The most accurate and relevant models to understand what happens under undisturbed physiological conditions are in vivo studies. Those studies are conducted within a whole living organism or cell. This is done by using a technique called intravital microscopy, where a vascular bed is dissected out of an animal, but remains connected to the feeding vessels, nerve cells and musculature. For this procedure the animals are placed under anaesthesia.

9.1 Intravital microscopy

Intravital microscopy uses the mesenteric capillary beds to observe changes in flow under various conditions of interest. Basically, any section of tissue can be used for those studies, as long as enough light can pass through it, which is why capillary beds are the best option, because they are relatively transparent. Changes in the vascular network, like vessel diameter, flow rate and cell volume can be investigated with intravital microscopy. The results that are collected heavily depend on the equipment, which is being used. Better optics and camera allow targeting smaller capillary bed segments and blood vessels more accurately. The usage of better anti-bodies/fluorescent probes allows targeting specific compounds, cell receptors and intracellular calcium compartments. Also, the relation between gene expression and physical changes can be observed.

A standard intravital microscopy apparatus consists of an epi-fluorescence microscope which is connected to a camera and feeds directly into a recording device and a live video monitor. To keep the tissue moist during the experiment, a perfusion system is necessary. Besides that, an anaesthesia controlling system makes sure, that the animal is kept under anaesthesia during the entire experiment, which can last up to 8h.

Intravital microscopy is a very important research tool because the vascular network that is under investigation remains connected to the animal and is still under its physiological regulation. It helps understanding the development of diseases and fundamental vascular physiology. Besides the applications in basic biology of microcirculation, intravital microscopy is used for designing drugs, implantable devices, and basically anything that comes in contact or interacts with the vascular system.

9.2 Doppler ultrasound

Basic three-dimensional ultrasound technology uses an ultrasound scanner to emit and collect sound waves, which are aimed at specific tissue locations. Besides that, an electromagnetic position and orientation measuring device is necessary to determine the spatial location of the probe. By sweeping the ultrasound probe over a tissue bed, multiple images can be obtained to observe the tissue from different locations.

Ultrasound imaging technology has multiple imaging modes connected with the ultra-sound probe. The most important one is called Doppler mode, because it makes it possible to obtain information about the flow of blood through a vascular network relatively close to skin. In addition to the general devices, used in ultrasound imaging technology, the Doppler ultrasound technique also uses a colour flow imaging module. The added colour codes the blood velocity and makes it possible to obtain a real-time depiction of the relative blood flow speed across the radial direction of the blood vessel and the direction of the blood flow.

Doppler ultrasound is useful to quantify the frequency of emboli or other large particulate matter by recording high-magnitude transient changes to the velocity profile. It is also a good research tool to characterize new cardiovascular devices. These de-vices can be implanted into an animal to investigate the changes in emboli formation for months or even years. Another use for ultrasound technology in general is to obtain vascular architectures. Its advantage is, that is neither harmful, nor expensive and can carry out real-time imaging and obtain flow information simultaneously to geometry information. The only problem is the limited scanning depth of ultrasound devices.

9.3 Magnetic resonance imaging (MRI)

Magnetic resonance imaging (MRI) is a technique used for imaging the entire human body. The basis of MRI technology is creating a large static magnetic field in which the water molecules in the human body align. This works because each hydrogen atom has a single proton and nucleus that spin continually and create a small magnetic field. When removing the magnetic field, information about the tissue composition can be obtained based on how rapidly the water molecules return to their “normal” spin rate. On their way back to the normal spin rate, two magnetic fields are generated which provide information about thermal tissue properties (longitudinal field) and the tissue inhomogeneities (transverse field).

With the collected frequency data, the analysing software can determine the tissue homogeneity/composition. Phase Contrast MRI (PC-MRI) can measure real time non-invasive blood flow.

MRI technology can detect changes within the tissue composition, brain activity, cerebral metabolism, blood flow and oxygenation. Changes in a single parameter affect all others because they are all linked, e.g. with increased metabolism the tissue oxygenation decreases, while it increases with increased blood flow. A special technology called phase contrast MRI can be used to obtain pictures of arteries and en-tire vascular networks. With this technique, the likelihood of cardiovascular diseases can be determined.

9.4 Magnetic resonance imaging (MRI)

Intravital microscopy is a powerful technique for understanding flow properties, cellular communication mechanisms, cell distributions among many other parameters as a function of cardiovascular antagonists, new therapeutics and new materials, among others. Research groups use this technique to visualise microcirculation and changes to microcirculatory parameters. This helps understanding the basic biology of micro-circulation in response to physiologically relevant stressors which is important to understand cardiovascular risk factors on disease progression.

Another research topic is the evaluation of new blood vessel growth in response to various biochemical and biomechanical stimuli relevant to the microcirculation. This allows research groups to augment and control the innate response and try to deter-mine what conditions are important during new vessel growth.

9.5 Areas of future research

One of the major current limitations of in vivo biofluid mechanics research is the availability of equipment and model systems that can more fully characterize systems of interest. It would be important to understand and correlate occurrences within the microcirculation and the microcirculation to fully understand disease progression. To accomplish his type of integrated system, it would be important to couple imaging techniques that can be used in parallel.

The most likely candidate for coupling two or more systems together is a system that includes a Doppler ultrasound imaging technique. The instrumentation associated with ultrasound techniques is minimal and would not interfere with other imaging modalities, such as intravital microscopy. The information gained from this type of system would be invaluable in understanding the real time effects of risk factors on both the microcirculation and the microcirculation.

Another advantage of systems designed with multiple imaging modalities at multiple length scale is that information gained from this work can be used to develop more comprehensive models. These improvements would help to gain an improved under-standing of the basic biology of these reactions while correlation them to modelling techniques and other predictive techniques that can be used to diagnosis and characterize the system of interest.