Novel optical methods for the study of dendritic integration in CNS neurons
A high-resolution optical recording method is presented that allows a spatially-resolved and quantitative characterization of membrane potential dynamics from small dendritic structures in CNS neurons. This approach is termed “quantitative, high-speed, random-access, laser-scanning fluorescence microscopy” and combines (i) a novel scanning microscope based on an acousto-optic deflection mechanism; and (ii) a ratiometric fluorescence recording scheme based on simultaneously sampling two emission wavelengths. This instrument has five main features that distinguish it from other comparable instruments currently in use. These advantages include a high-resolution random-access scanning capability (i.e., 12-bit spot positioning resolution), very high temporal resolution (<0.5 ms), high spatial resolution (2 μm spot size), high amplitude resolution (16-bit digitization) and multiple noise reduction features. Furthermore, this microscope is unique because it incorporates laser illumination in a manner compatible with long-term optical recording (i.e., high peak illumination intensity but low average intensity at a small number of recording sites). Together these properties facilitate experiments with voltage-sensitive dyes that require multi-site recording with high signal fidelity. The voltage-dependent spectral shift in the emission of di-8-ANEPPS, a voltage-sensitive dye, was used to generate a ratiometric and therefore quantitative measure of membrane potential. For this purpose, di-8-ANEPPS was excited at 476 nm and its emission simultaneously sampled in two spectral bands (540 ± 25 nm and 600 ± 30 nm) that were separated by secondary dichroic beamsplitting. Signals from each of these spectral bands exhibited a similar voltage sensitivity but varied in opposite directions. The ratio of these two signals demonstrated strong similarity to concurrent current-clamp recordings or voltage-clamp command waveforms. This ratiometric parameter was quantified with a predetermined calibration factor and an offset (determined empirically for each point). The accuracy, precision and holding potential independence of this type of membrane potential measurement, were experimentally verified with concurrent patch clamp recordings under conditions that maximized voltage-clamp control. In summary, this method allows quantitative determinations of both resting membrane potential and fast voltage transients with a better signal-to-noise ratio, higher temporal resolution and a superior level of voltage discrimination (∼5mV) than previous studies.
Neurology|Biophysics|Biomedical research|Electrical engineering
Bullen, Andrew Robert, "Novel optical methods for the study of dendritic integration in CNS neurons" (1999). Dissertations available from ProQuest. AAI9937705.