Name: scanning light scattering profiler (slps) based methodology to quantitatively evaluate forward and backward light scattering from intraocular lenses
Uploaded: 2017-06-06T15:30:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses at JoVE.com

The authors would like to thank the companies for the access of their monofocal and multifocal IOLs. This work was supported by Oak Ridge Institute for Science and Education (ORISE) and the Medical Device Fellowship Program (MDFP) and their contributions are appreciated. In addition, the authors would like to thank Samuel Song for his contributions in the laboratory.

1. SLSP Measurement Platform Preparation
NOTE: All alignment steps require precision and patience to ensure accurate quantitation when measuring light scatter. An overview of the SLSP setup in provided within Figure 1. Here, an illustration (Figure 1a) shows the basic concept of the SLSP setup. In addition, Figures 1b and 1c help define the various angles referenced within the discussion. Specifically, the following three angles are defined within Figures 1b and 1c: &#730;R (sensor rotational angle), &#730;S (sensor angle of measurement), and &#730;I (IOL angle of incidence).
<ol>
	<li>SLSP Alignment (Figure 2).
	<ol>
		<li>Focus a narrow-linewidth laser source (here, a 543 nm central wavelength) into a single-mode delivery optical fiber using a 10&#215; infinity corrected objective lens. 
		NOTE: Test the light source to ensure that the lumen output is steady or measurements will be difficult to quantify. A focused beam is determined by observing light passing through the fiber, this will not achieve 100% efficiency, but should be enough so that light can ultimately be detected by the sensor.</li>
		<li>Collimate the light source by integrating the single-mode optical fiber with a 10X infinity corrected objective lens so that the fiber is positioned on the focal point of the objective lens. The output light should result in a uniform Gaussian beam profile.</li>
		<li>Position an iris aperture in front of light source to adjust the diameter of Gaussian beam. 
		NOTE: Set the iris aperture diameter to be representative of a human eye (e.g. 1-6 mm diameter). As the light scatter type complaints are commonly associated with night driving, iris aperture diameters representative of a dilated iris may be preferable.</li>
		<li>Construct a goniophotometer by attaching a photodiode sensor to a motorized/programmable 360&#730; rotational stage with linear translation (x, y, and z direction) capabilities using an extendable arm (metal post with post clamp). 
		NOTE: Design a stage platform that enables translation as well as tilt adjustments. Design the sensor mount that enables 360&#730; of sensor rotational angle (&#730;R) and can be adjusted to at least 45&#730; of sensor angle rotation (&#730;S) to measure different planes of scatter. The distance of the extended arm is dependent on the sensitivity of the photodiode sensor and the desired angular precision.</li>
		<li>Adjust the sensor angle of detection (as needed) by angling the sensor face and adjusting the location of the arms.</li>
	</ol>
	</li>
	<li>IOL Alignment
	<ol>
		<li>Construct an IOL holding platform so that the IOL is positioned above the goniophotometer (Figure 2).
		<ol>
			<li>To accomplish this, build the IOL holding platform so that the IOL is suspended above the center of the goniophotometer (reversing the positions of the goniophotometer and IOL is also possible).
			<ol>
				<li>To construct the platform use four, 18&#34; long, &#189;&#34; diameter cylindrical posts and post stands and attach them to a 18 x 18&#34; breadboard. This breadboard is the base support for the platform.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Attach a translational stage (x, y, and z direction) with tilting and rotational (I&#730;) capabilities underneath the breadboard so that the stage is facing down. 
		NOTE: Translation stages with small step sizes (a few microns) enable higher precision during the alignment of the IOL and will improve goniophotometry accuracy. The specific dimensions of the platform can be customized to individual needs. As a result, the cylindrical posts and breadboard dimensions can be adjusted.
		<ol>
			<li>Securely attach the IOL to the IOL holding platform by clamping one of the IOL haptics. 
			NOTE: In this proof of purpose experiment, IOLs are tested in air; however, IOLs in solution and temperatures that best represent in vivo conditions would be ideal.</li>
		</ol>
		</li>
		<li>Align the IOL directly in front of the light source (with the IOL plane of focus perpendicular to the light source) using linear and tilt adjustments from the IOL holding platform stage to ensure that the direction of light does not change while passing through the center of the IOL. This position will constitute an angle of incidence (I&#730;) of 0&#730;.</li>
