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Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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Published on: March 15, 2018

Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator.

Carsten Tischbirek¹, Antje Birkner¹, Hongbo Jia¹

¹Institute for Neuroscience, Technische Universität München, 80802 Munich, Germany; Munich Cluster for Systems Neurology, 80802 Munich, Germany; Center for Integrated Protein Sciences, 80802 Munich, Germany.

Proceedings of the National Academy of Sciences of the United States of America

|August 26, 2015

Summary

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This study shows that the red-shifted Cal-590 dye enables deep-tissue in vivo Ca2+ imaging in all mouse cortex layers using two-photon microscopy. It also allows for multicolor imaging with other calcium indicators like OGB-1.

Area of Science:

Neuroscience
Biophysics
Optical Imaging

Background:

In vivo Ca2+ imaging in deep cortical layers is challenging due to photon scattering and absorption in brain tissue.
Two-photon microscopy's depth penetration is limited, hindering the study of deeper neuronal populations.
Red-shifted fluorescent dyes offer a potential solution by reducing photon scattering at longer wavelengths.

Purpose of the Study:

To evaluate the red-shifted fluorescent Ca2+ indicator Cal-590 for in vivo two-photon imaging in deep cortical layers of the mouse brain.
To assess Cal-590's suitability for recording action potential-evoked Ca2+ transients at significant depths.
To determine if Cal-590 can be used for simultaneous multicolor functional imaging with other Ca2+ indicators.

Main Methods:

Keywords:

calcium imaging mouse cortical circuits multicolor functional imaging neuronal activity

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Bulk loading of mouse cortex neurons with the acetoxymethyl (AM) ester version of Cal-590.

Combined two-photon imaging and cell-attached recordings in vivo.

Testing Cal-590 in combination with Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-1 (OGB-1) for multicolor imaging.

Main Results:

Cal-590 successfully detected single-action potential-evoked Ca2+ transients in neurons across all six cortical layers, reaching depths of up to -900 µm.
Good signal-to-noise ratios were achieved despite Cal-590's relatively low Ca2+ affinity (Kd=561 nM).
Simultaneous multicolor imaging was demonstrated by recording Ca2+ transients in Cal-590-labeled cells and an OGB-1-labeled neuron.

Conclusions:

The red-shifted Ca2+ indicator Cal-590 is effective for in vivo two-photon Ca2+ imaging throughout all layers of the mouse cortex.
Cal-590 facilitates deep-tissue imaging, overcoming limitations of photon scattering.
Cal-590 is suitable for multicolor functional imaging when combined with spectrally distinct indicators like OGB-1.