Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging.
Nat Protoc
; 13(3): 581-597, 2018 03.
Article
em En
| MEDLINE
| ID: mdl-29470463
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 takes â¼2 d, and OGB-1 loading, imaging, and data analysis take 2 d.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Cálcio
/
Imagem Óptica
/
Líquido Intracelular
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article