		<li>Identify the location of the focal spot of light from the IOL and position a small conical device at the focal spot to mitigate detection of defocused light (when necessary). Identify the focal spot of light by placing a piece of paper (such as a business card) behind the IOL and identifying where the light is most tightly focused. This can be a subjective measurement. 
		NOTE: This step is only necessary if wanting to measure purely non-ballistic light.</li>
		<li>Position the motorized stage for the photodiode sensor directly underneath the IOL to ensure that the IOL is located in the center of the goniophotometer trajectory. Align the goniophotometer so that it is approximately 12 cm away from the IOL. 
		NOTE: The relationship of the IOL and the goniophotometer will determine the resolution of the tests, where the further away the goniophotometer is located the greater resolution can be achieved. However, increased distance (and smaller step sizes) will result in lower signal and longer experimentation times.</li>
		<li>Adjust the angle of incidence (I&#730;) by rotating the IOL holding platform stage. 
		NOTE: Initial experiments should be conducted with an angle of incidence of 0&#730; to 80&#730;. Beyond 80&#730; will begin to near the grazing angle where all light will be reflected.</li>
	</ol>
	</li>
	<li>Programming
	<ol>
		<li>Build a software program to coordinate the mechanical movement of the sensor with its corresponding light measurement using system design software (see Supplemental file 1 and Table of Materials). 
		NOTE: When building the software program take into consideration the speed of the sensor to ensure that the physical location of the sensor accurately reflects its recorded measurement. The program designed for this experiment is provided in Supplemental file 1.</li>
	</ol>
	</li>
</ol>
2. SLSP Experimentation and Data Analysis
<ol>
	<li>Scanning (&#730;R)
	<ol>
		<li>Ensure that the IOL and the light source are properly aligned (see sections 1.1 and 1.2).</li>
		<li>Construct an enclosure around the photodiode sensor and the IOL using a container with non-reflective internal coating to minimize the detection of errant light. Ensure to provide an opening for the light source. 
		NOTE: The specific design of the enclosure should be customized based on an external light in the room. As a result, multiple designs are usable. However, the purpose of the enclosure is to mitigate all external light from being detected by the sensor.</li>
		<li>Turn off all light sources within the room, except for the programming computer.</li>
		<li>Run the SLSP software program (step 1.3.1) so that the sensor rotates around the IOL to measure scattered light at each degree of rotation (&#730;R).</li>
		<li>To measure scattered light at more than one plane, run the SLSP software program multiple times while manually adjusting the sensor&#39;s extended arm and sensor angle of measurement (&#730;S). 
		NOTE: The number of times the program is run is dependent on the desired outcome. The more angles of detection measured will result in more precision for identifying the directionality of scattered light.</li>
		<li>For studies on beam diameter, adjust the iris aperture to the desired diameter before running the SLSP program. 
		NOTE: Here, the laser beam diameters of 1, 2, 3, 4, and 4.64 mm were used to best mimic typical iris diameters. 4.64 mm was the largest diameter used as this was the diameter of the collimated beam without passing through the iris aperture.</li>
		<li>For studies on angle of incidence, rotate the IOL mount to the desired angle of incidence before running the SLSP program. Here, angles of incidence (I&#730;) of 0&#730;, 20&#730;, 45&#730;, and 80&#730; were studied. 
		NOTE: A scientific data processing package is needed for the analysis of the collected data.</li>
		<li>For three dimensional imaging, stich together the data from each scan at differing &#730;S with a data processing package. Stich the data by plotting a matrix book where the sensor angle of measurement (&#730;S) is plotted against the angle or rotation (&#730;R). 
		NOTE: To better represent in vitro conditions, the SLSP platform can be reversed so that the goniophotometer is above the IOL and the IOL can then be placed inside of a temperature controlled saline solution bath. However, in these conditions, sensor dwell times will need to be considerably longer to take into consideration the motion of the saline solution as the sensor is moved from position to position and displaces the medium.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Walker, B. N., James, R. H., Calogero, D., Ilev, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+full-angle+scanning+light+scattering+profiler+to+quantitatively+evaluate+forward+and+backward+light+scattering+from+intraocular+lenses.">A novel full-angle scanning light scattering profiler to quantitatively evaluate forward and backward light scattering from intraocular lenses.