3D-printed phantoms for characterizing SERS nanoparticle detectability in turbid media.

Recent advances in plasmonic nanoparticle synthesis have enabled extremely high per-particle surface-enhanced Raman scattering (SERS) efficiencies. This has led to the development of SERS tags for in vivo applications (e.g. tumor targeting and detection), providing high sensitivity and fingerprint-like molecular specificity. While the SERS enhancement factor is a major contributor to SERS tag performance, in practice the throughput and excitation-collection geometry of the optical system can significantly impact detectability. Test methods to objectively quantify SERS particle performance under realistic conditions are necessary to facilitate clinical translation. Towards this goal, we have developed 3D-printed phantoms with tunable, biologically-relevant optical properties. Phantoms were designed to include 1 mm-diameter channels at different depths, which can be filled with SERS tag solutions. The effects of channel depth and particle concentration on the detectability of three different SERS tags were evaluated using 785 nm laser excitation at the maximum permissible exposure for skin. Two of these tags were commercially available, featuring gold nanorods as the SERS particle, while the third tag was prepared in-house using silver-coated gold nanostars. Our findings revealed that the measured SERS intensity of tags in solution is not always a reliable predictor of detectability when applied in a turbid medium such as tissue. The phantoms developed in this work can be used to assess the suitability of specific SERS tags and instruments for their intended clinical applications and provide a means of optimizing new SERS device-tag combination products.

Application of optical coherence tomography and optical path length method for monitoring corneal thickness and refractive index change during corneal cross-linking.

Corneal cross-linking (CXL) using UVA irradiation with a riboflavin photosensitizer has emerged as a new treatment paradigm for corneal ectatic disorders. The thickness threshold for protection of intraocular structures has often been challenged with ongoing developments, and corneal thinning becomes an important safety concern, especially for patients with thin corneas. In this study with an ex vivo bovine eye model, we monitored corneal thinning and corneal refractive index changes using optical coherence tomography (OCT) integrated with an adaptation of the optical path length method. CXL experiments were performed based on the standard protocol that includes removal of the corneal epithelium to facilitate diffusion of riboflavin into the stroma. The corneal stromal thickness and group refractive index were measured by a 1310 nm Fourier-domain OCT imaging system at three critical points of the procedure: immediately after epithelial removal, after 30 min riboflavin instillation, and after 30 min UVA irradiation with continuing instillation. We found that the refractive index of the bovine cornea changed significantly from epithelial removal to riboflavin instillation and UVA irradiation, increasing from 1.377±0.005 (mean±standard deviation) after de-epithelization to 1.387±0.003 after 30 min instillation and 1.388±0.008 after subsequent irradiation. The corneas also underwent a considerable decrease (10%-20%) in stromal thickness with thinning of 95±29 µm (mean±standard deviation) after riboflavin instillation and a further decrease (∼5%) with thinning of 42±19 µm after UVA irradiation. Our study highlights the importance of corneal thickness monitoring during CXL, especially after riboflavin instillation when the decrease is the largest, to avoid delivering endothelial cytotoxic doses. An increase in refractive index heightens the concern for corneal thinning and the need for careful monitoring as a safety precaution.

Effect of therapeutic femtosecond laser pulse energy, repetition rate, and numerical aperture on laser-induced second and third harmonic generation in corneal tissue.

Clinical therapy incorporating femtosecond laser (FSL) devices is a quickly growing field in modern biomedical technology due to their precision and ability to generate therapeutic effects with substantially less laser pulse energy. FSLs have the potential to produce nonlinear optical effects such as harmonic generation (HG), especially in tissues with significant nonlinear susceptibilities such as the cornea. HG in corneal tissue has been demonstrated in nonlinear harmonic microscopy using low-power FSLs. Furthermore, the wavelength ranges of harmonic spectral emissions generated in corneal tissues are known to be phototoxic above certain intensities. We have investigated how the critical FSL parameters pulse energy, pulse repetition rate, and numerical aperture influence both second (SHG) and third harmonic generation (THG) in corneal tissue. Experimental results demonstrated corresponding increases in HG intensity with increasing repetition rate and numerical aperture. HG duration decreased with increasing repetition rate and pulse energy. The data also demonstrated a significant difference in HG between FSL parameters representing the two most common classes of FSL therapeutic devices.

Impact of environmental temperature on optical power properties of intraocular lenses.

