PMID-16050036 Imaging brain activity with voltage- and calcium-sensitive dyes.
- Voltage-sensitive dyes are well suited for measuring synaptic integration, as:
- Electrodes are too blunt to effectively record these fine, < 1um diameter structures.
- The surface area to volume ratio is highest in the dendrites
- Voltage-sensitive dyes also permeate internal membranes not subject to voltage gradients, hence this does not contribute to the signal, leading to a decreased .
- Dominant experimental noise is shot noise, statistical -- see {1181}.
- modern dyes and imagers can reliably record single action potentials; spatial averaging yields similar resolution as electrical recording.
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- They performed optical recording of Aplysia sensory ganglia, and discovered following light tail touch: "It is almost as if the Aplysia nervous system is designed such that every cell in the abdominal ganglion cares about this (and perhaps every) sensory stimulus. In addition, more than 1000 neurons in other ganglia are activated by this touch..."
- These results force a more pessimistic view of the present understanding of the neuronal basis of apparently simple behaviors in relatively simple nervous systems.
- Used calcium imaging on olfactory glomeruli of mice and turtles; measurements were limited by either shot-noise or heart/breathing artifacts.
- Confocal and two-photon microscopes, due to their exchange of spatial resolution for sensitivity, are not useful with voltage-sensitive dyes.
- The generation of fluorescent photons in the 2-photon confocal microscope is not efficient. We compared the signals from Calcium Green-1 in the mouse olfactory bulb using 2-photon and ordinary microscopy. In this comparison the number of photons contributing to the intensity measurement in the 2-photon confocal microscope was about 1000 times smaller than the number measured with the conventional microscope and a CCD camera.
- By the numbers, quote: Because only a small fraction of the 10e16 photons/ms emitted by a tungsten filament source will be measured, a signal-to-noise ratio of 10e8 (see above) cannot be achieved. A partial listing of the light losses follows. A 0.9-NA lamp collector lens would collect 0.1 of the light emitted by the source. Only 0.2 of that light is in the visible wavelength range; the remainder is infrared (heat). Limiting the incident wavelengths to those, which have the signal means, that only 0.1 of the visible light is used. Thus, the light reaching the
preparation might typically be reduced to 1013 photons/ms. If the light-collecting system that forms the image has high efficiency e.g., in an absorption measurement, about 1013 photons/ms will reach the image plane. (In a fluorescence measurement there will be much less light measured because 1. only a fraction of the incident photons are absorbed by the fluorophores, 2. only a fraction of the absorbed photons appear as emitted photons, and 3. only a fraction of the emitted photons are collected by the objective.) If the camera has a quantum efficiency of 1.0, then, in absorption, a total of 10e13 photoelectrons/ms will be measured. With a camera of 1000 pixels, there will be 10e10 photoelectrons/ms/pixel. The shot noise will be 10e5 photoelectrons/ms/pixel; thus the very best that can be expected is a noise that is 10e−5 of the resting light (a signal-to-noise ratio of 100 db). The extra light losses in a fluorescence measurement will further reduce the maximum obtainable signal-to-noise ratio.
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