Novel beam-based optical tweezers, employed in microfluidic devices, has enabled efficient and non-contact
actuation of microscale samples. We report development of an optical isolator based on pincushion distortion
introduced into astigmatic optical tweezers. While objects in the range of 1 to 5 microns (polystyrene particles
and bacteria) were transported away along the curvilinear trajectories of the pincushion profile, objects in 10-20
micron range (e.g. cells) could easily be trapped in the center of the pincushion profile. This enabled efficient
isolation of cell(s) from its surrounding with high spatial and temporal precision and thus opens up new
possibility to control and study interaction of cells (and other microscopic objects) with surrounding objects
without requiring presence or actuation of physical valves. The trapped and isolated cell(s) could be transported
by maneuvering the sample stage or beam. Further, optical clearing of wide microscopic area was achieved
using the distorted profile without requiring beam scanning or sample movement. The distorted tweezers was
used to clear floating microscopic particles near axonal networks as well as from the top surface of retina
explant. Theoretical simulation of force exerted by such beam profiles and experimental demonstration of its
potential in microfluidic isolation and manipulation is discussed.
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.
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.
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.
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