Optical power properties of lenses and materials in general can be influenced by thermal changes of the material and surrounding medium. In the case of an intraocular lens (IOL) implant, the spherical power (SP), cylinder power, (CP), astigmatism, and spherical aberration are the critical fundamental properties that can significantly impact its efficacy. Directly evaluating how changes in temperature can affect these optical properties may show the importance of considering temperature when evaluating IOL optical characteristics. In this paper, we present a quantitative study on evaluating the impact of environmental temperature changes on IOL fundamental optical properties by testing IOL samples with different materials (e.g., hydrophobic and hydrophilic) and designs (e.g., monofocal and toric) to better encompass types of IOLs in conventional use today. The results from this study demonstrate that significant changes are observed as temperatures are changed from room temperature (20°C) to slightly above body temperature (40°C). Findings indicate that evaluating optical properties at arbitrary temperatures could significantly affect the characterization of IOLs that are already near the tolerance thresholds.

Evaluation of standardized performance test methods for biomedical Raman spectroscopy (Erratum).

The erratum corrects an error in Fig. 4 of the published article.

Alternative method for measuring effective focal length of lenses using the front and back surface reflections from a reference plate.

We present a simple method for measuring the effective focal length without determining the location of principle plane of the lens. The method is based on the measurement of confocal backreflection axial responses from the front and back surfaces of a reference plate with known refractive index and thickness. We proved the concept by measuring the effective focal lengths of thin singlet lenses and complex microscope objectives. The theoretical limit of measurement precision varies depending on the numerical aperture of the lens. This method can provide an alternative focal length measurement method for complex lenses or lenses that are permanently attached to other structures. Measurement errors were analyzed theoretically and improvements in measurement accuracy were discussed.

Axial-scanning low-coherence interferometer method for noncontact thickness measurement of biological samples.

We investigated a high-precision optical method for measuring the thickness of biological samples regardless of their transparency. The method is based on the precise measurement of optical path length difference of the end surfaces of objects, using a dual-arm axial-scanning low-coherence interferometer. This removes any consideration of the shape, thickness, or transparency of testing objects when performing the measurement. Scanning the reference simplifies the measurement setup, resulting in unambiguous measurement. Using a 1310 nm wavelength superluminescent diode, with a 65 nm bandwidth, the measurement accuracy was as high as 11.6 µm. We tested the method by measuring the thickness of both transparent samples and nontransparent soft biological tissues.

Quantification of glistenings in intraocular lenses using a ballistic-photon removing integrating-sphere method.

An alternative method for quantification of glistenings in intraocular lenses (IOLs) using an integrating sphere with an adjustable back aperture to remove ballistic photons is presented. Glistenings in soft IOLs have been known for more than a decade; however, their severity and visual impact are still under investigation. A number of studies have been made to quantitatively describe glistenings in IOLs. Quantization and precise grading of IOLs will provide needed information to evaluate the severity and visual impact of glistenings in patients. We investigated the use of a simple modification of an integrating-sphere method to eliminate ballistic photons to quantitatively measure scattered light from glistenings in IOLs. The method described in this paper provides a simple and effective way to quantitatively characterize glistenings in vitro. It may be especially useful to quantify scattering associated with low-grade glistenings where the density of the scattering centers is low. Finally, the modified integrating-sphere method may also be generally applicable to quantitatively characterize scattering from other optical media.

A Novel Fiber-Optic Confocal Laser Caliper Approach for Non-Contact Optical Characterization of Silk Fibroin Thin Films Created by Riboflavin Photo-Crosslinking.

Silk fibroin with its attractive combination of advanced properties is promising for regenerative treatments of corneal disorders. Novel photonics approach is used to characterize the thickness and refractive index of silk fibroin thin films photo-crosslinked with a natural photosensitizer Riboflavin.

Evaluation of standardized performance test methods for biomedical Raman spectroscopy.

SIGNIFICANCE: Raman spectroscopy has emerged as a promising technique for a variety of biomedical applications. The unique ability to provide molecular specific information offers insight to the underlying biochemical changes that result in disease states such as cancer. However, one of the hurdles to successful clinical translation is a lack of international standards for calibration and performance assessment of modern Raman systems used to interrogate biological tissue. AIM: To facilitate progress in the clinical translation of Raman-based devices and assist the scientific community in reaching a consensus regarding best practices for performance testing. APPROACH: We reviewed the current literature and available standards documents to identify methods commonly used for bench testing of Raman devices (e.g., relative intensity correction, wavenumber calibration, noise, resolution, and sensitivity). Additionally, a novel 3D-printed turbid phantom was used to assess depth sensitivity. These approaches were implemented on three fiberoptic-probe-based Raman systems with different technical specifications. RESULTS: While traditional approaches demonstrated fundamental differences due to detectors, spectrometers, and data processing routines, results from the turbid phantom illustrated the impact of illumination-collection geometry on measurement quality. CONCLUSIONS: Specifications alone are necessary but not sufficient to predict in vivo performance, highlighting the need for phantom-based test methods in the standardized evaluation of Raman devices.

