Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein

Science's STKE : Signal Transduction Knowledge Environment
Satoshi ShimozonoAtsushi Miyawaki

Abstract

Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca(2+) imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca(2+) using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet las...Continue Reading

References

Aug 1, 1997·The Biochemical Journal·G R BrownP O Berggren
Oct 6, 1998·Annual Review of Biochemistry·R Y Tsien
Mar 3, 1999·Proceedings of the National Academy of Sciences of the United States of America·A MiyawakiR Y Tsien
Mar 15, 2001·Proceedings of the National Academy of Sciences of the United States of America·T NagaiA Miyawaki
Sep 5, 2001·The EMBO Journal·V RobertT Pozzan

❮ Previous
Next ❯

Citations

Oct 23, 2008·Brain Cell Biology·Takashi FukanoAtsushi Miyawaki
Mar 30, 2004·Biochemical and Biophysical Research Communications·Takashi FukanoAtsushi Miyawaki
Apr 15, 2011·Journal of Visualized Experiments : JoVE·Askar M Akimzhanov, Darren Boehning
Dec 4, 2013·Proceedings of the National Academy of Sciences of the United States of America·Pamela J SungMark R Philips
Jan 15, 2013·American Journal of Physiology. Heart and Circulatory Physiology·Jong J KimGuy Salama
Dec 20, 2005·Biochemical and Biophysical Research Communications·Takashi FukanoAtsushi Miyawaki
Jun 17, 2015·Current Opinion in Chemical Biology·Gary Yellen, Rebecca Mongeon
Aug 27, 2011·Chemistry & Biology·Hiroyuki KatayamaAtsushi Miyawaki
Jun 19, 2012·Current Biology : CB·Pawel E FerdekOleg V Gerasimenko
Dec 14, 2004·Biochimica Et Biophysica Acta·Katiuscia BianchiRosario Rizzuto
Apr 5, 2003·Science·Cornelis J Weijer

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Proceedings of the National Academy of Sciences of the United States of America
T NagaiAtsushi Miyawaki
Growth Hormone & IGF Research : Official Journal of the Growth Hormone Research Society and the International IGF Research Society
Thomas K Graves, Patricia M Hinkle
© 2022 Meta ULC. All rights reserved