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Abstract
2-photon fluorescence microscopy of NADH and flavoprotein as a measure
of neuronal activity in PC12 cells and in zebrafish olfactory bulb.
Keith, CH*,†, Lauderdale, JD*, and Sornborger, AT§,†. *Department
of Cellular Biology, †Faculty of Engineering, and §Department
of Mathematics.
Nicotine
adenine dinucleotide (NADH) is the most prevalent donor of reducing potentials
in all living cells, and it donates its reducing equivalents to a number
of flavoproteins (FP) so that they can be used to pump protons and generate
ATP in the electron transport chain. The reduced form of NADH and the
oxidized form of FP are both fluorescent, so variations in the NADH and
FP fluorescence signal can be used as sensitive indicators of the metabolic
load on cells. In the 2-photon fluorescence microscope, these fluorophores
can be excited simultaneously at low light levels, and can be used to
nondestructively detect energetic load on cells.
In neuronal
activation, a variety of physiological events contribute to an increased
metabolic load on cells. We are using 2-photon fluorescence microscopy
of excitable cells in culture, together with statistical image processing
techniques, to detect activation of PC12 cells in culture under a variety
of different conditions designed to isolate these physiological events,
and decipher their metabolic signatures. We are then applying these data
to analyze the response of the olfactory apparatus of living zebrafish
(D. rerio) embryos to mixtures of olfactants.
In this manner,
we intend to establish a functional imaging technique that can be used
to study task-specific activation of neural circuits at the cellular level,
and with vastly improved spatial and temporal resolution. It is anticipated
that this technique will be broadly applicable to functional studies of
neuronal systems in a variety of different animals.
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