Imaging Intracellular ATP Dynamics Using FRET-Based Sensors in an Organotypic Mouse Hippocampal Slice

Abstract

Source: Lerchundi, R., et. al. Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK. J. Vis. Exp. (2019)

This video demonstrates the use of FRET(Fluorescence Resonance Energy Transfer)-based sensors to measure intracellular ATP levels in organotypic mouse hippocampal slices, enabling real-time visualization of ATP dynamics in neurons and astrocytes under physiological and experimental conditions.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. FRET-based ATP Imaging (Figure 1)

1. Before the experiment, prepare E-ACSF and bubble it with 95% O₂/5% CO₂ for at least 30 min to obtain a pH of 7.4. Switch on the fluorescent light source (Xenon lamp) of the monochromator (Figure 1). St....

Results

Figure 1: Configuration of the FRET imaging setup. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-deoxyglucose	Alfa Aesar	L07338	Non-metabolizable glucose analog
Band pass filters 483/32	AHF Analysentechnik AG		Splitter compatible emmision filter
Band pass filters 542/27	AHF Analysentechnik AG		Splitter compatible emmision filter
Beamsplitter T 455 LP	AHF Analysentechnik AG		Excitation dichroic mirror
Beamsplitter T 505 LPXR	AHF Analysentechnik AG		Splitter dichroic
Data processing Origin Pro 9.0.0 (64-bit)	OriginLab corporation		Scientific graphing and data analysis software
D-glucose monohydrate	Caelo	2580-1kg
DPBS	GIBCO/Life	14190250	Dulbecco's phosphate-buffered saline
Eclipse E 600FN upright microscope	Nikon Microscope Solutions
Eclipse FN1 upright microscope	Nikon Microscope Solutions
Experimental chamber	custom build		Perfusion chamber for live-cell imaging
Hanks' Balanced Salt solution	Sigma-Aldrich	H9394	With Phenol Red for pH monitoring
Minimum Essential Medium Eagle	Sigma-Aldrich	M7278	Synthetic cell culture media
Monochromator Polychrome V	Thermo Scientific/FEI		Ultra fast switching monochromator
Nikon Fluor 40x / 0.80 W DIC M ∞/0 WD 2.0	Nikon Microscope Solutions		Water Immersion Microscope Objective
NIS Elements 4.50 advanced Research	Nikon Microscope Solutions		Imaging software. Upgraded version for FRET imaging
ORCA-Flash4.0	Hamamatsu Photonics		Digital CMOS camera
Perfusion tubing	Pro Liquid GmbH		Tygon tubing, 1.52 x 322 mm (Wd: 0.85)
Photoshop CS 6 Version 13.0	Adobe		Image processing software
ssAAV-2/2-hSyn1-ATeam1.03YEMK-WPRE-hGHp(A)	ETH Zürich	v244	Single-stranded AAV vector that induces the expression of ATeam1.03YEMK under the control of the human synapsin 1 promoter fragment hSyn1.
ssAAV-5/2-hGFAP-hHBbI/E-ATeam1.03YEMK-WPRE-bGHp(A)	ETH Zürich	v307	Single-stranded AAV vector that induces the expression of ATeam1.03YEMK under the control of the human glial fibrillary acidic protein promoter fragment ABC1D.