the microarchitecture of the vascular wall. The main advantage of the technique is that you don't have to label your sample for the imaging.

Though this method can provide insight into resistance artery mechanics, a modified version of the protocol can be applied to other tube-like organs like the bronchi or to strips from human bladder, or the intestinal wall. To begin, collect tissue samples of interest immediately upon their excision during surgery and prepare the vessels as described in the accompanying text protocol. Pressurize a cannulated artery to five millimeters mercury.

Then set up the optical path for the imaging by activating the two-photon laser and setting the excitation wavelength to 820 nanometers. Insert a 460 nanometer long pass dichroic mirror to split the emission light between the photo multipliers and collect the emission simultaneously in two channels. Use 30 to 60 nanometer wide bandpass filters centered at 520 nanometers to collect the elastin autofluorescence and another centered at 410 nanometers to collect the collagen second harmonic generation signal.

Now set the laser dwell time and pixel to 10 microseconds. Set the resolution to 512 by 512 pixels for 100%field of view. Enable continuous scanning and use as low excitation light power and pixel dwell time as possible to avoid photo damaging the live artery.

Then manually scan through the artery to find the plane with the maximum diameter. Next, choose a rectangular slice covering the artery's widest diameter and scan a single frame with a pixel size of 300 nanometers per pixel or less. Save this image for analysis at a later time.

To determine lumen diameter and wall thickness at the maximum diameter of the artery, first load the image in Fiji. Set the scale by clicking on main menu and then go to analyze followed by set scale. Here enter the micrometer per pixel and pixel ratio.

Next, choose the Fiji line tool and draw a line between the two internal elastic lamina at each side of the arterial lumen. Then click Control M to report the length in the results table. Continue to draw more lines to measure the thickness of each wall and save the final measurement.

Change to a 60X objective with a numerical aperture of one or greater. Image the collagen and elastin microarchitecture at five millimeters mercury by scanning the entire thickness of the arterial wall. Next, define the Z stack start and end depth and the Z step spacing and pixel density.

When you're designing your Z stacks, make sure your region of interest is small enough to avoid any inference of curvature on your subsequent analysis. When complete, set each channel to be saved separately. Lastly, find one to three different regions of interest and obtain 3D image stacks of good quality.

If your elastin autofluorescence is bleaching, you are using too high excitation light power. This should be avoided. In order to finish your ongoing experiment, you may add a dilute concentration of eosin to enhance the elastin autofluorescence.

When finished, repeat the entire process at pressures varying up to 100 millimeters mercury, replacing the buffer in the myograph chamber with fresh 37 degrees Celsius HBS after each pressure step. To begin image analysis, open the elastin image stack in Fiji. Then go to image, click on stacks, and select Z project to choose the images to work with and choose max intensity for projection type.

Save the max intensity projection as a TIFF image. Then choose as many internal elastic lamina fiber branching points as possible and measure them using the angle tool. Systematically work through each image and when finished, save the Fiji results sheet.

To measure the waviness of the adventitial collagen, open the collagen image stack in Fiji. Next, go to image down to stacks and select Z project to choose images to work with and to set the max intensity projection. Now change the image type to an eight-bit TIFF image by clicking image, type, and then eight-bit.

Save the max intensity projection as a TIFF image. Open the Neuron J plugin by clicking on Fiji and selecting Neuron J under plugins. Then open the eight-bit TIFF file in Neuron J.Once open, click on add tracings and select fibers to analyze by clicking on the start and the end of each fiber.

Next, go to measure tracings and choose display tracing and vertex measurements in the dialogue box. To calculate collage fiber straightness, first copy the values in the Fiji tracings window to Excel. The length values in the tracings window are measures of the full lengths of the analyzed collagen fibers Lf.Second, copy for each collagen fiber bundle analyzed the first and last XY coordinates of the Fiji vertices window to Excel.

Then calculate the length of the straight line connecting each end of the analyzed collagen fiber L0 using Pythagoras Theorem. Collagen fiber straightness is finally calculated as L0/Lf. Following the protocol just shown, the widest part of the artery was identified and at this point the artery was scanned using label-free fluorescence microscopy to measure the maximum vessel diameter and vessel wall thickness.

Utilizing a higher magnification to high numerical aperture objective, not only can the vessel diameter and wall thickness be measured, but also the microarchitectures of collagen and elastin. The microarchitectures of elastin fiber branching angles and collagen straightness can be determined in consecutive image sections using the appropriate image analysis software. This allows a direct analysis of the relationship between the pressure induced changes in the microarchitectures of collagen and elastin and the incremental elastic modulus at different pressures.

Following this procedure, other information can be extracted from the images as well. This could be the orientation angels of adventitial collagen along the vessel longitudinal axis or it could be the microarchitecture of the extracellular matrix in tunica media. The quantitative measures extracted from the images using this method can be fed into mathematical models in order to elucidate how the healthy and deceased human circulation respond to different conditions.

After watching this video, you should have a thorough understanding on how to design your imaging experiments and subsequent image analysis in order to extract information on how pressure induces changes in the microarchitecture of the resistance artery wall. Remember that you have to avoid any exposure to visible and invisible light when working with two-photon excitation fluorescence microscopy.