Confocal live cell imaging is a powerful tool for studying central neurones and well as glial and vascular cells abundant in brain. Expression of fluorescent proteins allows imaging of particular cellular phenotypes or intracellular elements which can be in this way distinguished from irrelevant background structures. Viral vectors are one of the best ways of introducing genes encoding for fluorescent constructs into differentiated postnatal brain neurones and glia. Using various promoter sequences it proves possible to selectively target at least certain cell types and to express a variety of fluorescent constructs. Several popular viral gene delivery systems are currently in use. These include vectors derived from adenovirus, lentivirus and adeno-associated virus genomes which all have their specific advantages for gene delivery in vivo and in vitro. Viral vectors can be introduced in vivo and fluorescent neurones imaged a few days later in either fixed or living tissue. In this way, certain neuronal groups can be transduced in the retrograde manner and cells which have certain axonal projections visualised specifically. However, the most favourable conditions for imaging which at the same time are compatible with viral gene delivery are provided by organotypic brain slice cultures. Such cultures can be transfected using one or more viral vectors and then remain viable and suitable for imaging experiments for several weeks. An adenoviral vector encoding for EGFP under the control of 3.7 kb of the GAD67 promoter has been used to fluorescently target GABAergic neurones in the nucleus tractus solitarii. These cells were then recorded in whole cell patch clamp mode using a red-shifted Ca2+ indicator Rhod-2 and the effects of nitric oxide on intracellular Ca2+ concentration in somata, dendrites and axons were studied in great detail. For imaging of noradrenergic neurones we use vectors based on an artificial PRSx8 promoter (Teschemacher et al., 2005). Both, EGFP and monomeric Red Fluorescent Protein (mRFP)-expressing vectors have been generated. mRFP distribution in many neurones is far from homogeneous and it frequently forms small aggregates which puts in question claims of its suitability as an intracellular tag. However as it leaves the green part of the spectrum open it is possible to patch mRFP-expressing neurones using fast Ca2+ dyes (such as Fluo-4) and study dynamics of the fast Ca2+ events in different cellular compartment including the putative release sites in axonal varicosities. These methods will be demonstrated in the course of the meeting.