Significant efforts are being made for control on axonal guidance due to its importance in nerve regeneration and in the
formation of functional neuronal circuitry in-vitro. These include several physical (topographic modification, optical
force, and electric field), chemical (surface functionalization cues) and hybrid (electro-chemical, photochemical etc)
methods. Here, we report comparison of the effect of linear flow versus microfluidic flow produced by an opticallydriven
micromotor in guiding retinal ganglion axons. A circularly polarized laser tweezers was used to hold, position and
spin birefringent calcite particle near growth cone, which in turn resulted in microfluidic flow. The flow rate and
resulting shear-force on axons could be controlled by a varying the power of the laser tweezers beam. The calcite
particles were placed separately in one chamber and single particle was transported through microfluidic channel to
another chamber containing the retina explant. In presence of flow, the turning of axons was found to strongly correlate
with the direction of flow. Turning angle as high as 90° was achieved. Optofluidic-manipulation can be applied to other types of mammalian neurons and also can be extended to stimulate mechano-sensing neurons.
Controlled release of ATP can be used for understanding extracellular purinergic signaling. While coarse
mechanical forces and hypotonic stimulation have been utilized in the past to initiate ATP release from cells,
these methods are neither spatially accurate nor temporally precise. Further, these methods cannot be utilized
in a highly effective cell-specific manner. To mitigate the uncertainties regarding cellular-specificity and
spatio-temporal release of ATP, we herein demonstrate use of optogenetics for ATP release. ATP release in
response to optogenetic stimulation was monitored by Luciferin-Luciferase assay (North American firefly,
photinus pyralis) using luminometer as well as mesoscopic bioluminescence imaging. Our result
demonstrates repetitive release of ATP subsequent to optogenetic stimulation. It is thus feasible that
purinergic signaling can be directly detected via imaging if the stimulus can be confined to single cell or in a
spatially-defined group of cells. This study opens up new avenue to interrogate the mechanisms of purinergic
signaling.
KEYWORDS: Optogenetics, Thalamus, Brain, Chemical analysis, Modulation, Neurons, In vivo imaging, Optical fibers, Neurological disorders, Animal model studies
Here, we introduce a computational procedure to examine whether optogenetically activated neuronal firing recordings
could be characterized as multifractal series. Optogenetics is emerging as a valuable experimental tool and a promising
approach for studying a variety of neurological disorders in animal models. The spiking patterns from cortical region of
the brain of optogenetically-stimulated transgenic mice were analyzed using a sophisticated fluctuation analysis method
known as multifractal detrended fluctuation analysis (MFDFA). We observed that the optogenetically-stimulated neural
firings are consistent with a multifractal process. Further, we used MFDFA to monitor the effect of chemically induced
pain (formalin injection) and optogenetic treatment used to relieve the pain. In this case, dramatic changes in parameters
characterizing a multifractal series were observed. Both the generalized Hurst exponent and width of singularity
spectrum effectively differentiates the neural activities during control and pain induction phases. The quantitative nature
of the analysis equips us with better measures to quantify pain. Further, it provided a measure for effectiveness of the
optogenetic stimulation in inhibiting pain. MFDFA-analysis of spiking data from other deep regions of the brain also
turned out to be multifractal in nature, with subtle differences in the parameters during pain-induction by formalin
injection and inhibition by optogenetic stimulation. Characterization of neuronal firing patterns using MFDFA will lead
to better understanding of neuronal response to optogenetic activation and overall circuitry involved in the process.
For intervention of cardiovascular diseases, biodegradable and biocompatible, poly(lactic-co-glycolic acid)
(PLGA) nanoparticles (NP) are emerging as agents of choice for controlled and targeted drug delivery. Therefore
development of PLGA-NP with optimal physico-chemical properties will allow efficient binding and thus delivery of
drug to targeted cells under various patho-physiological conditions. The force kinetics and its dependence on size of the
NPs will be crucial for designing the NPs. Since optical tweezers allow non-contact, highly sensitive force measurement
with high spatial and temporal resolution, we utilized it for studying interaction forces between magnetic
PLGA nanoparticles with smooth muscle cells (SMC). In order to investigate effect of size, interaction force for 200 to
1100nm PLGA NP was measured. For similar interaction duration, the force was found to be higher with increase in
size. The rupture force was found to depend on time of interaction of SMC with NPs.
