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Many topically applied drugs are naturally fluorescent, but quantifying their uptake into skin via fluorescence emission is complicated by both weak fluorescence and often-overwhelming skin autofluorescence. Fluorescence lifetime imaging microscopy (FLIM) has been found capable of identifying and separating the fluorescence of multiple drugs from skin autofluorescence via their different spectral and lifetime properties. This investigation was focused on combining epi-fluorescence microscopy with deep learning for the quantification of naturally fluorescent topically applied drugs. This combination of deep learning and fluorescence imaging may allow for straightforward relative quantification of fluorescent drugs in tissue using only simple, readily available imaging tools.

Our group has recently developed a method for characterizing distribution of a topical drug within skin using two-photon fluorescence lifetime imaging (FLIM) and phasor analysis. Here, we expand on this work by describing a multimodal approach for rapidly visualizing multiple components in tissue using FLIM and coherent Raman imaging (CRI). By employing a non-Euclidian FLIM phasor analysis for a three-component system informed with the vibrational signature of one of the components retrieved with CRI, we were able to semi-quantitatively describe the spatial distribution of drugs in tissue with molecular specificity and cellular resolution.

We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean phasor analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.

Oral minocycline has been the standard of care for the treatment of non-nodular moderate to severe inflammatory acne vulgaris due to its inhibitory effects on the acne-causing Propionibacterium acnes bacterium and its anti-inflammatory properties, Despite the availability of an oral dosage form since 1966, a commercial topical minocycline remains elusive because of the challenges in stabilizing the active pharmaceutical ingredient (API) in a liquid/semisolid while ensuring sufficient uptake into targeted lesions. Recently, an investigative topical minocycline gel (BPX-01) has been developed to address the unmet needs for localized and targeted delivery while minimizing the risks of systemic side effects. Earlier preclinical studies pertaining to transepidermal delivery of the API had depended on semi-infinite doses of the 1%, 2% and 4% formulations to elicit enough fluorescence yield. We have subsequently shown evidence of minocycline delivery of 1% and 4% BPX-01 into the pilosebaceous unit of ex vivo human facial skin specimens dosed with about 2.5× daily dose using two-photon excitation fluorescence microscopy. In this study, we demonstrated another novel approach to identifying minocycline fluorescence signature using fluorescence lifetime imaging microscopy (FLIM) with phasor analysis. It was found that for a single daily dose and with FLIM, minocycline was consistently noted in the epidermis and hair follicle, with some incidence in the sebaceous gland for both 1% and 2% BPX-01. These observations corroborated with the recent success of a Phase 2b dose-finding study, with 2% BPX-01 meeting the primary endpoint of lesion reduction at week 12 with statistical significance over the vehicle.

Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a phasor approach to fluorescence lifetime microscopy (FLIM). In phasor analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the phasor approach to FLIM. The unique signature of minocycline in FLIM phasor analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.

The safety and efficacy of an investigational topical minocycline gel (BPX-01) has recently been studied in a Phase 2b trial for the treatment of acne vulgaris. As part of the drug development process, there was a need to determine if minocycline was delivered to the target tissue compartments, including the epidermis, hair follicle, and the sebaceous gland. While it was easier to demonstrate delivery on an ex vivo human skin model with an infinite dose, it was initially challenging to verify low-dose delivery with conventional fluorescence microscopy due to the high autofluorescence inherent to human skin. An integrating sphere screening approach was implemented along with conventional fluorescence microscopy to quantitatively and qualitatively assess endogenous fluorescence concurrently from numerous human facial skin specimens. Donor tissues were cut into 50-µm frozen sections, mounted onto microscope slides, and positioned on an inverted fluorescence microscope, sandwiched between the microscope’s 40x high NA objective lens and an external integrating sphere. The tissue sections were illuminated with UV excitation centered at 386 nm. For the first time, it was found that random samples from >40 human facial skin donors produced at least 5× differential in measurable autofluorescence. This observation has significant implications for the use of 2PEF microscopy and FLIM to visualize/quantify drug distribution; the endogenous autofluorescence may limit the detectability of the minocycline signature. Our studies indicated that a single daily dose of BPX-01 was detected in low autofluorescence skin specimens with FLIM, thus validating a novel imaging modality for future pharmacokinetic studies.

