Dataset Information

Spectral unmixing: analysis of performance in the olfactory bulb in vivo.

ABSTRACT:

Background

The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.

Methodology/principal findings

Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180-250 microm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.

Conclusions/significance

Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.

SUBMITTER: Ducros M

PROVIDER: S-EPMC2635473 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Publications

Spectral unmixing: analysis of performance in the olfactory bulb in vivo.

Ducros Mathieu M Moreaux Laurent L Bradley Jonathan J Tiret Pascale P Griesbeck Oliver O Charpak Serge S

PloS one 20090209 2

<h4>Background</h4>The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.<h4>Methodology/principal findings</h4>Using fl ...[more]

PMID: 19198655

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Project description:Here we present a detailed statistical analysis of the discharge characteristics of mitral cells of the main olfactory bulb of urethane-anesthetized rats. Neurons were recorded from the mitral cell layer, and antidromically identified by stimuli applied to the lateral olfactory tract. All mitral cells displayed repeated, prolonged bursts of action potentials typically lasting >100 sec and separated by similarly long intervals; about half were completely silent between bursts. No such bursting was observed in nonmitral cells recorded in close proximity to mitral cells. Bursts were asynchronous among even adjacent mitral cells. The intraburst activity of most mitral cells showed strong entrainment to the spontaneous respiratory rhythm; similar entrainment was seen in some, but not all nonmitral cells. All mitral cells displayed a peak of excitability at ~25 msec after spikes, as reflected by a peak in the interspike interval distribution and in the corresponding hazard function. About half also showed a peak at about 6 msec, reflecting the common occurrence of doublet spikes. Nonmitral cells showed no such doublet spikes. Bursts typically increased in intensity over the first 20-30 sec of a burst, during which time doublets were rare or absent. After 20-30 sec (in cells that exhibited doublets), doublets occurred frequently for as long as the burst persisted, in trains of up to 10 doublets. The last doublet was followed by an extended relative refractory period the duration of which was independent of train length. In cells that were excited by application of a particular odor, responsiveness was apparently greater during silent periods between bursts than during bursts. Conversely in cells that were inhibited by a particular odor, responsiveness was only apparent when cells were active. Extensive raw (event timing) data from the cells, together with details of those analyses, are provided as supplementary material, freely available for secondary use by others.

Dataset Information

Spectral unmixing: analysis of performance in the olfactory bulb in vivo.

Background

Methodology/principal findings

Conclusions/significance

Publications

Spectral unmixing: analysis of performance in the olfactory bulb in vivo.

Similar Datasets

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