Recent advent of optogenetics has enabled activation of genetically-targeted neuronal cells using low intensity blue light
with high temporal precision. Since blue light is attenuated rapidly due to scattering and absorption in neural tissue,
optogenetic treatment of neurological disorders may require stimulation of specific cell types in multiple regions of the
brain. Further, restoration of certain neural functions (vision, and auditory etc) requires accurate spatio-temporal
stimulation patterns rather than just precise temporal stimulation. In order to activate multiple regions of the central
nervous system in 3D, here, we report development of an optogenetic prosthetic comprising of array of fibers coupled to
independently-controllable LEDs. This design avoids direct contact of LEDs with the brain tissue and thus does not
require electrical and heat isolation, which can non-specifically stimulate and damage the local brain regions. The
intensity, frequency, and duty cycle of light pulses from each fiber in the array was controlled independently using an inhouse
developed LabView based program interfaced with a microcontroller driving the individual LEDs. While the
temporal profile of the light pulses was controlled by varying the current driving the LED, the beam profile emanating
from each fiber tip could be sculpted by microfabrication of the fiber tip. The fiber array was used to stimulate neurons,
expressing channelrhodopsin-2, in different locations within the brain or retina. Control of neural activity in the mice
cortex, using the fiber-array based prosthetic, is evaluated from recordings made with multi-electrode array (MEA). We
also report construction of a μLED array based prosthetic for spatio-temporal stimulation of cortex.
While pulsed laser beams have been used for stimulation of neurons, cellular specificity during optical stimulation is
achieved by photo-sensitization of genetically-targeted cells by optogenetic means. However, till date, the process of
optogenetic-sensitization primarily involves use of viral vectors. In rare occasions, electroporation has been used. Here,
we report an all-optical method in which pulsed laser beam is used for delivery of genes, encoding optogenetic probes, to
spatially-targeted cells, followed by optogenetic stimulation and optical detection of the activation process. Use of laser
microbeam enabled highly precise spatially-patterned delivery of optogenes, as confirmed by expression of conjugated
fluorescent protein. Light-activation of opsin-expressing cells was confirmed by calcium-imaging. The laser-assisted
expression of optogenetic probes in spatially-targeted regions in combination with light-assisted activation and optical
detection of neural activity will help in better understanding of the neuronal circuitry.
KEYWORDS: Optogenetics, Bessel beams, In vivo imaging, Monte Carlo methods, Brain, Gaussian beams, Neurons, Electrodes, Near infrared, Optical simulations
Since development of optogenetic stimulation paradigm, there has been several attempts to red shift the excitation
maximum of the efficient blue-sensitive opsins. While there has been some success at the cost of altered light-activation
kinetics, near-infrared optogenetic probe will be ideal for in-depth cell-specific stimulation of excitable cells in an organ.
However, single-photon near-infrared optogenetics based stimulation will still limit precise probing and modulation of
in-vivo neural circuits. In contrast, by virtue of non-linear nature of ultrafast light-matter interaction, high spatial
precision in optogenetic activation can be achieved in addition to inherent cellular specificity and temporal resolution
provided by the opsins. Here, we report use of non-linear optogenetics for stimulation of neurons in-vivo in mouse
models. Advantage of using non-linear optogenetics for probing neuronal circuitry is discussed. Further, effectiveness of
the non-diffracting optogenetic Bessel beam over classical Gaussian beam in a layered mouse-brain geometry is
demonstrated using Monte Carlo (MC) simulation. This is corroborated by electrophysiological measurements in in-vivo
mouse models. The large propagation distance, characteristics of Bessel beam is better suited for in-depth single as well
as two-photon optogenetic stimulation.