Acne vulgaris is a chronic inflammatory skin condition commonly resulting in negative aesthetic and social impacts on those affected. Minocycline, currently available as an oral antibiotic for moderate to severe acne, has a known minimum inhibitory concentration (MIC) for the acne-causing bacterium Propionibacterium acnes (P. acnes) in vitro, with its anti-inflammatory properties also eliciting inhibitory effects on pro-inflammatory molecules. A novel topical gel composition containing solubilized minocycline (BPX-01) has been developed to directly deliver the drug to the skin. Because minocycline is a known fluorophore, fluorescence microscopy and concurrent quantitative measurements were performed on excised human facial skin dosed with different concentrations, in order to determine the spatial distribution of the drug and quantification of its local concentration in the epidermis and the pilosebaceous unit where P. acnes generally reside. Local minocycline delivery confirmed achievement of an adequate therapeutic dose to support clinical studies. Subsequently, a 4-week double-blind, randomized, vehicle controlled clinical study was performed to assess the safety and efficacy of 1% minocycline BPX-01 applied daily. No instances of cutaneous toxicity were reported, and a greater than 1 log reduction of P. acnes count was observed at week 4 with statistical significance from baseline and vehicle control. In addition, no detectable amounts of minocycline in the plasma were reported, suggesting the potential of this new formulation to diminish the known systemic adverse effects associated with oral minocycline. Follow-on clinical plans are underway to further establish the safety of BPX-01 and to evaluate its efficacy against inflammatory acne lesions in a 225 patient multi-center dose-finding study.

Minocycline is an antibiotic regularly prescribed to treat acne vulgaris. The only commercially available minocycline comes in an oral dosage form, which often results in systemic adverse effects. A topical minocycline composition (BPX-01) was developed to provide localized and targeted delivery to the epidermis and pilosebaceous unit where acne-related bacteria, Propionibacterium acnes (P. acnes), reside. As minocycline is a known fluorophore, fluorescence microscopy was performed to investigate its potential use in visualizing minocycline distribution within tissues. BPX-01 with various concentrations of minocycline, was applied topically to freshly excised human facial skin specimens. Spatial distribution of minocycline and its fluorescence intensity within the stratum corneum, epidermis, dermis, and pilosebaceous unit were assessed. The resulting fluorescence intensity data as a function of minocycline concentration may indicate clinically relevant therapeutic doses of topical BPX-01 needed to kill P. acnes and reduce inflammation for successful clinical outcomes.

An innovative High Resolution Maskless Lithography System (Hi-Res MLS) was designed using Texas Instruments’ SVGA DMD, which employs additional micro-optics with a combination of low and high NA projection lens systems. A low power mercury-arc lamp filtered for G-line (λ = 435.8 nm) was used as the light source. Exposure experiments were performed using Ball patented Point Array scrolling or scanning method and proprietary data conversion, extraction and transfer software algorithms. In each scan, the field-width (W) was approximately 8.47 mm with the field-length (longitudinal field) only limited by memory capacity. DMD frame rates of up to 5 kHz (kframe/s) were achievable, which were synchronized to the stage motion. In this experiment, TSMR-8970XB10 photo-resist, diluted to 3.8 cP with PR thinner was employed. The photo-resist was spin-coated on a glass substrate to 1.0-μm thickness with 0.1-μm uniformity. A 0.4-μm step-size was used and 27000 DLP frames were extracted and transferred to the DMD driver. Results indicated consistent 1.8 μm L/S resolved across the entire field-width of 8.47 mm. At certain locales, 1.5-μm L/S was also resolved. The potential of this maskless lithography system is substantial. Even at the current level of performance, the system is sufficient for applications in MEMS, MOEMS, photomasking, high resolution LCD, high density PCB, etc. Higher productivity is predicted by lens system designed for H-line (λ = 405 nm), by using Ball’s violet diode laser systems, and the development of real-time driver.

