Fiber-optic TPOS technology will allow
in-depth probing of neural circuitry in
vivo since it permits minimally invasive
and more precise anatomical delivery
of stimulation. Since the same NIR fs
laser microbeam can be used for targeted
transfection of opsin-coding genes and
for two-photon imaging of the opsin-expression pattern, targeted modulation of
their activity by the NIR TPOS will allow
in-depth probing of the neural circuitry
in a minimally invasive manner. 1, 3, 4 NIR
phase-sensitive measurements and voltage-sensitive or calcium imaging of the neural
activity will allow all-optical evaluation of
the functional connectivity of the complex
neural circuitry of the brain. 1, 5
Functional connections between billions of neurons in central and peripheral nervous
systems enable thousands of specialized
functions, from sensory to motor activities.
This has created significant priorities in the
scientific community to develop precise
stimulation and imaging tools for probing
complex neural circuitry. We demonstrated a
two-photon optogenetic stimulation (TPOS)
of excitable cells and brain slices using scanning near-infrared (NIR) femtosecond laser
beams five years ago. 1 Since then, applications of this method for probing neural
circuitry have been growing.
TPOS using NIR laser beams provide
deeper penetration than conventional
single-photon optogenetic stimulation due to
low absorption and scattering coefficients of
tissue in the NIR spectral region. 2 Further,
high spatial precision, important for activating sub-cellular structures, is achieved by
virtue of the non-linear nature of ultrafast
light interaction with the opsins. However,
bulky microscope objectives and scanning
methods used for a TPOS restricts application
of this technology to in vitro preparations.
In order to modulate and probe neural
circuitry in freely behaving animals, we
developed a multimode fiber-optic TPOS and
demonstrated it in a non-scanning manner. 3
The dependence of a two-photon-induced
inward current as a function of incident
average-power densities has been measured
using a patch clamp. In contrast to the
expected nonlinear response of the inward
current, the fiber-optic TPOS-induced current
in channelrhodopsin-2-sensitized cells was
found to be linearly dependent on the incident laser-power density as it is a response to
activation of channelrhodopsin- 2 rather than
a direct measure of emitted photons, unlike
two-photon fluorescence emission.
Conventional two-photon optogenetic stimulation (TPOS) scanning pattern
delivered by objective. (b) Fiber-optic TPOS. (c) Composite image of two-photon
fluorescence (green) and excitation intensity pattern (red) from fiber-optic beam.
(d) Comparison of in-depth and precise stimulation by TPOS (left) vs. invasive
and less-accurate visible beam (right).
Kamal Dhakal, Ling
Gu, Bryan J. Black and
Samarendra K. Mohanty
University of Texas at Arlington, Texas, U.S.A.
1. S.K. Mohanty et al. Bio-phys. J. 95, 3916 (2008).
2. E.S. Boyden et al. Nat.
Neurosci. 8, 1263 (2005).
3. K. Dhakal et al. Opt. Lett.
38, 1927 (2013).
4. L. Gu et al. J. Biomed.
Opt. 16, 128003 (2011).
5. N. Choudhury et al.
Comp. Biomed. 1, DOI:
Non-Scanning Multimode Beam
Led to Optogenetic Stimulation