Abstract
High resolution, in vivo optical imaging of the mouse brain over time often requires anesthesia, which necessitates maintaining the animal's body temperature and level of anesthesia, as well as securing the head in an optimal, stable position. Controlling each parameter usually requires using multiple systems. Assembling multiple components into the small space on a standard microscope stage can be difficult and some commercially available parts simply do not fit. Furthermore, it is time-consuming to position an animal in the identical position over multiple imaging sessions for longitudinal studies. This is especially true when using an implanted gradient index (GRIN) lens for deep brain imaging. The multiphoton laser beam must be parallel with the shaft of the lens because even a slight tilt of the lens can degrade image quality. In response to these challenges, we have designed a compact, integrated in vivo imaging support system to overcome the problems created by using separate systems during optical imaging in mice. It is a single platform that provides (1) sturdy head fixation, (2) an integrated gas anesthesia mask, and (3) safe warm water heating. This THREE-IN-ONE (TRIO) Platform has a small footprint and a low profile that positions a mouse's head only 20 mm above the microscope stage. This height is about one half to one third the height of most commercially available immobilization devices. We have successfully employed this system, using isoflurane in over 40 imaging sessions with an average of 2 h per session with no leaks or other malfunctions. Due to its smaller size, the TRIO Platform can be used with a wider range of upright microscopes and stages. Most of the components were designed in SOLIDWORKS® and fabricated using a 3D printer. This additive manufacturing approach also readily permits size modifications for creating systems for other small animals.
Highlights
Laser scanning confocal microscopy through cortical windows has opened upper layers of the mouse brain for high resolution, time-course, in vivo imaging (Didion et al, 2005; Brown et al, 2010) and multiphoton microscopy (MPM) has facilitated in vivo imaging of the olfactory bulb (Adam and Mizrahi, 2011) and lower layers of the cortex, up to several 100 microns deep (Levene et al, 2004)
We have developed a low profile, multifunctional, in vivo brain imaging support system capable of positioning a mouse in precise alignment with an upright microscope, while simultaneously providing heat and anesthesia
Through measuring the mean temperatures of the base plate surface across the length of the plate that the silicone adhesive applied to seal the heated plate acts as an insulator when applied in a sufficiently thick layer
Summary
Laser scanning confocal microscopy through cortical windows has opened upper layers of the mouse brain for high resolution, time-course, in vivo imaging (Didion et al, 2005; Brown et al, 2010) and multiphoton microscopy (MPM) has facilitated in vivo imaging of the olfactory bulb (Adam and Mizrahi, 2011) and lower layers of the cortex, up to several 100 microns deep (Levene et al, 2004). Transgenic mice with fluorescent proteins expressed under specific genetic promoters have permitted celltype specific identification of targeted cell populations By using these mice for in vivo imaging, investigators have observed processes such as changes in dendritic spine density (Stetter et al, 2013), migration and activation of microglia (Nimmerjahn et al, 2005), growth of brain tumors (Brown et al, 2010; Barretto et al, 2011) and the dynamics of a brain tumor microenvironment (Ricard and Debarbieux, 2014). The means by which mice are positioned and maintained for imaging has relied primarily on old technology and non-standardized custom made components
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