In-Depth Activation of Channelrhodopsin 2-Sensitized Excitable Cells with High Spatial Resolution Using Two-Photon Excitation with a Near-Infrared Laser Microbeam

We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a...

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Bibliographic Details
Published in:Biophysical journal 2008-10, Vol.95 (8), p.3916-3926
Main Authors: Mohanty, Samarendra K., Reinscheid, Rainer K., Liu, Xiaobin, Okamura, Naoe, Krasieva, Tatiana B., Berns, Michael W.
Format: Article
Language:English
Subjects:
Activation
Animals
Atoms & subatomic particles
Biophysics
Cell Line
Cellular
Cellular biology
Channelrhodopsins
Excitation
Fluorescence
Humans
Infrared radiation
Infrared Rays
Ion Channel Gating - radiation effects
Lasers
Mathematical models
Mice
Mice, Inbred C57BL
Microorganisms
Microscopy, Confocal
Neurons - cytology
Neurons - metabolism
Organic Chemicals - metabolism
Photobiophysics
Photons
Spatial resolution
Wavelengths
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Summary:We used two-photon excitation with a near-infrared (NIR) laser microbeam to investigate activation of channelrhodopsin 2 (ChR2) in excitable cells for the first time to our knowledge. By measuring the fluorescence intensity of the calcium (Ca) indicator dye, Ca orange, at different wavelengths as a function of power of the two-photon excitation microbeam, we determined the activation potential of the NIR microbeam as a function of wavelength. The two-photon activation spectrum is found to match measurements carried out with single-photon activation. However, two-photon activation is found to increase in a nonlinear manner with the power density of the two-photon laser microbeam. This approach allowed us to activate different regions of ChR2-sensitized excitable cells with high spatial resolution. Further, in-depth activation of ChR2 in a spheroid cellular model as well as in mouse brain slices was demonstrated by the use of the two-photon NIR microbeam, which was not possible using single-photon activation. This all-optical method of identification, activation, and detection of ChR2-induced cellular activation in genetically targeted cells with high spatial and temporal resolution will provide a new method of performing minimally invasive in-depth activation of specific target areas of tissues or organisms that have been rendered photosensitive by genetic targeting of ChR2 or similar photo-excitable molecules.
ISSN:0006-3495
1542-0086
DOI:10.1529/biophysj.108.130187