Evaluation of Potential Optical Radiation Hazards from LED Flashlights.

We performed optical radiation safety evaluations of LED flashlights to determine if they pose potential ocular hazards. Six commercially available flashlight samples were randomly selected from various vendors online. They were evaluated in accordance with specifications provided in the American National Standards Institute/Illuminating Engineering Society of North America (ANSI/IESNA) Standards RP 27.1 and RP 27.3. Four of the flashlights were found to have relatively high blue-light-weighted radiance values with short times (40 to 50 s) to reach the exposure limit specified in RP 27.1. These flashlights are in Risk Group 2 and present a moderate risk for retinal damage. Two of the flashlights are in Risk Group 1 and present a low risk for retinal damage. None of the flashlights present an ultraviolet (UV) radiation hazard or a retinal thermal hazard. Cautionary labeling on the packaging as required by RP 27.3 and on the flashlight handle is recommended for flashlights and on other handheld light sources that are in Risk Group 2 or Risk Group 3.

Experimental and analytical quantification of light scattering from vacuoles in intraocular lenses.

PURPOSE: To develop an advanced test methodology for quantification of scattered light from intraocular lenses (IOLs) and to evaluate the correlation between IOL vacuole characteristics and measured scattered light. SETTING: U.S. Food and Drug Administration, Optical Therapeutics and Medical Nanophotonics Laboratory, Silver Spring, Maryland, USA. DESIGN: Experimental and analytical study. METHODS: Twenty-four IOLs containing vacuoles were evaluated using a digital microscopy approach for identifying and characterizing the vacuoles present. A scanning light scattering profiler (SLSP) was used to evaluate and quantify the amount of scattered light from each IOL and from a 25th control IOL without any vacuoles. A variety of IOLs and vacuoles were also modeled in a Zemax simulation of the SLSP, and the simulated scattered light was modeled. RESULTS: The scattered light as measured with SLSP was well correlated with vacuole characteristics, specifically density and size, as measured under the digital microscope for the 24 vacuole-containing IOLs. Additional correlations were found between vacuole sizes, orientations, and the angle at which light was scattered most severely. These correlations were also present in the Zemax model. CONCLUSIONS: Vacuole optical characteristics can be well correlated with measured scatter, demonstrating an ability to predict scattered light based solely on microscope evaluation. Furthermore, the quantitative amount of scatter predicted with Zemax simulations trended closely with the experimentally measured trends.

3D Bioprinting with UVA1 Radiation and Photoinitiator Irgacure 2959: Can the ASTM Standard L929 Cells Predict Human Stem Cell Cytotoxicity?

3D bioprinting often involves human mesenchymal stem cells (hMSC) that are differentiated into the desired cells to replace body parts like ears. Scaffolds of crosslinked hydrogels offer structural support during differentiation. Different photoinitiators are used to make free radicals that photocrosslink these hydrogels; the more penetrating ultraviolet A1 (UVA1) (340-400 nm) wavelengths can be used because Irgacure 2959 only absorbs in the UV (100-400 nm) region. We questioned if the L929 mouse fibroblast cells used in the American Society for Testing Materials standard cytotoxicity assays (F895&F813) can predict the viability of hMSC after exposure to UVA1 radiation alone and in combination with Irgacure 2959 (0.05-0.5% w/v usual range). We exposed both cell types to a high dose of LED UVA1 (370 ± 5 nm; 788 kJ m-2 ) and side by side to increasing UVA1 doses from a glass-filtered black light source combined with either 0.05% (w/v) or 0.5% (w/v) of Irgacure 2959 and monitored their viabilities using flow cytometry. We found UVA1 radiation alone killed ~50% of the hMSC cells compared to ~8% of the L929 cells and significantly more hMSC than L929 died after UVA1 with Irgacure 2959. Thus, L929 cannot be used to accurately predict the viability of hMSC after these specific 3D bioprinting conditions.

Quantitative Multiparameter Evaluation of Vacuoles in Intraocular Lenses Employing a High-Magnification Digital Microscopy Method.

As small imperfections with micrometric sizes, fluid-filled vacuoles, also referred to as glistenings, in intraocular lenses (IOLs) have been known to induce significant unwanted light scattering that in several cases presumably cause complaints and sometimes lead to IOL explantation and replacement. This unwanted scatter is of particular concern for patients viewing bright light in reduced-light conditions such as when driving at night, as the scattered light toward the retina can cause temporary blindness. In this study, we have developed and implemented an accurate test methodology based on a high-magnification digital microscopy approach for quantitative multiparameter evaluation and classification of IOL vacuoles depending on their critical optical characteristics including vacuole size, density, shape, and orientation within the IOL material. Using the multiparameter database developed by evaluating vacuole characteristics, we established a classification grading system that can be used to evaluate vacuole effects on light scattering.

