Imaging skeletal growth plates using in vivo multiphoton microscopy
Figure 3. In vivo multiphoton image of blood vessels in the plexus surrounding the tibial growth plate of a live, anesthetized 5-week-old mouse. Orientation matches Figure 1. Vessels were visualized using a multiphoton microscope after an intravenous injection of fluorescein. Plasma is red and blood cells appear as dark shadows within the vessels. The collagen-rich perichondrium around the growth plate (green-gray pseudocolor) was visualized by second harmonic generation (SHG), a robust signal from unstained collagen that is unique to multiphoton excitation. SHG allows collagenous structures to be identified without injecting stains or dyes. Imaging was done by Maria Serrat on a Leica TCS SP5 II Multiphoton Microscope housed in the Molecular and Biological Imaging Center at Marshall University (image modified from Serrat, 2014, Comprehensive Physiology, 4:621-55, Copyright 2014 American Physiological Society).
Multiphoton microscopy is an emerging technology for live animal imaging that offers exciting possibilities for the study of growth plate dynamics in vivo. In collaboration with colleagues at Cornell University, we developed a unique platform for imaging intact skeletal growth plates that we use to assess how systemic regulators arrive at and move within the cartilage matrix of the growth plate under various experimental conditions. This system provides a new mechanism for understanding the physiological regulation of bone growth through the ability to dynamically measure changes in molecular transport to the growth plate of a living animal (Fig. 3). Using biologically-inert dextrans as size-proxies for systemic regulators, we have shown that heat can increase real-time entry of large molecules into growth plate cartilage in vivo (Serrat, Efaw, and Williams, 2014). We are now using fluorescently-labeled proteins to quantify heat-enhanced access of biologically-active molecules into growth plates of live, intact mice.
Unilateral heating to lengthen extremities of growing mice
Fig. 4 Thermal Image of Mice during a Limb Heating Experiment (Serrat Lab) Limb heating experiments are being done in the Serrat Lab to study mechanisms underlying heat-enhanced bone elongation. Thermal image of juvenile mice on a heating pad (left) shows the temperature differential between heat-treated and non-treated sides. Limbs are equilibrated to 40C during the daily treatments. Digital image (right) shows foam separators that are used to keep the non-treated side at a cooler 30C. Images captured June 2014 by Ph.D. student Holly Tamski and Maria Serrat, Ph.D.