For typically small volume production of MEMS, MOEMS, fine feature PCB, high density chip packaging and display panels, especially for lab tests, low cost and the capability to change the original design easily and quickly are very important for customers and researchers. BALL Semiconductor Inc.'s Maskless Lithography Systems (MLS) feature the Digital Mirror Device (DMD) as the pattern generator to replace photo-masks. This can remove masks from UV lithography, and dramatically reduce the running cost and save time for lab tests and small volume production. At Ball Semiconductor Inc, 1.5μm line/space, 10μm line/space, and 20μm line/space Maskless Lithography Systems were developed. In our MLS, an 848×600 microlens and spatial filter array (MLSFA) was used to focus the light and to filter the noise. In order to produce smaller line-space than 16μm the MLSFA was used to get smaller UV light pad (compared with the SVGA DMD’s micro-mirror: 17μm×17μm) and to filter the noise produced from the DMD, optical lens system, and micro lens array. This MLSFA is one of the key devices for our Maskless Lithography System, and determines the resolution and quality of maskless lithography. A novel design and fabrication process of a single-package MLSFA for our Maskless Lithography System will be introduced. To avoid problems produced by misalignment between a two-piece spatial filter and microlens array, MEMS processing is used to integrate the microlens array with the spatial filter array. In this paper, the self-alignment method used to fabricate exactly matched MLSFA will be presented.

The potential application of an Erbium:YAG (Er:YAG) laser (Q 0 = 50 mJ/pulse; τ p = 275 μs; rep. rate = 2, 10 Hz) with a sapphire delivery fiber for intracorporeal laser lithotripsy was explored. Preliminary measurements on calculus mass-loss and fragmentation efficiency were conducted and results were compared with that of Ho:YAG laser lithotripsy. Laser induced bubble and lithotripsy dynamics were investigated to assess the mechanism(s) involved in the fragmentation process. Results showed that the fragmentation efficiency (mass-loss/H o - g.μm 2 /J) in Er:YAG laser lithotripsy was about 2.4 times that of Ho:YAG laser lithotripsy (used: Q 0 = 500 mJ/pulse; Tp = 250 μs; rep. rate = 10 Hz). Acoustic transients were found to have minimal effect during Er:YAG laser lithotripsy. Schlieren flash images suggested a predominantly photothermal mechanism due to direct laser energy absorption, which resulted in recrystallization and plume formation. These events indicated melting and chemical decomposition of the calculus composition. Another observation led to the possibility of a plasma-mediated photothermal mechanism. The 'Moses effect' facilitating pulsed mid-infrared laser delivery appeared more efficient for the Er:YAG laser than for the Ho:YAG laser. With the sapphire fiber, experimental results suggested the potential of an improved treatment modality by the Er:YAG laser for intracorporeal laser lithotripsy.

In this study, we used a technique that alters the optical properties of tissue to study the effects of tissue scattering on the detected fluorescence from a target placed under the tissue sample. By using hyper-osmotic solutions of glycerol or dimethyl sulfoxide, scattering in tissue samples was reduced substantially. A fluorescent film was placed underneath in vitro and in vivo samples of hamster skin. Excitation of the fluorescent film was achieved through the tissue and the fluorescent signal from the film monitored over time as an osmotically active agent was added to the tissue. The film had an absorption peak at 542 nm and an emission peak at 585 nm. The reduction in tissue scattering caused by the optical clearing agents led to an increase in the detected fluorescent signal (up to double the original signal). The increase was greater for 14 M dimethyl sulfoxide than for 13 M glycerol, and greater for in vivo skin than for in vitro skin. The study serves as a demonstration of the benefits of optical tissue clearing by osmotically active agents to the detected signal in an optical application. Temporary reduction of scattering in tissue will be useful in therapeutic and diagnostic applications that can benefit from an increase in the penetration depth of light.