Photothermal therapy with assistance of nanoparticles offers a solution for the destruction of cancer cells without significant collateral damage to otherwise healthy cells. However, minimizing the required number of injected nanoparticles is a major challenge. Here, we introduce the use of magnetic carbon nanoparticles (MCNPs), localizing them in a desired region by applying an external magnetic-field, and irradiating the targeted cancer cells with a near-infrared laser beam. The MCNPs were prepared in benzene, using an electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements showed the size distribution of these MCNPs to be in the range of 5 to 10 nm. For photothermal irradiation, a tunable continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely guide photothermal therapy.
The laser microbeam has enabled highly precise noncontact delivery of exogenous materials into targeted cells without compromising cell viability, which has been a highly challenging task for traditional methods. Here, we report targeted delivery of impermeable substances into mammalian cells and goldfish retinal explants subsequent to ultrafast laser microbeam assisted injection. Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time constant for injection. Using optimized laser intensity, exposure, and a spatial irradiation pattern, desired spatial transfection patterns in goldfish retina explants were achieved as confirmed by the expression of injected plasmids encoded for light-activable channelrhodopsin-2 ion-channel, tagged with fluorescent protein. Laser assisted delivery of exogenous material into a specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the functioning of neuronal circuitry of normal and degenerated retina.
Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.
Here, we report development of an integrated system for co-registration of patch-clamp measurements with calcium
imaging during two-photon stimulation (TPS) of excitable cells sensitized with optogenetic probe, chanelrhodopsin-2
(ChR2). Comparison of calcium changes due to focused two-photon micro-irradiation of excitable cells with and without
optogenetic sensitization, revealed wavelength-insensitive injection of extra-cellular calcium via pore formation at high
laser beam powers. However, use of defocused/weakly-focused beam allowed sub-threshold stimulation of the excitable
cells, revealed by both calcium imaging and whole-cell patch-clamping. Irregular calcium spiking was observed for
continuous two-photon defocused micro-irradiation. Even at high extra-cellular calcium conditions, since presence of alltrans-
retinal (ATR) was necessary even for detectable calcium increase (and inward current) under defocused twophoton
irradiation, role of ChR2 was confirmed as opposed to optoporation, for defocused condition. In the subthreshold
stimulation regime, while peak-photocurrents variation with TPS wavelength followed ChR2 two-photon
activation spectrum, the power dependence of the current was highly non-linear. Though defocused two-photon beam
may cause minimal photo damage while stimulating the cells, the threshold average power required for generating action
potential in the ChR2-sensitized cells is higher than that used for routine two-photon imaging.
Re-orientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled
interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and
reorientation of adherent cells. The cell gets detached after irradiation for few seconds, followed by vertical orientation.
The detached cell was transported along axial direction by scattering force and trapped at a higher plane inside the media
using the same laser beam by Gravito-optical trap. The trapped cell could then be repositioned by movement of the
sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was
found to depend on the laser beam power. The cell could be brought back to the substrate by reducing the laser beam
power using a polarizer or blocking the laser beam. Viability of the detached and manipulated cell was found not to be
compromised as confirmed by PI fluorescence exclusion assay. The
re-oriented cell was allowed to re-attach to the
substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape
even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for non-contact
modification of cellular orientations that will enable study of
inter-cellular interactions and design of engineered
tissue.
Laser microbeam has enabled highly precise non-contact delivery of exogenous materials into targeted cells, which has
been a highly challenging task while using traditional methods without compromising cell viability. We report distinct
spatial localization of impermeable substances into mammalian cells and goldfish retinal cells in explants subsequent to
ultrafast laser microbeam assisted injection, realized by focusing a near infrared tunable Ti: sapphire laser beam.
Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at
the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect
optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time
constant for injection. Using optimized laser intensity, exposure and spatial irradiation pattern, desired spatial
transfection patterns in goldfish retina explants were achieved as confirmed by expression of injected plasmids encoded
for light-activable channelrhodopsin-2 (ChR2) ion channel tagged with fluorescent protein. Laser assisted delivery of
exogenous material into specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the
functioning of neuronal circuitry of normal and degenerated retina.
