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L. Jin,
B. J. Baker,
Lawrence B. Cohen,
H. Mutoh,
D. Dimitrov,
A. Perron,
Y. Iwamoto,
E. Y. Isacoff,
V. A. Pieribone,
T. Hughes,
T. Knöpfel, W. Akemann
[show abstract]
[hide abstract]
ABSTRACT: Imaging activity of neurons in intact brain tissue was conceived several decades ago, and, after many years of development,
voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living
brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors
of membrane potential. These fluorescent protein voltage sensors overcome some drawbacks of organic voltage-sensitive dyes,
such as nonspecificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a
genetically encoded sensor could, in principle, be expressed specifically in any cell type and would have the advantage of
staining only the cell population determined by the specificity of the promoter used to drive expression. Here we, critically
review the current status of these developments.
11/2009: pages 27-43;
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B J Baker,
H Mutoh,
D Dimitrov, W Akemann,
A Perron,
Y Iwamoto,
L Jin,
L B Cohen,
E Y Isacoff,
V A Pieribone,
T Hughes,
T Knöpfel
[show abstract]
[hide abstract]
ABSTRACT: Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.
Brain Cell Biology 09/2008; 36(1-4):53-67. · 3.25 Impact Factor
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[show abstract]
[hide abstract]
ABSTRACT: New variants of green fluorescent protein (GFP) can be engineered by circular permutation of their amino acid sequence. We characterized a series of permuted enhanced GFP (PEGFP) with new termini introduced at N144-Y145 and linkers of 1, 3, 5 and 6 residues inserted between G232 and M1, as well as a variant with an extended 7-residues linker between K238 and M1. A minimum linker length of 3 residues was necessary for a functional chromophore to be formed, and linkers exceeding 4 residues yielded almost the same fluorescence quantum yield as enhanced GFP (EGFP). PEGFP exhibited dual-wavelength absorption and fluorescence excitation with peaks at 395 and 490 nm but single-wavelength emission at 512 nm. Fluorescence emission increased with increasing pH for all excitation wavelengths with a pKa of 7.7. Between the pH values of 6 and 8 optical absorption showed an isobestic point at 445 nm. PEGFP rapidly denatured in urea between 50 and 60 degrees C. Renaturation proceeded with a short (approximately 29 s) and a longer (> 150 s) time constant. Transient transfection of HEK293 and HeLa cells revealed the expression dynamics of PEGFP to be similar to that of EGFP. Laser-scanning microscopy of HeLa cells demonstrated that the PEGFP are particularly well suited as fluorescent indicators in two-photon imaging.
Photochemistry and Photobiology 09/2001; 74(2):356-63. · 2.41 Impact Factor
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[show abstract]
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ABSTRACT: Interest in non-invasive methods for optical probing of neuronal electrical activity has been ongoing for several decades and methods for imaging the activity of single or multiple individual neurons in networks composed of thousands of neurons have been developed. Most widely used are techniques that use organic chemistry-based dyes as indicators of calcium and membrane potential. More recently a new generation of probes, genetically encoded fluorescent protein sensors, have emerged for use by physiologists studying the operation of neuronal circuits. In this review we describe the advance of these emerging optical techniques and compare them with more conventional approaches.
Neurosignals 08/1970; 16(4):289-299. · 2.11 Impact Factor
