Diving head-first into brain intravital microscopy

Abstract

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation.

Keywords: brain; imaging; intravital microscopy; neuroinflammation; stroke.

Figure 1

Intravital microscopy significant discoveries and developments. A timeline of major discovery milestones of the hardware and associated tools of brain intravital imaging. Image created with BioRender.com.

Figure 2

Common intravital microscopes. The light excitation and emission pathways, and intravital images of mouse brain taken from (A) Widefield Microscopy, (B) Confocal Microscopy, (C) Multiphoton Microscopy, (D) OR-PAM, and (E) AR-PAM. The cell type or label used are in the corresponding colour font for each image. Representative images were our own work (confocal and 2PM) or adapted from published work with permission [Wong et al. for Widefield (16); Wang et al. for 3PM (17); Zhu et al. for AR-PAM (18)]. Created with BioRender.com.