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The spatial and temporal activation of NF-kB (p65) was monitored in the retina of a transgenic mouse model (cis-NFkB-EGFP) in vivo after receiving varying grades of laser induced thermal injury in one eye. Baseline images of the retinas from 26 mice were collected prior to injury and up to five months post-exposure using a Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope (cSLO) with a spectral domain optical coherence tomographer (SDOCT). Injured and control eyes were enucleated at discrete time points following laser exposure for cryosectioning to determine localization of NF-kB dependent enhanced green fluorescent protein (EGFP) reporter gene expression within the retina using fluorescence microscopy. In addition, EGFP basal expression in brain and retinal tissue from the cis-NFkB-EGFP was characterized using two-photon imaging. Regions of the retina exposed to threshold and supra-threshold laser damage evaluated using fluorescence cSLO showed increased EGFP fluorescence localized to the exposed region for a duration that was dependent upon the degree of injury. Fluorescence microscopy of threshold damage revealed EGFP localized to the outer nuclear region and retinal pigment epithelial layer. Basal expression of EGFP imaged using two-photon microscopy was heterogeneously distributed throughout brain tissue and confined to the inner retina. Results show cis-NF-kB-EGFP reporter mouse can be used for in vivo studies of light induced injury to the retina and possibly brain injury.

Balb/c wild type mice were used to perform in vivo experiments of laser-induced thermal damage to the retina. A Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope with a spectral domain optical coherence tomographer was used to obtain fundus and cross-sectional images of laser induced injury in the retina. Sub-threshold, threshold, and supra-threshold lesions were observed using optical coherence tomography (OCT), infrared reflectance, red-free reflectance, fluorescence angiography, and autofluorescence imaging modalities at different time points post-exposure. Lesions observed using all imaging modalities, except autofluorescence, were not visible immediately after exposure but did resolve within an hour and grew in size over a 24 hour period. There was a decrease in fundus autofluorescence at exposure sites immediately following exposure that developed into hyper-fluorescence 24-48 hours later. OCT images revealed threshold damage that was localized to the RPE but extended into the neural retina over a 24 hour period. Volumetric representations of the mouse retina were created to visualize the extent of damage within the retina over a 24 hour period. Multimodal imaging provides complementary information regarding damage mechanisms that may be used to quantify the extent of the damage as well as the effectiveness of treatments without need for histology.

A study of retinal damage thresholds in non-human primates (NHP) in the near-infrared (NIR) wavelengths of 1110, 1130, 1150, and 1319 nm has recently been reported. The progression of damage in retinal areas that received exposures below, greater than, and at threshold values for each respective wavelength are compared. Subjects were imaged using an Adaptive Optics (AO) enhanced Spectral Domain Optical Coherence Tomographer (SD-OCT) a year post laser exposure to examine damage characteristics and localization. The subject's retinas within the study exhibited a delayed response to NIR exposures in that many of the lesions that were not visible at the 1-hour observation period continued to grow in size over the 24-hour period and or became visible. Thermal lensing is believed to play a significant role in the formation or retinal lesions in the NIR and may explain the delayed response.