Photothermal therapy offers a solution for the destruction of cancer cells without significant collateral damage to
otherwise healthy cells. Several attempts are underway in using carbon nanoparticles (CNPs) and nanotubes due to their
excellent absorption properties in the near-infrared spectrum of biological window. However, minimizing the required
number of injected nanoparticles, to ensure minimal cytotoxicity, is a major challenge. We report on the introduction of
magnetic carbon nanoparticles (MCNPs) onto cancer cells, localizing them in a desired region by applying an external
magnetic field and irradiating them with a near-infrared laser beam. The MCNPs were prepared in Benzene, using an
electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by
use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements
showed the size distribution of these MCNPs to be in the range of 5-10 nm. For photothermal irradiation, a tunable
continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence
microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence
of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion
assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely
guide photothermal therapy.
Here, we report in situ formation of microstructures from the regular constituents of culture media near live cells
using spatially-structured near infrared (NIR) laser beam. Irradiation with the continuous wave (cw) NIR laser
microbeam for few seconds onto the regular cell culture media containing fetal bovine serum resulted in
accumulation of dense material inside the media as evidenced by phase contrast microscopy. The time to form the
phase dense material was found to depend on the laser beam power. Switching off the laser beam led to diffusion of
phase dark material. However, the proteins could be stitched together by use of carbon nanoparticles and continuous
wave (cw) Ti: Sapphire laser beam. Further, by use of spatially-structured beam profiles different structures near live
cells could be formed. The microfabricated structure could be held by the Gravito-optical trap and repositioned by
movement of the sample stage. Orientation of these microstructures was achieved by rotating the elliptical laser
beam profile. Thus, multiple microstructures were formed and organized near live cells. This method would enable
study of response of cells/axons to the immediate physical hindrance provided by such structure formation and also
eliminate the biocompatibility requirement posed on artificial microstructure materials.
Stimulation of retinal neuronal cells using optogenetics via use of chanelrhodopsin-2 (ChR2) and blue light has
opened up a new direction for restoration of vision with respect to treatment of Retinitis pigmentosa (RP). In addition
to delivery of ChR2 to specific retinal layer using genetic engineering, threshold level of blue light needs to be
delivered onto the retina for generating action potential and successful behavioral outcome. We report measurement
of intensity distribution of light reaching the retina of Retinitis pigmentosa (RP) mouse models and compared those
results with theoretical simulations of light propagation in eye. The parameters for the stimulating source positioning
in front of eye was determined for optimal light delivery to the retina. In contrast to earlier viral method based
delivery of ChR2 onto retinal ganglion cells, in-vivo electroporation method was employed for retina-transfection of
RP mice. The behavioral improvement in mice with Thy1-ChR2-YFP transfected retina, expressing ChR2 in retinal
ganglion cells, was found to correlate with stimulation intensity.
We have found that ultrafast laser microbeam inducing bubble(s) can lead to condensation of molecules in solution. The
laser microbeam was generated by coupling a near infrared mode-locked femtosecond (fs) laser beam onto an inverted
fluorescence microscope and focusing it with a 100X objective. Fluorescence imaging revealed that collapse of
cavitation micro-bubbles created high-concentration regions of dye molecules in the aqueous solution. Further, twophoton
excitation of the molecules under the ultrafast laser microbeam showed significant increase in fluorescence
intensity as a function of laser exposure time without micro-bubble formation. This may be attributed to nano-bubble
formation and or conformational change in the molecules under intense laser intensity at the focused spot to significantly
enhance the molar absorptivity (extinction coefficient) or fluorescence excitation cross-section. High-concentration
regions of the dye molecules are found to be retained for a longer period of time and therefore provide an opportunity for
collection of these condensed molecules using microcapilary and/or for further analysis.
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