In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

The overall goal of the following experiment is to quantify microcirculation in a murn colon tumor model using a confocal fluorescence video microscopy system. This is achieved by first intravenously injecting a contrast agent and recording movies of the tumor microvessels. Then four quadrants of the tumor are imaged, allowing quantification of the functional capillary density.

Next, the passage of the contrast agent from the vessels into the interstitium is observed for quantification of the capillary index leakage reflecting the endothelial wall permeability. Ultimately, morphological and functional characteristics of the tumor vessels can be quantified in order to detect differences between tumor models or after targeted therapy. This dynamic contrast inance optical imaging technique allows the analysis of tumor micro circulation.

It reflects the architecture and function of the tumor vessels by quantifying the density and the permeability of the capillaries. Though this method could provide insight into tumor physiology, it can also be applied to other systems such as anaphylactic shock, inflammation, cardiovascular diseases, and diabetes. Begin by shaving the anesthetized mouse at the location of interest.

Then incise the skin, facing the organ to be imaged when the bleeding has stopped. Inject the contrast agent to quantify the functional capillary density. First place the probe in front of the organ to be imaged.

Next turn on the laser illuminating the organ. Then maintaining a steady hand. Slowly move the probe recording as the fluorescent capillary network is visualized.

To stabilize the dynamic acquisition over time, place a bit of ultrasound gel on the tip of the probe, and then place the probe in handmaid support. After placing the probe in contact with the organ of interest, stabilize the probe. Once the position is secure, set the laser to record three images every 30 seconds for 20 minutes to detect the presence of capillary leakage.

Turning the laser off between each recording to reduce bleaching of the contrast agent after recording five second movies in each of the four quadrants of the organ of interest, to visualize the capillary network process, the movies, using the appropriate software, performing an automated segmentation of the vessels in the images around a chosen diameter to quantify the functional capillary density, that is the ratio between the total vessel area and the total area of the image. Then from the dynamic acquisition performed for detecting the presence of capillary leakage, draw three regions of interest in the capillaries and three regions of interest in the interstitium at the 0, 5, 10, and 20 minute time points. Finally, average the signal intensities within the three different capillaries and contiguous interstitial areas, and calculate the index leakage percentage.

According to this formula, where IP is the perivascular or interstitial intensity, and II is the intravascular intensity, the functional capillary density parameter is a surrogate marker of microvascular density, usually measured using pathology tools. Here an example of the types of images obtained and the result of vessel segmentation are shown. In this example, the functional capillary density was measured at 36%In these images, an example of contrast agent leakage in the interstitium with an index leakage of 1.47 is shown at 0, 5, 10, and 20 minutes after injection.

In each of the images, the vessels appear as high signal linear structures immediately after injection. No signal is seen in the interstitium. Over time, however, the contrast agent can be observed leaking into the interstitium through the abnormal tumor.

Endothelial barrier Experience is necessary to master this procedure. However, once mastered, the technique can be completed in 30 minutes if it's performed properly.