Pulsed laser damage of gold nanorods in turbid media and its impact on multi-spectral photoacoustic imaging.

Innovative biophotonic modalities such as photoacoustic imaging (PAI) have the potential to provide enhanced sensitivity and molecule-specific detection when used with nanoparticles. However, high peak irradiance levels generated by pulsed lasers can lead to modification of plasmonic nanoparticles. Thus, there is an outstanding need to develop practical methods to effectively predict the onset nanoparticle photomodification as well as a need to better understand the process during PAI. To address this need, we studied pulsed laser damage of gold nanorods (GNRs) using turbid phantoms and a multi-spectral near-infrared PAI system, comparing results with spectrophotometric measurements of non-scattering samples. Transmission electron microscopy and Monte Carlo modeling were also performed to elucidate damage processes. In the phantoms, shifts in PAI-detected spectra indicative of GNR damage were initiated at exposure levels one-third of that seen in non-scattering samples, due to turbidity-induced enhancement of subsurface fluence. For exposures approaching established safety limits, damage was detected at depths of up to 12.5 mm. Typically, GNR damage occurred rapidly, over the course of a few laser pulses. This work advances the development of test methods and numerical models as tools for assessment of nanoparticle damage and its implications, and highlights the importance of considering GNR damage in development of PAI products, even for exposures well below laser safety limits.

Experimental investigation of parameters influencing plasmonic nanoparticle-mediated bubble generation with nanosecond laser pulses.

Plasmonic nanoparticles (PNPs) continue to see increasing use in biophotonics for a variety of applications, including cancer detection and treatment. Several PNP-based approaches involve the generation of highly transient nanobubbles due to pulsed laser-induced vaporization and cavitation. While much effort has been devoted to elucidating the mechanisms behind bubble generation with spherical gold nano particles, the effects of particle shape on bubble generation thresholds are not well understood, especially in the nanosecond pulse regime. Our study aims to compare the bubble generation thresholds of gold nanospheres, gold nanorods, and silica-core gold nanoshells with different sizes, resonances, and surface coatings. Bubble generation is detected using a multimodality microscopy platform for simultaneous, nanosecond resolution pump-probe imaging, integrated scattering response, and acoustic transient detection. Nanoshells and large (40-nm width) nanorods were found to have the lowest thresholds for bubble generation, and in some cases they generated bubbles at radiant exposures below standard laser safety limits for skin exposure. This has important implications for both safety and performance of techniques employing pulsed lasers and PNPs.

Author Correction: Quantitative Evaluation of Nanosecond Pulsed Laser-Induced Photomodification of Plasmonic Gold Nanoparticles.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Noncontact method for sensing thickness and refractive index of intraocular lens implants using a self-calibrating dual-confocal laser caliper.

We present a fiber-optic dual-confocal laser caliper method for noncontact high-precision sensing and measuring thickness and refractive index of intraocular lens (IOL) implants. The principle of the method is based on sensing and measuring the confocal intensity response of the laser beam reflection from the opposite object surfaces, which provides the advanced feature of having no limitations on the object shape, thickness, and transparency. Using single-mode optical fibers and a 658-nm laser source, the thickness measurement accuracy was assessed to be as high as 5 µm. In addition, refractive index of a transparent object with thickness smaller than the working distance of the focusing lenses can be measured. The thickness and refractive index of a planoconvex IOL were measured with a high accuracy.

Simple fiber-optic confocal microscopy with nanoscale depth resolution beyond the diffraction barrier.

A novel fiber-optic confocal approach for ultrahigh depth-resolution (

Quantitative Evaluation of a Time-Dependent Eye Hazard Posed by Laser Pointers.

A novel test methodology was developed for quantitative evaluation of critical radiant power characteristics as a function of time for diode pumped solid state (DPSS) laser pointers. It is based on a simultaneous measurement of time-dependent radiant power characteristics of multi-wavelength spectral components emitted by DPSS laser pointers. The authors tested green DPSS laser pointers, which emit three spectral components at the fundamental near-infrared (1064-nm), pumping near-infrared (808-nm), and second-harmonic green (532-nm) wavelengths. The obtained experimental results are employed for performing eye hazard evaluation according to U.S. and International laser safety standards. All tested green laser pointers demonstrated significant variability of radiant power as a function of time and wavelength. Thus, the severity of the potential eye hazard from DPSS laser pointers for a given exposure time depends on when a person was exposed after the pointer was turned on. Most laser pointers emitted radiation in excess of their classification limits, including unwanted infrared radiation that is not necessary for their intended use as laser pointers.