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Related Experiment Video

Updated: Mar 9, 2026

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice
08:10

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Published on: March 13, 2008

29.7K

Improved deep two-photon calcium imaging in vivo.

Antje Birkner1, Carsten H Tischbirek1, Arthur Konnerth1

  • 1Institute of Neuroscience, Technical University of Munich, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy) and Center for Integrated Protein Sciences (CIPSM), Munich, Germany.

Cell Calcium
|December 29, 2016
PubMed
Summary

This study presents a simple method to improve depth penetration for two-photon calcium imaging in the mouse brain. The optimized protocol allows functional imaging of all cortical layers in awake, behaving animals.

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Area of Science:

  • Neuroscience
  • Biophysics
  • Optical Imaging

Background:

  • Two-photon laser scanning calcium imaging is crucial for studying neural function and structure in vivo.
  • Current limitations include shallow depth penetration (few hundred micrometers) due to light scattering and aberrations.
  • Existing enhancement methods often require new instrumentation or complex protocols.

Purpose of the Study:

  • To present a simple, readily implementable approach to enhance depth penetration for standard two-photon microscopes.
  • To enable functional imaging of neuronal populations across all cortical layers in vivo.
  • To demonstrate the feasibility of deep-layer recordings in awake, behaving animals.

Main Methods:

  • Utilized an optimized protocol for depth-restricted labeling with the red-shifted fluorescent calcium indicator Cal-590.
Keywords:
Calcium signalingMouse visual cortexMulti-photon microscopyNeuronal activity

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Related Experiment Videos

Last Updated: Mar 9, 2026

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice
08:10

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

Published on: March 13, 2008

29.7K
Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices
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Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window
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In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

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  • Leveraged ultra-short laser pulses for improved imaging.
  • Applied the method to standard two-photon microscopes without requiring new hardware.

    • Main Results:

    • Achieved in vivo functional imaging of neuronal populations with single-cell resolution in all six layers of the mouse cortex.
    • Demonstrated stable recordings in deep cortical layers of awake, behaving mice.
    • The approach overcomes limitations of shallow penetration in standard two-photon microscopy.

    Conclusions:

    • The presented method offers a simple and effective way to enhance depth penetration in two-photon calcium imaging.
    • This technique allows for comprehensive functional analysis of the entire cortical depth in vivo.
    • The improved depth penetration is expected to benefit future studies in larger animal models, including non-human primates.