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Deep two-photon brain imaging with a red-shifted fluorometric Ca2+ indicator
Contributed by Bert Sakmann, July 27, 2015 (sent for review May 20, 2015)

Significance
We introduce a two-photon imaging method with improved depth penetration for the recording of neuronal activity with single-cell resolution in the intact brain of living animals. This method relies on the use of the fluorometric Ca2+-sensitive dye Cal-590, which is effectively excited by infrared light (1,050 nm). By combining population Ca2+ imaging and electrical recordings in vivo, we demonstrate that neuronal activity can be monitored in all six layers of the mouse cortex. In combination with spectrally different Ca2+ indicators, Cal-590 can be used for the simultaneous imaging of neuronal activity in distinct neuronal populations.
Abstract
In vivo Ca2+ imaging of neuronal populations in deep cortical layers has remained a major challenge, as the recording depth of two-photon microscopy is limited because of the scattering and absorption of photons in brain tissue. A possible strategy to increase the imaging depth is the use of red-shifted fluorescent dyes, as scattering of photons is reduced at long wavelengths. Here, we tested the red-shifted fluorescent Ca2+ indicator Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving bulk loading of neurons with the acetoxymethyl (AM) ester version of Cal-590, combined two-photon imaging and cell-attached recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ (Kd = 561 nM), single-action potential-evoked Ca2+ transients were discernable in most neurons with a good signal-to-noise ratio. Action potential-dependent Ca2+ transients were recorded in neurons of all six layers of the cortex at depths of up to −900 µm below the pial surface. We demonstrate that Cal-590 is also suited for multicolor functional imaging experiments in combination with other Ca2+ indicators. Ca2+ transients in the dendrites of an individual Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-1 (OGB-1)-labeled neuron and the surrounding population of Cal-590-labeled cells were recorded simultaneously on two spectrally separated detection channels. We conclude that the red-shifted Ca2+ indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of mouse cortex. In combination with spectrally different Ca2+ indicators, such as OGB-1, Cal-590 can be readily used for simultaneous multicolor functional imaging experiments.
Footnotes
↵1C.T. and A.B. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: bsakmann{at}neuro.mpg.de or arthur.konnerth{at}tum.de.
Author contributions: C.T., A.B., H.J., B.S., and A.K. designed research; C.T., A.B., and A.K. performed research; A.B. and H.J. contributed new reagents/analytic tools; C.T., A.B., H.J., B.S., and A.K. analyzed data; and C.T., A.B., H.J., B.S., and A.K. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1514209112/-/DCSupplemental.
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