Two-photon polymerization and crosslinking are commonly used methods for microfabrication of three-dimensional structures with applications spanning from photonic microdevices, drug delivery systems, to cellular scaffolds. However, the use of two-photon processes for precise, internal modification of biological tissues has not yet been reported. One of the major challenges has been a lack of appropriate tools to monitor and characterize crosslinked regions nondestructively.
Here, we demonstrate spatially selective two-photon collagen crosslinking (2P-CXL) in intact tissue for the first time. Using riboflavin photosensitizer and femtosecond laser irradiation, we crosslinked a small volume of tissue within animal corneas. Collagen fiber orientations and photobleaching were characterized by second harmonic generation and two-photon fluorescence imaging, respectively. Using confocal Brillouin microscopy, we measured local changes in longitudinal mechanical moduli and visualized the cross-linked pattern without perturbing surrounding non-irradiated regions. 2P-CXL-induced tissue stiffening was comparable to that achieved with conventional one-photon CXL. Our results demonstrate the ability to selectively stiffen biological tissue in situ at high spatial resolution, with broad implications in ophthalmology, laser surgery, and tissue engineering.
As the most abundant cell type in the central nervous system, astrocyte has been one of main research topics in neuroscience. Although various tools have been developed, at present, there is no tool that allows noninvasive activation of astrocyte in vivo without genetic or pharmacological perturbation. Here we report a noninvasive label-free optical method for physiological astrocyte activation in vivo using a femtosecond pulsed laser. We showed the laser stimulation robustly induced astrocytic calcium activation in vivo and further verified physiological relevance of the calcium increase by demonstrating astrocyte mediated vasodilation in the brain. This novel optical method will facilitate noninvasive physiological study on astrocyte function.
Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular parameters including vascular density, perfusion rate, and permeability using spatiotemporal profiles of indocyanine green. Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal tissues with high spatial resolution. The multimodality of this method allowed us to find negative spatial correlations between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic drug. We suggest that DyFI could be a useful tool for the preclinical development of anti-angiogenic drugs.
Even though catheterization or electric stimulation are used for treatment of neurogenic bladder, invasiveness and
inconvenience of these approaches prompt us to develop a new possible therapeutic method to control urination by using
optical stimulation. The optical method using femtosecond pulsed laser (FSPL) has advantages of focused and
subsurface stimulation. Irradiation of FSPL induced a rapid increase of intracellular calcium level followed by
contraction of primary cultured human bladder smooth muscle cells. Short exposure of bladder detrusor ex-vivo to FSPL
also induced a controlled contraction of detrusor. Collectively, we propose that FSPL can be considered as a potential
therapeutic approach for intractable neurogenic bladder.
For a systemically administered drug to act, it first needs to cross the vascular wall. This step represents a bottleneck for
drug development, especially in the brain or retina, where tight junctions between endothelial cells form physiological
barriers. Here, we demonstrate that femtosecond pulsed laser irradiation focused on the blood vessel wall induces
transient permeabilization of plasma. Nonlinear absorption of the pulsed laser enabled the noninvasive modulation of
vascular permeability with high spatial selectivity in three dimensions. By combining this method with systemic
injection, we could locally deliver molecular probes in various tissues, such as brain cortex, meninges, ear, striated
muscle, and bone. We suggest this method as a novel delivery tool for molecular probes or drugs.
Even though electrical stimulation is generally used for induction of smooth muscle cell contraction, it is very hard to
obtain fine control and also very invasive for inserting electrodes. Herein, we developed a new optical technology to
control smooth muscle cell contraction. This optical method using femtosecond pulsed laser (FSPL) has advantage of
focused stimulation and fine control of stimulation intensity. Upon brief exposure to FSPL, smooth muscle cells showed
a rapid increase of intracellular calcium levels followed by cell contraction. Collectively, we suggest that FSPL can be a
useful tool for control of smooth muscle cell contraction.
Blood vessels, especially in the brain, dynamically change the diameters over time to provide sufficient blood supply
where needed. At present, there is no technique that allows noninvasive control of vascular diameter in vivo. Here we
report that label-free irradiation of femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to
laser irradiation, cultured vascular smooth muscle cell showed a rapid increase in calcium concentration followed by the
cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused
localized vascular contraction followed by recovery. Nonlinear nature of the pulsed laser allowed highly specific
targeting of subcortical vessels without affecting the surrounding region. We propose that femtosecond pulsed laser
irradiation will be a useful experimental tool in the field of vascular biology.
The diameters of blood vessels, especially in the brain, change dynamically over time to provide sufficient blood supply as needed. No existing technique allows noninvasive control of vascular diameter in vivo. We report that label-free irradiation with a femtosecond pulsed laser can trigger blood vessel contraction in vivo. In response to laser irradiation, cultured vascular smooth muscle cells showed a rapid increase in calcium concentration, followed by cell contraction. In a murine thinned skull window model, laser irradiation focused in the arterial vessel wall caused localized vascular contraction, followed by recovery. The nonlinear nature of the pulsed laser allowed highly specific targeting of subcortical vessels without affecting the surrounding region. We believe that femtosecond pulsed laser irradiation will become a useful experimental tool in the field of vascular biology.
Angiogenesis is essential for tumor growth and a promising target for cancer therapy. Blood vessel monitoring is an
indispensable tool for evaluation and development of anti-angiogenic drugs. Here, we report a new noninvasive in vivo
imaging tool, named dynamic fluorescence imaging (DyFI), for the simultaneous measurement of multiple vascular
parameters, such as vascular density, perfusion rate, and permeability, using spatiotemporal profiles of indocyanine green.
Using DyFI in a tumor xenograft model, we quantitatively measured multiple vascular parameters in tumors and normal
tissues with high spatial resolution. The multimodality of this method allowed us to find a negative spatial correlation
between perfusion and permeability. Moreover, DyFI was effective for revealing the early effects of an anti-angiogenic
drug; these findings were validated using two-photon microscopy. We suggest that DyFI could be a useful tool for the
preclinical development of anti-angiogenic drugs.
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