3D Volumetric Series of a Glomerulus

This is a series of images of a glomerulus—one of about two million renal corpuscles typically found within the human kidney. It resembles a hollow-walled cup, and inside it nests a series of capillaries. The glomeruli filter the blood for waste materials that are ultimately excreted by the kidneys as urine.

Modeled images, such as these created by Dr. Roger C. Wagner of the University of Delaware, depict anatomic structures in ways that traditional light microscopy can not. One advantage is that scientists and students can manipulate the structures and observe them from any angle, unlike scanning microscope images that only depict a single surface. And, because the modeled data is rendered in voxels, they can provide precise quantitative information about anatomical structures that would be difficult if not impossible to determine otherwise.

Apple: Dr. Wagner, how do you create images like these?

Dr. Wagner: We use Amira, by Mercury Computer Systems. It's a system for analyzing three-dimensional data sets, whether they’re from confocal microscopy or from CAT scans. Early in 2007, the company developed a version for Macintosh.

For this series of renal images, we used a Zeiss confocal microscope to gather the data. A confocal microscope is one in which information is gathered from only one focal plane at the same time. It has its own storage system and software cordoned in a file format called LSM, which stands for Laser Scanning Microscope. We then import that into the Mac running Amira, which reads and interprets the LSM data files.

Apple: How do you prepare the samples prior to the microscopy?

Dr. Wagner: We administer a rapidly hardening fluorescent plastic into the capillaries, which you can see in these images as red. The green circles in the first image are the nuclei of cells that surround the capillaries and are also found in the surrounding tissue.

The casting plastic happens to be fluorescent, so we scan the corrosion cast with a laser, which activates the fluorescent material in the plastic and gives us a signal. This is scanned one optical section at a time through perhaps several hundred optical sections, which builds up a three-dimensional data set that we call a Z series. This is our raw data that we take into Amira.

Apple: Tell us what we're seeing in this series of images.

Dr. Wagner: In the first image (Click here to enlarge ), what you see is a cast of the capillaries in that little glomerulus, shown without the tissue being removed or corroded away.

The second image (Click here to enlarge ) is a cast of those capillaries after the tissue has been corroded away with alkali, so all that's left is the plastic cast material.

Using this model, we can gather quantitative information about the surface area of this cavity, of the capillaries, and also about their volume. With Amira, everything is rendered in voxels, so if you enter the appropriate calibration figures, you get a direct readout of the mathematical parameters.

So the surface area and volume of those capillaries pertain directly to the filtration capacity of the glomerulus. From the model, you can derive the total surface area in volume of the blood space inside the capillaries, because that’s the data that matters most.

Apple: What else can researchers tell about glomeruli using these images?

Dr. Wagner: Well, you can’t easily see through the red model, however you would want to view what’s at the center to see the relative amount of branching of the capillaries in there. In order to do that, we apply a technique called skeletonization, and that's shown in the third image. (Click here to enlarge )

It shows a slice through that glomerulus from a confocal microscope, and superimposed upon that, you see the skeleton derived from that model. Now, a skeleton is what we call a medial axis transform, and it takes the original model and it erodes the surface, voxel by voxel, until you arrive at a central voxel—the contiguous voxels that actually form the skeleton of that structure.

Here you can see a moving image I've created of that, also using Amira, and when it rotates around, you can see what that capillary system looks like from the inside. You can also zoom in on it for more detail. (Watch QuickTime Video )

What you get from a skeleton like this is immediate information indicating the total length of the capillary and the number of intersects between the capillaries. The number of intersects gives you quantitative information about the blood vascular system. These data are immediately available from the model, and something that would have otherwise taken many, many hours to compute.

In every kidney, there are approximately two million of these little glomeruli. And in each glomeruli, the total length of the capillaries is about 1.3 centimeters. If you multiply that by two million, it almost comes out to about 19 miles of capillaries in a single kidney. So that’s kind of amazing, because that kind of data was not quantifiable before by any other means.

The final image that you see – this is the one with the skeleton of various different types of colors – that’s the skeleton superimposed inside an image of the original model (Click here to enlarge ). You can see the model from which the skeleton was derived, and the colors are proportional to the thickness of the vessel from which the skeleton was derived. In other words, there’s a big, thick vessel coming out, that’s got a skeleton in red, and you see the other colors, and they pertain to the cross-sectional diameter of the vessel from which the skeleton was derived.

Apple: Are there any particularly intriguing uses for this imaging technology in medical research?

Dr. Wagner: Yes, one area of great interest to my colleagues is to model the vasculature in tumors to determine how these systems grow, what kind of capacity is there for nourishing tumor cells, and how we can measure this process. So the things we’re learning here using non-tumor systems will soon become very valuable in analyzing the growth of blood vessels inside tumors.

Apple: Are there currently any limitations to this technology, and how do you envision this kind of imaging evolving?

Dr. Wagner: We’d like to be able to view whole organ systems, but we're currently limited by the enormous file sizes, and with confocal microscopy we're limited to very small data sets. In order to do whole organs, or even tumors, we'd have to have a device that will capture much larger volumes of information, such as a mini-CAT scan. We're attempting to add heavy metals to the samples, which will make them more X-ray dense and give us a much better model with which to work.