Functional Imaging with Mitochondrial Flavoprotein Autofluorescence:

While great strides have been made in the development of novel optical imaging techniques in the past two decades, these methods still either measure coarsely or require relatively invasive procedures. The ideal optical measure would faithfully represent local neural activity, both spiking and nonspiking, at subcellular resolution, with persistent, reliable responses without the use of exogenous agents. Practically, there are several opportunities to measure endogenous signals that could serve as indirect measures of neural activity: most commonly, metabolic processes which take advantage of the tight coupling between neural activity and ATP production. Unfortunately, the most robust metabolic signals (such as blood deoxygenation) are only weakly linked to neural activity, and can be spatially diffuse, putting strict limitations on the interpretation of these data. Recently, the endogenous fluorescence of mitochondrial flavoproteins (FA, flavoprotein autofluorescence) has been exploited for functional imaging in a variety of preparations, both in vivo and in vitro. These flavoproteins are oxidized during aerobic metabolism, which is closely coupled to neuronal signaling, and their fluorescence has been shown to follow neural activity in response to pharmacological, electrical, and sensory stimulation. They have been used to confirm functional maps in several cortical areas, and are supported by a growing body of in vitro work. As these signals (1) are highly spatially localized, (2) can be measured by a variety of optical techniques both in vitro and in vivo, and (3) require no exogenous dyes, they offer substantial improvement in our ability to map functional activity and trace neural circuits. Here, we describe the underlying biochemistry of the flavoprotein signal, the experimental details of using it for functional imaging in vivo, and outline example studies and future directions for this promising new approach to visualizing neural activity patterns.