The objectives of this study were to determine if the optical absorption properties of urinary calculi affect thethreshold fluence for ablation or fragmentation and the ablation efficiency due to laser irradiation. The Vanderbiltfree electron laser (FEL) was tuned to selected wavelengths based on the absorption spectrum of various types ofurinary calculi. The threshold fluences for ablation of the calculi were measured at different wavelengths. Apreliminary study of the ablation efficiency (ablation depth per unit incidence fluence) was performed. The resultswere found to be in agreement with a thermal ablation model for which the threshold fluences were proportional to 1/J.ta. The ablation efficiencies were higher in regions of the infrared spectra in which absorption was higher. For afixed laser irradiation, the lower threshold fluences within regions of high optical absorption allowed more energy toenhance calculus ablation. This study provided insight into determining the optimum wavelengths for ablation andlaser lithotripsy.Keywords: ablation efficiency, absorption coefficient, fragmentation efficiency, free electron laser, infrared, kidneystone, optimum wavelength, threshold radiant exposure, tissue ablation.

Because of the >= 250 microsecond(s) pulsewidth emitted by the Ho:YAG laser used in clinical lithotripsy, it is unlikely that stress confinement occurs within the irradiated stones. Experimental data supports a thermal mechanism for Ho:YAG laser stone ablation. Previous work has shown that stone fragmentation occurs soon after the onset of the laser pulse, is uncorrelated to cavitation bubble formation or collapse, and is associated with low pressures. Moreover, lithotripsy proceeds fastest with desiccated stones in air (data based on laser ablation of calcium oxalate monohydrate stones), indicating that direct absorption of the laser radiation by the stone material is required for the most efficient ablation. Lowering the initial temperature of calculi reduces the stone mass-loss following 20 J of delivered laser energy: 2.2 +/- 1.1 mg vs 5.2 +/- 1.6 mg for calcium oxalate monohydrate (COM) stones (-80 vs 23 degree(s)C), and 0.8 +/- 0.4 mg vs 2.2 +/- 1.1 mg for cystine stones (-80 vs 23 degree(s)C), p

As the design of Micro-Electro-Mechanical Systems (MEMS) devices matures and their application extends to critical areas, the issues of reliability and long-term survivability become increasingly important. This paper reviews some general approaches to addressing the reliability and qualification of MEMS devices for space applications. The failure modes associated with different types of MEMS devices that are likely to occur, not only under normal terrestrial operations, but also those that are encountered in the harsh environments of space, will be identified.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Because of the greater than or equal to 250 microsecond pulsewidth emitted by the Ho:YAG laser used in clinical lithotripsy, it is unlikely that stress confinement occurs within the irradiated stones. Experimental data supports a thermal mechanism for Ho:YAG laser stone ablation. Stone fragmentation occurs soon after the onset of the laser pulse, is uncorrelated to cavitation bubble formation or collapse, and is associated with low pressures (cf. part I). The mass- loss of desiccated calcium oxalate monohydrate (COM) stones exposed to 150 J from the Ho:YAG laser in air was 40 plus or minus 12 mg (mean plus or minus 1 s.d.); for hydrated stones in air was 25 plus or minus 9 mg; and for hydrated stones in water was 17 plus or minus 3 mg, p less than .001. These differences indicate that direct absorption of the laser radiation by the stone is required for the most efficient ablation. Lowering the initial temperature of COM or cystine stones also reduced the stone mass-loss following 20 J of delivered laser energy: 2.2 plus or minus 1.1 mg vs 5.2 plus or minus 1.6 mg for COM stones (-80 vs 23 degrees Celsius), and 0.8 plus or minus 0.4 mg vs 2.2 plus or minus 1.1 mg for cystine stones (-80 vs 23 degrees Celsius), p less than or equal to .05. Finally, chemical analysis of the laser-induced stone fragments revealed the presence of thermal breakdown products: CaCO 3 from COM; free sulfur and cysteine from cystine; Ca 2 O 7 P 2 from calcium hydorgen phosphate dihydrate, and cyanide from uric acid.

