Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging

Nature Protocols
Kaiyu ZhengDmitri A Rusakov

Abstract

Nanomolar-range fluctuations of intracellular [Ca2+] are critical for brain cell function but remain difficult to measure. We have advanced a microscopy technique to monitor intracellular [Ca2+] in individual cells in acute brain slices (also applicable in vivo) using fluorescence lifetime imaging (FLIM) of the Ca2+-sensitive fluorescent indicator Oregon Green BAPTA1 (OGB-1). The OGB-1 fluorescence lifetime is sensitive to [Ca2+] within the 10-500 nM range but not to other factors such as viscosity, temperature, or pH. This protocol describes the requirements, setup, and calibration of the FLIM system required for OGB-1 imaging. We provide a step-by-step procedure for whole-cell OGB-1 loading and two-photon FLIM. We also describe how to analyze the obtained FLIM data using total photon count and gated-intensity record, a ratiometric photon-counting approach that provides a highly improved signal-to-noise ratio and greater sensitivity of absolute [Ca2+] readout. We demonstrate our technique in nerve cells in situ, and it is adaptable to other cell types and fluorescent indicators. This protocol requires a basic understanding of FLIM and experience in single-cell electrophysiology and cell imaging. Setting up the FLIM system take...Continue Reading

References

Apr 6, 1990·Science·W DenkW W Webb
Dec 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·J Lisman
Jan 1, 1989·Annual Review of Neuroscience·R Y Tsien
Mar 1, 1968·The Journal of Physiology·B Katz, R Miledi
Jul 1, 1980·Brain Research Bulletin·T J Teyler
Oct 1, 1994·Journal of Neuroscience Methods·W DenkR Yuste
May 20, 1999·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·S A SchnellM W Wessendorf
Nov 29, 2001·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·W BaschongR H Laeng
Feb 19, 2002·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Michael Neumann, Detlef Gabel
Aug 26, 2003·Nature Neuroscience·Ryohei YasudaKarel Svoboda
Feb 12, 2004·Science's STKE : Signal Transduction Knowledge Environment·Ryohei YasudaKarel Svoboda
Dec 1, 2004·Journal of Biomedical Optics·A V AgronskaiaH C Gerritsen
May 13, 2006·Cell Calcium·Christian D WilmsJens Eilers
Jun 30, 2006·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Ricardo Scott, Dmitri A Rusakov
Apr 5, 2007·Nature Protocols·Jenny T DavieMichael Häusser
May 10, 2007·Nature Protocols·Josef BischofbergerPeter Jonas
Aug 3, 2007·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Christoph Schmidt-HieberJosef Bischofberger
Nov 9, 2007·Annual Review of Physiology·Donald M Bers
Dec 14, 2007·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Philip A WilliamsBen W Strowbridge
Aug 1, 2008·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Ricardo ScottDmitri A Rusakov
Oct 22, 2008·Methods : a Companion to Methods in Enzymology·R Madelaine ParedesJames D Lechleiter
Jul 21, 2009·Trends in Neurosciences·Gertrudis PereaAlfonso Araque
Nov 26, 2009·Nature Methods·Christian D Wilms, Michael Häusser
Feb 13, 2010·Annual Review of Physiology·Michael M Halassa, Philip G Haydon
May 25, 2010·Nature Neuroscience·Eiji ShigetomiBaljit S Khakh
Dec 21, 2010·Nature Neuroscience·Jason M ChristieCraig E Jahr
Jan 25, 2011·Nature Neuroscience·Guido C FaasIstvan Mody
Jan 29, 2011·Aging Cell·Ulyana LaloYuriy Pankratov
Sep 13, 2011·Nature Neuroscience·Maria Amalia Di CastroAndrea Volterra
Mar 13, 2012·Neuron·Christine Grienberger, Arthur Konnerth
Jan 29, 2013·Biochimica Et Biophysica Acta·Vadim Pérez Koldenkova, Takeharu Nagai
Mar 9, 2013·Proceedings of the National Academy of Sciences of the United States of America·Sergiy SylantyevDmitri A Rusakov
Jul 23, 2013·Nature·Tsai-Wen ChenDouglas S Kim
Oct 18, 2013·Frontiers in Neural Circuits·Yoshibumi UedaYasunori Hayashi

❮ Previous
Next ❯

Citations

Oct 11, 2019·The Journal of General Physiology·Jan MeyerChristine R Rose
Aug 4, 2018·Frontiers in Veterinary Science·Laura Lossi, Adalberto Merighi
Jan 22, 2019·Frontiers in Bioengineering and Biotechnology·Hideaki FujitaMadoka Suzuki
Feb 6, 2020·Biomedical Optics Express·Rajannya SenDmitri Papkovsky
Nov 25, 2020·Current Protocols in Neuroscience·Daniel J LiputSteven S Vogel
Feb 17, 2021·Journal of Muscle Research and Cell Motility·S O KarakhimD V Sinko
Dec 11, 2021·Nature Communications·Franka H van der LindenJoachim Goedhart

❮ 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.