This study experimentally evaluates the self-targeting ability of asiaticoside-loaded nanoemulsions compared with nontargeted nanoemulsions in ex vivo experiments with porcine skin samples. Homebuilt two-photon and confocal laser-scanning microscopes were employed to noninvasively examine the transdermal delivery of two distinct nanoemulsions. Prior to the application of nanoemulsions, we noninvasively observed the morphology of porcine skin using two-photon microscopy. We have successfully visualized the distributions of the targeted and nontargeted nanoemulsions absorbed into the porcine skin samples. Asiaticoside-loaded nanoemulsions showed an improved ex vivo transdermal delivery through the stratum corneum compared with nonloaded nanoemulsions. As a secondary measure, nanoemulsions-applied samples were sliced in the depth direction with a surgical knife in order to obtain the complete depth-direction distribution profile of Nile red fluorescence. XZ images demonstrated that asiaticoside-loaded nanoemulsion penetrated deeper into the skin compared with nontargeted nanoemulsions. The basal layer boundary is clearly visible in the case of the asiaticoside-loaded skin sample. These results reaffirm the feasibility of using self-targeting ligands to improve permeation through the skin barrier for cosmetics and topical drug applications.
In the present study, we employed the laser scanning confocal microscope to image entire blood flow with accurate
red blood cell imaging of 0.001 mm spatial resolution. In vitro blood flow of rat with different hematocrit ratios was
simulated inside a 100and 300-micron opaque tube. The scanning rate of confocal microscope was 30 fps with 500 x 500
pixels of image. As a result, we can obtain clear images of RBCs to which is enough to be used as tracer particle directly
to get the velocity vector field of blood flow by performing particle image velocimetry (PIV) technique non-invasively.
Based on the present novel optical application, we can easily indicate the presence of cell depleted layer of blood flow in
vitro and its boundaries.
Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 µs ex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED50,d) and cavitation (ED50,c) increased with pulse duration and were approximately equal for pulses of up to 10 µs. For 20 µs, the ED50,d was about 10% lower than the ED50,c; the difference increased with 40-µs pulses. Cells were killed predominantly by cavitation (up to 10-µs pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting.
Purpose: An in vivo flow cytometer was developed recently, providing quantification of fluorescently labeled cells in live animals without extracting blood samples. This non-invasive procedure allows continuously tracking a cell population of
interest over long periods of time to examine its dynamic changes in the circulation. However, it has not been shown
that counting signals arise from individual cells. Furthermore, cell morphology and cell-cell interaction in the blood
stream (e.g. aggregation) are not visualized. Here we describe an imaging in vivo flow cytometer.
Material and Methods: Fluorescence images are obtained simultaneously with quantitative information on a DiD-labeled cell population. As
fluorescent cells pass through the slit of light focused across a blood vessel, the excited fluorescence is detected
confocally. This cell counting signal triggers a strobe beam and an intensified CCD camera to capture a snapshot image
of the cell as it moves down-stream from the slit.
Results: Nearly all peaks counted as circulating T-cells originate from individual cells, while cell aggregates were rarely
observed (<2%). Counting signal amplitude variation is attributed to uneven dye-loading among cells. We identify
non-T-cells by their abnormal shape and size. Cell velocity was measured by determining the traveled distance from the
slit within the delay of the strobe pulse or by applying multiple strobe pulses during the integration time of the CCD
camera. Conclusions: An improved in vivo imaging flow cytometer can be a useful tool for studying cell populations in circulation.
Stone retropulsion during Ho:YAG (λ = 2.12 μm) laser lithotripsy with various pulse
durations (τp: 250 ~ 495 μsec) was investigated. Depending on pulse energy, optical pulse
durations were divided into two regimes: short pulse (250~350 μsec) and long pulse (315~495
μsec). Retropulsion distance was measured as a function of pulse energy from 0.4 J to 1.2 J.
