Dataset Information

Highlighted Ca²? imaging with a genetically encoded 'caged' indicator.

ABSTRACT: Genetically encoded fluorescent indicators for bioimaging are powerful tools for visualizing biological phenomena in specified cell types or cellular compartments. However, available gene promoters or localization sequences are not applicable for visualizing all expression events. Furthermore, a visualization technique focusing on single cells or cellular compartments is required for characterizing specific cellular properties including individuality of cells in the cell population. To address these limitations, we developed a genetically encoded caged Ca(2+) indicator for which expression timing and location could be controlled. This indicator, PA-TNXL, comprises a Ca(2+)-binding protein and troponin between a photoactivatable FRET donor (PA-GFP) and a FRET quencher (dim variant of YFP). Ultraviolet irradiation activates the FRET Ca(2+) indicator. Using this indicator, we successfully imaged Ca(2+) dynamics in a given set of HeLa cells and cultured hippocampal neurons. This technology can be applied for developing other photoactivatable indicators, thereby opening a new area of biological research.

SUBMITTER: Matsuda T

PROVIDER: S-EPMC3593221 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Highlighted Ca²⁺ imaging with a genetically encoded 'caged' indicator.

Matsuda Tomoki T Horikawa Kazuki K Saito Kenta K Nagai Takeharu T

Scientific reports 20130101

Genetically encoded fluorescent indicators for bioimaging are powerful tools for visualizing biological phenomena in specified cell types or cellular compartments. However, available gene promoters or localization sequences are not applicable for visualizing all expression events. Furthermore, a visualization technique focusing on single cells or cellular compartments is required for characterizing specific cellular properties including individuality of cells in the cell population. To address t ...[more]

PMID: 23474844

Similar Datasets

Project description:SignificanceGenetically encoded calcium ion (Ca2+) indicators (GECIs) are powerful tools for monitoring intracellular Ca2+ concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca2+ concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited. Expanding the selection of available GECIs to include new colors and distinct photophysical properties could create new opportunities for in vitro and in vivo fluorescence imaging of neuronal activity. In particular, blue-shifted variants of GECIs are expected to have enhanced two-photon brightness, which would facilitate multiphoton microscopy.AimWe describe the development and applications of T-GECO1 - a high-performance blue-shifted GECI based on the Clavularia sp.-derived mTFP1.ApproachWe used protein engineering and extensive directed evolution to develop T-GECO1. We characterize the purified protein and assess its performance in vitro using one-photon excitation in cultured rat hippocampal neurons, in vivo using one-photon excitation fiber photometry in mice, and ex vivo using two-photon Ca2+ imaging in hippocampal slices.ResultsThe Ca2+-bound state of T-GECO1 has an excitation peak maximum of 468 nm, an emission peak maximum of 500 nm, an extinction coefficient of 49,300 M-1cm-1, a quantum yield of 0.83, and two-photon brightness approximately double that of EGFP. The Ca2+-dependent fluorescence increase is 15-fold and the apparent Kd for Ca2+ is 82 nM. With two-photon excitation conditions at 850 nm, T-GECO1 consistently enabled detection of action potentials with higher signal-to-noise (SNR) than a late generation GCaMP variant.ConclusionT-GECO1 is a high performance blue-shifted GECI that, under two-photon excitation conditions, provides advantages relative to late generation GCaMP variants.

Dataset Information

Highlighted Ca²? imaging with a genetically encoded 'caged' indicator.

Publications

Highlighted Ca²⁺ imaging with a genetically encoded 'caged' indicator.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets