Bone imaging interview

Please outline microCT as an imaging modality for analysing bone structure.

Bone structure is something that researchers and clinicians need to be familiar with for a wide range of reasons. Perhaps the biggest and longest standing application is in the condition of osteoporosis, which usually affects people in their old age, especially women.

There are some special cases of pediatric or drug-related to anorexia, and it's been one of the big applications. Like many areas of medical research, the term preclinical refers to the use of rats and mice for the models. MicroCT is a really user friendly and effective way of measuring bone structure.

You can non-destructively get the three-dimensional architecture of bone, any site of the small animal, in a lot of detail and the advantage is that it's non-destructive. The sample is still intact at the end, allowing the scientists to go and do their genetic and histological analysis.

The image itself is just the start of it, because then you can select the part that you want and make measurements of the structure. Those measurements are what give the scientists their data that forms the substance of their research.

MicroCT is a physical method, and it's exactly the same as a CT scan that you go to the hospital for; the same technology, x-rays taken from multiple angles synthesized into a 3D image, and it works on the basis of x-ray contrast. It gives you structure in any case where your sample has different materials and different x-ray absorption. In biological tissues, bone is the easiest tissue to image by x-ray because it has calcium and minerals in it so you can see the bone structure very easily.

MicroCT, in bone remains, will probably always be the biggest application in the life sciences because of the fact that it physically lends itself to imaging because of the calcification of the tissue.

How does microCT for bone imaging compare to other methods?

MicroCT has a definite time advantage over histological slicing, which can take a day or two while you imbed the sample in a paraffin or plastic matrix. Then you need to slice up the entire sample, which takes another couple of hours, followed by staining it for another couple of hours, to see the sample in a microscope.

Next you do the analysis slice by slice and take a picture of each individual cross section or choose a few. Then you will make an analysis on 2D slices resulting in 2D information, which sometimes is extrapolated to 3D situations because our organs are 3D objects.

With micro scan for a bone volume analysis, we typically take up to 10-15 minutes for a medium resolution or up to a few hours for the very highest resolution. After that, you will do a reconstruction for a few minutes, and the analysis can be done in a fully automated way in 3D. Whereas, for histology, you would spend a week putting everything together.

What makes microCT so suited to bone analysis? Does this mean it is limited to hard tissue?

It's not limited to hard tissue only, but what makes it especially interesting for bones is that the whole basis of microCT is to measure absorption of x-rays in organs or tissues. Based on that absorption, you make a 3D image of the entire structure, both the outside but also the inside in a non-destructive way.

Bones are easy to image in this way because they are calcified, hence why they show up as radio dense. However if you do go for other types of tissues, you can still scan and get a good image. Sometimes you will need to pre-treat the samples and/or apply an x-ray staining like a PTA, acid staining or iodine staining.

If we talk about in vivo or ex vivo analysis, with in vivo you can discriminate between fat tissues, lean tissues, the muscles, the liver, the blood, the heart, and the bones. With ex vivo, you have less limitations, so we can apply more staining and extract information about other organs as well.

What are the other preclinical capabilities of microCT?

In mineralized tissues we have bone and dental imaging, and some pre-clinical zoology work within that as well.

After bone and dental, we have lung imaging, which can be done both in vivo and ex vivo. Typically, the resolution for ex vivo will be a lot higher compared to in vivo studies. The resolution range we are working in is in the micrometer scale, that's where the name microCT name comes from.

Some other applications will require a contrast agent to be injected, for example in cardiac imaging and vascularization.

Please outline the methodology behind microCT.

There are several steps involved with microCT. The first step is to scan the sample, and to do this you put an extracted bone, tissue or a live animal in a microCT system. Here, we discriminate between two types of microCT systems. You have the in vivo scanners for whole animals., which include a temperature sensor, ECG sensors and breathing detectors. You put the mouse on a bed and then enter with a camera and an x-ray source rotating around the sample.

A very similar technology, but different from an architectural point of view, is the ex vivo scanners. The camera doesn't rotate around the sample - rather, it's the sample which will rotate on the sample stage in between the source and the camera.

To acquire images, it uses the same technology. During your scan, you will shoot these radiographs as you would in the clinic, at several angles around the object. This will give you a stack of radiographs, projection images, which are then step reconstructed. That's a back-projection algorithm, which will combine all this information and give you a stack of cross sectional images.

You can then create visualizations using the images, by loading them in a 3D rendering software package, also provided by Bruker to support the images created using Brukers microCT instrumentation.

Important to note here is that all the software we use is developed in house in Bruker MicroCT, compared to some other companies who rely on third party software. Either the 3D rendering or the analysis can be done.

After the scan and reconstruction comes the third and final part of microCT - what do we want to do with this 3D information?