Near threshold retinal lesions were created in the eyes of non-human primate (NHP) subjects in the near infrared (NIR) wavelength range of 1100 to 1319 nm, with 80 to 100 ms laser exposures. Two new in vivo imagining techniques, Adaptive Optic enhanced-Spectral Domain Optical Coherence Tomography (AO-SDOCT) and Adaptive Optic enhanced confocal Scanning Laser Ophthalmoscope imagery (AOcSLO) we re utilized to pinpoint areas of chronic damage within the retinal layers resulting from laser e xposure. Advantages and limitations of each technology with regard to the study of laser retinal tissue interaction are highlighted. Keywords: Adaptive Optics, MVL, NIR, laser threshold, in vivo imaging, confocal, scanning laser ophthalmoscope, SLO, Spectral Domain, OCT 1. INTRODUCTION High-fidelity imaging of lesions in the NHP retina is vital for determining damage thresholds for laser exposure. Retinal damage thresholds are traditionally determined from direct funduscopic observation with an ophthalmoscopic camera. The ability to discern a change from healthy to denatured retinal tissue in vivo is limited primarily by the optical transfer function (OTF) of the eye, with observable lesions of 25 um in extent (or larger) typically reported as being a minimally visible lesion (MVL).[1] However, damage widths which correspond to the extent of a single cell at the RPE and photoreceptor layers in the retina for ultrashort laser exposures have been reported with histological confirmation by Toth.[2] Recent observations of changes in autofluorescence in the retina also suggest that unresolved damage as a result of subthreshold exposure levels can exist without detection via traditional imaging methods in threshold studies. [3-5] These observations and the lack of data reported for similarly sized lesions resulting from photothermal exposures accentuate the possibility that damage which is not directly observable through standard funduscopic observation is present at exposure levels which are “subthreshold.” Given the 25-um size purported to be the smallest repeatedly observable lesion in thermal studies, the observation of small lesions in ultrashort laser studies and the subthreshold detection of changes in autofluorescence of RPE cells it is apparent that a method of observation which is both more sensitive (improved spatial resolution) and specific (able to isolate damaged tissue within the layers of the retina) is needed. Therefore, imaging techniques which overcome the li mitations of the OTF of the eye and which enable imaging at depth in turbid media are highly desired for use in retinal laser threshold studies. Two adaptive-optic-enabled retinal imaging systems were delivered to Brooks City-Base as the product of Air Force Research Laboratory sponsored Small Business Initiative for Re search (SBIR) technology de velopment contracts. The first system developed by PSI (Andover, MA) is a spectral domain optical coherence tomography (AO-SDOCT) system. Details of this system are published elsewhere.[6]. The second system, developed by IRIS Electro-Optics (Berkeley, CA), is a confocal scanning ophthalmoscope (AOcSLO). [7] A schematic for each system is shown in Figure 1. This paper details the initial use of these imaging systems to observe laser damage in the NHP retina which were produced by exposure to NIR laser wavelengths.

An assessment of skin damage caused by near-IR laser exposures is reported. The damage from two distinct laser-tissue temporal regimes is compared at two wavelengths (1.3 mm and 1.5 mm). Skin damage caused by thermal effects from single laser pulses is compared to damage caused by LIB (laser induced breakdown) using histological examinations. Modeling applications are explored to determine crossover points between thermal and photomechanical damage thresholds.

The delivery of therapeutic, detection and imaging agents for the diagnosis and treatment of cancer patients has improved dramatically over the years with the development of nano-carriers such as liposomes, micelles, dendrimers, biomolecules, polymer particles, and colloidal precipitates. While many of these carriers have been used with great success in vitro and in vivo , each suffers from serious drawbacks with regard to stability, flexibility, or functionality. To date, there has been no general particle fabrication method available that afforded rigorous control over particle size, shape, composition, cargo and chemical structure. By utilizing the method we has designed referred to as P article R eplication I n N on-wetting T emplates, or PRINT , we can fabricate monodisperse particles with simultaneous control over structure ( i.e. shape, size, composition) and function ( i.e. cargo, surface structure). Unlike other particle fabrication techniques, PRINT is delicate and general enough to be compatible with a variety of important next-generation cancer therapeutic, detection and imaging agents, including various cargos (e.g. DNA, proteins, chemotherapy drugs, biosensor dyes, radio-markers, contrast agents), targeting ligands (e.g. antibodies, cell targeting peptides) and functional matrix materials (e.g. bioabsorbable polymers or stimuli responsive matrices). PRINT makes this possible by utilizing low-surface energy, chemically resistant fluoropolymers as molding materials and patterned substrates to produce functional, harvestable, monodisperse polymeric particles.

The reflectance and absorption of the skin plays a vital role in determining how much radiation will be absorbed by human tissue. Any substance covering the skin would change the way radiation is reflected and absorbed and thus the extent of thermal injury. Hairless guinea pigs ( cavia porcellus ) in vivo were used to evaluate how the minimum visible lesion threshold for single-pulse laser exposure is changed with a topical agent applied to the skin. The ED 50 for visible lesions due to an Er: glass laser at 1540-nm with a pulse width of 50-ns was determined, and the results were compared with model predictions using a skin thermal model. The ED50 is compared with the damage threshold of skin coated with a highly absorbing topical cream at 1540 nm to determine its effect on damage pathology and threshold. The ED 50 for the guinea pig was then compared to similar studies using Yucatan minipigs and Yorkshire pigs at 1540-nm and nanosecond pulse duration. 1,2 The damage threshold at 24-hours of a Yorkshire pig for a 2.5-3.5-mm diameter beam for 100 ns was 3.2 Jcm -2 ; very similar to our ED 50 of 3.00 Jcm -2 for the hairless guinea pigs.