Fiber-optic two-photon optogenetic stimulation
Spotlight summary: Optogenetics is a rapidly evolving field that enables optical control of genetically targeted biological systems at both high temporal and spatial resolution. The combination of optics, genetics, and bioengineering technology can be used to stimulate or inhibit cellular activity through light-sensitive membrane proteins termed opsins. Optical control of cellular activity has significant advantages over conventional techniques such as electrical stimulation and other manipulation techniques with the most distinct advantage being that optogenetic cellular control is far less invasive to the biological system under study. Most opsins have excitation maximum in the visible spectrum, making tissue penetration difficult due to significant absorption and scattering from the tissue chromophores. In 2008 two-photon optogenetic stimulation (TPOS) of opsin containing cells and brain slices was demonstrated by Mohanty, et al. TPOS has much greater penetration depth than single photon optogenetic stimulation as near-infrared (NIR) light is used for excitation of the opsins, improving precision of the optical stimulation. Since the technique was published, numerous applications for probing neural circuitry have been published, but to date TPOS requires use of a microscope objective and complex scanning beam geometries. This method is not advantageous for TPOS of large tissue areas as raster scanning is relatively slow and microscope objectives are bulky.
In the work by Dhakal, et al a method was developed for TPOS using a non-scanning method based on a multimode fiber and was demonstrated to facilitate fiber-optic two-photon optogenetic stimulation (FO-TPOS) of cells. Cells were transfected with channelrhodopsin-2 (ChR2), which has been estimated to have a two-photon cross section larger than most fluorophores, and thus has the potential for efficient stimulation by TPOS in dense tissues. A full description of the multi-modal fiber construction and characterization is given in the manuscript. Following validation of the multi-modal fiber for two-photon excitation and intensity-dependent characterization HEK 293 cells containing ChR2 were utilized to test the FO-TPOS capabilities of the instrument. Nonlinear interaction of the laser beam within the ChR2 expressing cells was confirmed and the peak two-photon activation spectrum was observed to be around 850 nm. Initial characterization of the multimode fiber two-photon activation efficacy at different NIR laser intensities was completed, but further studies will be required for optimization of the multimode FO-TPOS strategy. However, the significant two-photon cross-section of ChR2 should enable use of a nanosecond and possibly a microsecond compact NIR laser source for FO-TPOS. This technology will permit in depth probing experiments of neural circuitry in vivo as FO-TPOS permits minimally invasive, precise anatomical delivery of stimulation.
Technical Division: Optics in Biology and Medicine
ToC Category: Medical Optics and Biotechnology
|OCIS Codes:||(120.3890) Instrumentation, measurement, and metrology : Medical optics instrumentation|
|(170.0170) Medical optics and biotechnology : Medical optics and biotechnology|
|(170.0180) Medical optics and biotechnology : Microscopy|
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