For the analysis, we will split up between either morphometric analysis where you will extract information about shapes, volumes, thicknesses, separation, voracities, or a density analysis where you will get information about how radio dense certain areas are.

Are there different types of microCT? If so, how do they differ in the measurements they make?

First of all we have in vivo versus ex vivo, whether you set up the system to scan live animals or something not living. Then we'll specify a system and the resolution, and you can insert different sources and cameras.

The source will determine the penetrating power from the x-ray, so the more energy you have, the bigger and denser the objects you can scan. Very low voltages would be sufficient to scan soft tissues, for example, whereas with higher voltages you can scan bones. The whole idea behind it is that the x-rays must make it through the object to reach the camera. If all the x-rays are stopped in the object, there will not be any image on the camera. That's one side of the scanner, the x-ray source and the voltage.

You can either use CCD cameras or flat panel cameras; CCD refers to more high-resolution cameras, whereas the flat panel cameras might have bigger pixels and slightly lower resolution.

For the ex vivo specimen samples, we have scanners which use those two types of cameras. The CCD allows a smaller area to be scanned at very high resolutions, going down to the micron level. The flat panel doesn't have such high resolution but allows a larger area to be imaged more quickly and it's also able to go to higher energies and penetrate through denser objects. These two broad types have different ranges and both in the ex vivo and the in vivo scanners, we either have the CCD focusing on high resolution or the flat panels for high energy, larger samples and high speed.

However where all of our Bruker microCT systems are alike, is the focus on user friendliness, which is appreciated in many parts of the life sciences field.

Please give an overview of the Bruker SkyScan systems used in preclinical bone research.

Our most commonly used system is called the SykScan 1272. That is a CCD based, high resolution, freestanding desktop system with a small footprint. The systems we sell are used with a complete solution; the scanner, the software for the analysis and the visualization are all together. The 1272 covers a wide range of applications including bone, dental, zoological and all the life science applications.

We also provide another scanner in ex vivo called the 1275. The number 12 at the beginning indicates the generation, where the number at the end represents the type of scanner. 72 has the CCD high resolution desktop system. In contrast, the 75 has a panel base and a desktop system footprint, but with a larger camera for bigger samples and faster scans. Those are the two most important ones for desktop imaging of bones. You could use the 72 if you wanted the mouse bones at the highest resolution, and you'd pick a 75 for images of larger bones, teeth, or higher density human bone samples.

We also have the 1174 which is a smaller desktop system - this serves as our entry-level system.

As for in vivo, we have 2 scanners; One is a high resolution scanner with a CCD camera, the 1276, and the 1278 is a lower resolution scanner with a flat panel camera.

Speed is not too different anymore between those scanners, as was the case in the past. Low resolution was in the order of seconds versus minutes for the high-resolution scanner. Now, in both systems, it came down to a few seconds at the highest speed.

For the 1278, you can scan a whole animal with a resolution down to 50 micrometers. Whereas for the 1276, the highest resolution you can achieve there is in vivo down to about 5 micrometers. You can also scan ex vivo samples in an in vivo scanner, if you carry out anesthesia etc, and you can go down to 2.8 micrometer pixel size.

Something that we are very proud of in all of the in vivo SkyScan instruments, is the need for only a low radiation dose. When you're getting an x-ray image of an animal, it's important not to give too large a radiation dose so you don't damage the tissues that you're trying to study. Very special technologies in our in vivo scanners, especially in terms of the high-performance cameras but also very high performance data processing and speed, allow scans to be done with exceptionally low radiation dose.

How has Bruker technology helped or advanced microCT development?

We were one of the very first companies to invest in the commercialization of microCT as far back as 1996. We've played a role in developing the market, both in the bone field and in generally in zoology and material science industry. We've played an important role in really bringing this technology to the scientific community and developing it in many areas longer than most of our competitors.

What direction so you see microCT going in the next few years? What do you see as the next big milestone?

Things are always going towards higher resolution and faster scanning, and also of course automation. We also believe that user friendliness is important. MicroCT is 3D, and because you can manipulate images and make movies, once people get into it, it becomes quite a compelling technology. However sometimes it can be intimidating to people who haven't used this technology before, because it looks quite technically demanding which puts people off. We want to pull more people into it by removing that barrier and providing straightforward, intuitive training. We have a lifelong relationship with users in terms of ongoing training and support.

One of the things we'd like to try to do, in addition to the technology that we want to continue moving forward, is to stay connected to the research community, and the bone community in particular to provide our support, service and overall act as an important tool to them.

Wherever the bone and dental research field goes, we want to go there right with them and be available to help out with the direction of our research itself. We're in a position to do it and want to emphasize that we wish to remain partners with researchers in the bone field in whatever direction bone research go in or with whatever technology they're using.