SHORT-TERM COMPUTER-ASSISTED QUANTIFICATION OF PLUS DISEASE AFTER TREATMENT OF TYPE 1 RETINOPATHY OF PREMATURITY WITH INTRAVITREAL BEVACIZUMAB OR RETINAL LASER PHOTOCOAGULATION.

PURPOSE: To compare objectively measured changes in plus disease after bevacizumab and laser for Type 1 retinopathy of prematurity. METHODS: ROPtool (a computer program) analyzed fundus images at baseline, 1 week, and subsequent examinations. RESULTS: Six infants (9 eyes) were treated with bevacizumab and 7 (12 eyes) with laser. One week after treatment, bevacizumab compared with laser resulted in a greater median percent change from pretreatment in tortuosity (-53.8 vs. -0.2%, P < 0.001) and overall plus disease (-20.1 vs. -3.1%, P < 0.001). Change in dilation did not differ (-3.5 vs. -5.5%, P = 0.48). After week 1, all median ROPtool parameters continued to decrease for both groups. At last follow-up (median 3, range: 2-10 weeks), both bevacizumab and laser significantly decreased tortuosity (-54.3 and -23.8%), dilation (-8.7 and -7.5%), and overall plus disease (-27.3 and -10.3%). CONCLUSION: Improvement in plus disease, in particular tortuosity, likely occurs more quickly after bevacizumab compared with laser, particularly at 1 week after treatment. These results guide clinical expectations for plus disease resolution after both treatment modalities.

Detection of counterfeit U.S. paper money using intrinsic fluorescence lifetime.

Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning two-photon laser excitation and the time-correlated single photon counting (TCSPC) method to sample a approximately 4 mm(2) region. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

Multiphoton fluorescence lifetime imaging of intrinsic fluorescence in human and rat brain tissue reveals spatially distinct NADH binding.

Two-photon fluorescence lifetime imaging (FLIM) of molecules can reveal important information on the local microenvironment. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic co-enzyme, has a lifetime that depends strongly on enzymatic binding. We present a custom image-processing algorithm for raw fluorescence lifetime and amplitude data that produces an image showing spatially distinct NADH fluorescence lifetimes in slices of rat and human brain. NADH FLIM images were collected in control and epileptic rat tissue. Differences in spatial patterns of NADH lifetimes support the hypothesis that NADH binding, and thus metabolic capacity, is significantly different between groups. This type of analysis can provide information on metabolic states in pathological material.

Evaluation of Risk Factors for Glaucoma Drainage Device-related Erosions: A Retrospective Case-Control Study.

PURPOSE: To identify risk factors for glaucoma drainage device (GDD) erosions. PATIENTS AND METHODS: In a retrospective comparative case series, medical records of 1013 patients who underwent GDD surgery performed by 5 surgeons between 2006 and 2011 were reviewed. The outcome measures assessed included age, race, sex, contact lens wear, seasonal allergies, medical comorbidities, glaucoma diagnosis, preoperative oral and topical medications, type and number of preoperative surgeries and lasers, concomitant surgeries, tube type and position, patch graft material, and intraoperative use of Avastin, mitomycin-C, or Triescence. The association of variables with erosion status was evaluated using the Fisher exact test for categorical variables and the exact Wilcoxon rank-sum test for continuous variables. RESULTS: Charts were included from 339 eyes that had complete data sets and at least 6 months of follow-up. Twenty-eight eyes (8.3%) developed conjunctival erosions. The median follow-up time was 2.03 years for the erosion group and 1.71 years for nonerosion group. Erosion was only associated with the presence of concomitant surgical procedures at the time of GDD implantation (35.7% erosion group vs. 17.4% nonerosion group, P=0.02, OR=2.64). The majority of concomitant surgeries were composed of pars plana vitrectomy (35.0%) and cataract surgery (32.0%). Variables that were suggestive of association with erosion (P<0.20) included smoking (OR=2.14), pseudoexfoliation glaucoma (OR=2.71), and history of dry eye syndrome (OR=2.22). CONCLUSION: History of concomitant intraocular surgery with GDD implantation may be a potential risk factor for future erosions.

Multiphoton microscopy of cleared mouse organs.

Typical imaging depths with multiphoton microscopy (MPM) are limited to less than 300 mum in many tissues due to light scattering. Optical clearing significantly reduces light scattering by replacing water in the organ tissue with a fluid having a similar index of refraction to that of proteins. We demonstrate MPM of intact, fixed, cleared mouse organs with penetration depths and fields of view in excess of 2 mm. MPM enables the creation of large 3-D data sets with flexibility in pixel format and ready access to intrinsic fluorescence and second-harmonic generation. We present high-resolution images and 3-D image stacks of the brain, small intestine, large intestine, kidney, lung, and testicle with image sizes as large as 4,096 x 4,096 pixels.

In vivo imaging of deep cortical layers using a microprism.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism. Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light. The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations. Images are collected with a large field-of-view (approximately 900 microm). In addition, we provide details on the non-survival surgical procedure and microscope setup. Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts. Resolution was sufficient to image dendritic spines near the soma of layer V neurons. A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex. Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

Microprisms for in vivo multilayer cortical imaging.

Cortical slices allow for simultaneous imaging of multiple cortical layers. However, slices lack native physiological inputs and outputs. Although in vivo, two-photon imaging preserves the native context, it is typically limited to a depth of <500 microm. In addition, simultaneous imaging of multiple cortical layers is difficult due to the stratified organization of the cortex. We demonstrate the use of 1-mm microprisms for in vivo, two-photon neocortical imaging. These prisms enable simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. Images were collected from the mouse motor and somatosensory cortex and show a nearly 900-microm-wide field of view. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution is sufficient to resolve dendritic spines on layer V neurons. Images collected using the microprism are comparable to images collected from a traditional slice preparation. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. H&E staining shows the surrounding tissue remains in its native, stratified organization. Estimation of neuronal damage using propidium iodide and a fluorescent Nissl stain reveals cell damage is limited to <100 microm from the tissue-glass interface. Microprisms are a straightforward tool offering numerous advantages for into neocortical tissue.