The Ho:YAG laser commonly used for clinical lithotripsy of urinary stones typically emits 250-microsecond pulses at a wavelength of 2.12 micrometer and repetition rates of up to 10 Hz. This pulse duration is longer than the time required for a pressure wave to propagate beyond the optical penetration depth of this wavelength in water. Fast-flash photography was used to study the dynamics of urinary stone fragmentation by the Ho:YAG laser. Stone ablation began approximately 50 microseconds after the onset of the laser pulse, long before the collapse of the cavitation bubble at about 350 microseconds. Pressure measurements, made with a PVDF needle- hydrophone and correlated with the fast-flash images, indicated that the peak acoustical transient was less than 2 bars. Regardless of fiber orientation to the stone, no shockwaves were recorded at the beginning of the bubble, and the maximum pressure waves recorded at bubble collapse were approximately 20 bars. However, no fragmentation occurred during or subsequent to the bubble collapse. The measurements indicated that stone ablation was not due to a photomechanical effect.

The development of thermal damage during pulsed Holmium:YAG (2 — 2. 12 pm, t = 250 s) irradiation of albumen was analyzed experimentally and nmnencally with the intent of validating the Arrhemus integral and investigating the influence of beam profile and dynamic changes in absorption. Fast flash videography wasperformed to document the transient development of coagulation during and after the laser pulse. A two- dimensional dynamic optical-thermal model was developed.The optical component involved a discretized Beer's law method in which absorption is a function of temperature.The thermal component was comprised of 1) a cylindrical coordinate (r,z) finite difference formulation of the heatconduction equation in which the output from the optical model was used as the source term and 2) a routine forcalculating thermal damage using the Arrhenius relation. At the end of each time step, the temperature thstributionwas used to recalculate the absorption coefficient array, which in turn is used in the optical component.The model showed good agreement with experimental data and the literature. The Arrhemus integral was shown tobe valid for the sub-vaporization pulsed laser regime. Experimental and numerical results indicated that spatialbeam profile has a significant impact on thermal damage. Simulations predicted that at higher radiant exposuresdynamic changes in absorption become more significant, accounting for a 10% increase in coagulation depth.Keywords: thermal damage, albumen, pulsed holmium laser, laser-tissue interaction, high speed imaging, heattransfer, dynamic optical properties, numerical modeling, finite difference, Arrhemus integral

A fluorescence-based temperature probe (optrode) was designed to measure temperature within laser-induced vapor bubbles. The optrode consisted of a 400-micrometer optical fiber with a rhodamine B-doped polyurethane film attached to the fiber tip. The film exhibited a fluorescence decay time of 4 ns, and the measured fluorescence yield was temperature dependent in the range from 20 to 110 degrees Celsius. A Ho:YAG laser ((lambda) equals 2.12 micrometer, (tau) p equals 250 microseconds) with a pulse energy of 256 plus or minus 7 mJ delivered through a fiber was used to produce the vapor bubbles. The bubbles reached maximum expansion 200 microseconds after the onset of the laser pulse and had an average lifetime of 350 microseconds. The temperature was measured by positioning the optrode within the vapor bubble and exciting its fluorescent film-coated tip with a nitrogen dye laser ((lambda) equals 540 nm, (tau) p equals 500 ps). At maximum expansion, the temperatures in the vapor bubbles were approximately 61 degrees Celsius, indicating sub-atmospheric saturated vapor pressures. The vapor pressure at maximum expansion was confirmed by Rayleigh's equation.