Mar 10, 2025

Public workspaceDIO-SPOTlight Live 2-Photon Imaging v.250227 

DOI
dx.doi.org/10.17504/protocols.io.bp2l6dj35vqe/v1
  • Matt Oliver1,2,
  • Victoria Hall1,2
  • 1Calakos Lab;
  • 2Duke University
Matt Oliver
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DOI: dx.doi.org/10.17504/protocols.io.bp2l6dj35vqe/v1
Protocol Citation: Matt Oliver, Victoria Hall 2025. DIO-SPOTlight Live 2-Photon Imaging v.250227 . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dj35vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 27, 2025
Last Modified: March 10, 2025
Protocol Integer ID: 123548
Keywords: ASAPCRN, DIO-SPOTlight 2-photon
Funders Acknowledgements:
ASAP
Grant ID: ASAP-020607
Abstract
This protocol contains the steps taken for live tissue preparation and two-photon imaging.
Prepare acute brain slices
Prepare acute brain slices
Anesthetize mouse and perform intracardial perfusion with ice cold N-methyl-D-glucamine (NMDG)-based artificial cerebrospinal fluid (ACSF).
a. ACSF (90 mM NMDG, 2.5 mM KCl, 1.2 mM NaH2PO4, 35 mM NaHCO3, 20 mM HEPES, 25 mM glucose, 5 mM sodium ascorbate, 2 mM thiourea, 3 mM sodium pyruvate, 10 mM MgSO4 heptahydrate, 0.5 mM CaCl2 dihydrate, and saturated with 95% O2 and 5% CO2)
Quickly remove the brain and slice into 250 um sagittal slices using a vibratome.
Immediately transfer slices to a recovery chamber containing NMDG ACSF for 10 to 15 min at 32°C.
Transfer heat recovered slices to a holding chamber containing ACSF and let rest for approximately 1 hour
a. ACSF (124 mM NaCl, 2.5 mM KCl, 1 mM MgCl2,2 mM CaCl2, 26 mM NaHCO3, 1.2 mM NaH2PO4, and 10 mM glucose, saturated with 95% O2 and 5% CO2, pH 7.4, 300 mOsm/l)
Two-photon imaging
Two-photon imaging
Place the slices into a perfusion chamber and superfuse with oxygenated ACSF.
Image slices using a Bruker Investigator Multiphoton Imaging System or equivalent.
Set the emitted light wave-length to 920 nm on the Coherent Chameleon I Ti:Sapphire femtosecond laser.
a. Keep laser power between 35 and 50 mW
Set up image stack using galvo-galvo scanning with frame rate of 1 Hz with 3-4 µm depth increments at either 1X or 4X optical zoom.
Collect fluorescence signal using a 16X W objective and Quad GaAsP photomultiplier tubes (PMTs).
Split the SPOTlight red and green channels using a 565lpxr dichroic mirror followed by a 525/70 bandpass PMT filter.