Below is a schematic overview of our intravital microscope setup in our lab, which is used to visualize the microcirculation in the Extensor Digitorus Longus (EDL) muscle in the rat. The EDL preparation is an intravital tissue preparation, with which our lab has extensive experience.

To surgically exteriorize the muscle, the animal is anesthetized, and the lateral side of the right hindlimb is shaven and disinfected. An incision is made in the skin, and the underlying fascia is separated from the muscle. The EDL muscle is a thick bellied muscle with several extensions and tendons leading towards the toes. After surgically exposing the muscle, the distal tendon (just proximal of the lateral malleolus) is tied with a suture and subsequently cut so the muscle can be extended away from the paw, while the proximal side remains connected (1, 2, 3).  In this way, the muscle can be extended away from the hind paw while leaving the blood supply to the observed tissue intact.

The EDL muscle is placed onto the microscope, and saran wrap is placed onto the muscle to prevent any tissue desiccation. The muscle is gently stretched to its approximate original in-situ length and secured to avoid movement, and a thin microscopy object cover glass is placed on the tissue.

 
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New machine learning algorithms were developed to expedite the analysis of video data (4). Registering all pixel intensities in the consecutive images allows visualizing the actively perfused micro-vessels in the microscopic field of view. Quantifying the intensity changes in all images will enable creating a geometric vascular structure based on the Sum of Absolute intensity Differences (SAD). These SAD images depict all actively perfused micro-vessels in the microscopic field of view. Since the same area is observed at subsequent time points throughout the experiment, changes over time in the number of perfused blood vessels can be quantified.

 
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1. Balice-Gordon, R. J. & Thompson, W. J. The organization and development of compartmentalized innervation in rat extensor digitorum longus muscle. The Journal of Physiology 398, 211–231 (1988).
2. Holmberg, H., Schouenborg, J., Yu, Y.-B. & Weng, H.-R. Developmental Adaptation of Rat Nociceptive Withdrawal Reflexes after Neonatal Tendon Transfer. J. Neurosci. 17, 2071–2078 (1997).
3. Tyml, K. & Budreau, C. H. A new preparation of rat extensor digitorum longus muscle for intravital investigation of the microcirculation. Int J Microcirc Clin Exp 10, 335–343 (1991).
4. Mahmoud, O., Janssen, G. & El-Sakka, M. R. Machine-Learning-Based Functional Microcirculation Analysis. AAAI 34, 13326–13331 (2020).