Maintaining live cells and tissue slices in the imaging setup.

The development of new fluorescent probes and green fluorescent protein (GFP) reporter animal models, coupled with improved access to and convenience of imaging technologies, has generated a good deal of excitement in the field of live imaging. One of the challenges that would-be users face is the application of these technologies to live specimens that must remain viable in the imaging setup. Although some preparation methods permit high-resolution imaging of live cells in vivo, in many cases, it is still necessary or desirable to isolate the cells and tissues so that they may be stained and, subsequently, observed over time. Such procedures can compromise the health of the cells and tissues. The length of time needed to make the required observations will clearly dictate the kinds of measures taken to maintain the specimens. Experimental observations made over the course of many hours require a different level of specimen maintenance than those made over only a few minutes. Under optimal conditions, it is possible tomaintain viable tissues on the stage of amicroscope for many hours—even days— while collecting valuable information on dynamic events in live cells and tissues. How can biological samples bemounted and bemaintained to last long enough tomakemeaningful observations? This article offers some practical advice based on the investigators’ experiences imaging neurons (e.g., O’Rourke et al. 1992; Dailey and Smith 1994, 1996; Dailey et al. 1994; Marrs et al. 2001; Zha et al. 2005; Ahmed et al. 2006), astrocytes (Benediktsson et al. 2005), and microglia (Dailey and Waite 1999; Stence et al. 2001; Dailey 2002; Grossmann et al. 2002; Petersen and Dailey 2004; Kurpius et al. 2006, 2007) in rodent brain tissue slices, as well as neurons in excised chick neural retinal preparations (Marrs et al. 2006). Although the specific requirements of different samples will vary, there are some general principles to consider when attempting to maintain viable specimens in the imaging setup (Dailey et al. 2006). The emphasis here is on simple low-cost approaches for maintaining live mammalian brain cells and tissue slices in imaging setups.