Calculus phantoms made from plaster of Paris were ablated with a free running Ho:YAG laser
using various optical fibers (200, 400, 600 μm) in water. In order to examine the ablation
efficiency of two different pulse durations, a single pulse was applied, and the dynamics of the
recoil action of a calculus phantom was monitored using a high-speed camera. The correlation
among laser-induced topography, ablation volume, and retropulsion was evaluated. Higher pulse
energy and larger fibers resulted in larger ablation volume and retropulsion. At a given pulse
energy, optical pulses with different durations yielded comparable ablation volumes. The
shorter duration pulses induced more retropulsion than longer pulses did at the same pulse
energy. Larger retropulsion with the shorter pulse is thought to be induced by higher
temperature at the vapor-solid interface, subsequently resulting in faster plume ejection with
higher recoil momentum. The results suggest that a longer pulse could minimize retropulsion of
the stone during lithotripsy.
KEYWORDS: In vitro testing, Angiography, Luminescence, In vivo imaging, Eye, Continuous wave operation, Retina, Scanners, Laser scanners, Frequency modulation
Selective targeting of the retinal pigment epithelium (RPE) is a new strategy for treating certain retinal disorders while preserving adjacent photoreceptors. The treatment currently relies on a complex laser system to produce the required microsecond pulse structure. In our new approach, we scan the focus of a continuous-wave (cw) laser beam with acousto-optic deflectors to produce microsecond-long exposures at each RPE cell. Experiments were performed in vitro with a bench-top scanner on samples of young bovine RPE and in vivo on Dutch belted rabbits with a slit-lamp adapted scanner. Effective dose 50% (ED50) for RPE damage was determined in vitro by fluorescence cell viability assay and in vivo by fluorescein angiography. Damage to individual RPE cells was achieved with laser power on the order of 100 mW. Using separated scan lines, we demonstrate selectivity in the form of alternating lines of dead and surviving cells that resemble the scan pattern. Selectivity is also shown by the absence of retinal thermal coagulation in vivo. Selective RPE damage is feasible by rapidly scanning a cw laser beam. The scanning device is an attractive alternative to conventional laser coagulation and pulsed laser targeting of the RPE.
We compared urinary calculus fragmentation with long pulsed Ho:YAG (λ= 2.12 μm) versus Er:YAG (λ = 2.94 μm) lasers. We measured the ablation width, depth, volume and efficiency as a function of pulse energy from calculus threshold energy to clinical energy typically used for Ho:YAG laser lithotripsy. Ablation effects were evaluated for three types of urinary calculi (calcium oxalate monohydrate, cystine, and uric acid), for single and multiple pulses applied at various optical energy levels. By means of comparing laser-induced crater topography and ablation volume for each stone type, the feasibility of Er:YAG laser lithotripsy was appraised. The Er:YAG laser pulse energy generated deeper and narrower crater shapes with relatively smooth contours whereas the Ho:YAG laser produced shallower and wider craters with irregular shapes. In terms of multiple pulses ablation, the Er:YAG produced larger ablation volume than Ho:YAG. The deeper crater induced by the Er:YAG was attributed to the higher absorption coefficient of stones at the 2.94 μm wavelength, and widening of crater by Ho:YAG was perhaps caused by lateral expansion of ablated material. Comparing the ablation efficiency, Er:YAG was superior to Ho:YAG for both single and five-pulses.
Imaging of the in vivo murine myocardium using optical coherence tomography (OCT) is described. Application of conventional techniques (e.g. MRI, Ultrasound imaging) for imaging the murine myocardium is problematic because the wall thickness is less than 1.5mm (20g mouse), and the heart rate can be as high as six-hundred beats per minute. To acquire a real-time image of the murine myocardium, OCT can provide sufficient spatial resolution (10 micrometers ) and imaging speed (1000 A-Scans/s). Strong light scattering by blood in the heart causes significant light attenuation making delineation of the endocardium-chamber boundary problematic. By replacing whole blood in the mouse with an artificial blood substitute we demonstrate significant reduction of light scattering in the murine myocardium. The results indicate a significant reduction in light scattering as whole blood hematocrit is diminished below 5%. To measure thickness change of the myocardium during one cycle, a myocardium edge detection algorithm is developed and demonstrated.
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