</a> Rev. Sci. Instrum. 86 (9), (2015).</li><li>Vandenberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+relation+between+glare+and+straylight.">On the relation between glare and straylight.</a> Doc. Ophthalmol. 78 (3-4), 177-181 (1991).</li><li>De Hoog, E., Doraiswamy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+impact+of+light+scatter+from+glistenings+in+pseudophakic+eyes.">Evaluation of the impact of light scatter from glistenings in pseudophakic eyes.</a> J. Cataract. Refract. Surg. 40 (1), 95-103 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Global Intraocular Lens Market 2013-18: Industry Nanotechnology Analysis, Size, Share, Strategies, Growth, Trends and Forecast Research Report. , (2013).</li><li>Congdon, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+cataract+and+pseudophakia/aphakia+among+adults+in+the+United+States.">Prevalence of cataract and pseudophakia/aphakia among adults in the United States.</a> Arch. Ophthalmol. 122 (4), 487-494 (2004).</li><li>Mester, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Personality+Characteristics+on+Patient+Satisfaction+After+Multifocal+Intraocular+Lens+Implantation:+Results+From+the+&amp;#34;Happy+Patient+Study&amp;#34.">Impact of Personality Characteristics on Patient Satisfaction After Multifocal Intraocular Lens Implantation: Results From the &amp;#34;Happy Patient Study&amp;#34.</a> J. Refractive Surg. 30 (10), 674-678 (2014).</li><li>Ferrer-Blasco, T., Montes-Mico, R., Cervino, A., Alfonso, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+Scatter+and+Disability+Glare+After+Intraocular+Lens+Implantation.">Light Scatter and Disability Glare After Intraocular Lens Implantation.</a> Arch. Ophthalmol. 127 (4), 576-577 (2009).</li><li>Landry, R. J., Ilev, I. K., Pfefer, T. J., Wolffe, M., Alpar, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+reflections+from+intraocular+lens+implants.">Characterizing reflections from intraocular lens implants.</a> Eye. 21 (8), 1083-1086 (2007).</li><li>Kim, D. H., James, R. H., Landry, R. J., Calogero, D., Anderson, J., Ilev, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+glistenings+in+intraocular+lenses+using+a+ballistic-photon+removing+integrating-sphere+method.">Quantification of glistenings in intraocular lenses using a ballistic-photon removing integrating-sphere method.</a> Appl. Opt. 50 (35), 6461-6467 (2011).</li><li>Portney, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IOL+with+Square-Edged+Optic+and+Reduced+Dysphotopsia.">IOL with Square-Edged Optic and Reduced Dysphotopsia.</a> Optom. Vis. Sci. 89 (2), 229-233 (2012).</li><li>Choi, J., Schwiegerling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+performance+measurement+and+night+driving+simulation+of+ReSTOR,+ReZoom,+and+Tecnis+multifocal+intraocular+lenses+in+a+model+eye.">Optical performance measurement and night driving simulation of ReSTOR, ReZoom, and Tecnis multifocal intraocular lenses in a model eye.</a> J. Refractive Surg. 24 (3), 218-222 (2008).</li><li>Artigas, J. M., Felipe, A., Navea, A., Carmen Garcia-Domene, M., Pons, A., Mataix, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+scattering+in+intraocular+lenses+by+spectrophotometric+measurements.">Determination of scattering in intraocular lenses by spectrophotometric measurements.</a> J. Biomed. Opt. 19 (12), (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmic+implants+-+Intraocular+lenses+Part+2:+Optical+properties+and+test+methods.">Ophthalmic implants - Intraocular lenses Part 2: Optical properties and test methods.</a> The International Organization for Standardization (ISO). ISO 11979 (2), (2014).</li></ol>

The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.

The results from the SLSP platform experiments have found that using simple goniophotometry principles can lead to a powerful tool for evaluating the properties of light scatter associated with unique IOL designs and materials. Specifically, the SLSP platform has observed a direct correlation between the amount of detectable scattered light and the beam diameter of the light source. In addition, the multiple scattered peaks found in multifocal IOLs were easily observed with the SLSP. Furthermore, as the source of light a...

The scanning light scattering profiler (SLSP) approach was first introduced to address the need to quantitatively evaluate light scattering characteristics of intraocular lenses (IOLs) in a non-clinical setting1. Developing a test methodology to evaluate the light scattering tendencies of IOL designs and materials is of significant interest in order to help identify potential unwanted light scattering problems. Light scatter is commonly reported by patients and observed as glare, glistening, optical imperfections, and other forms of dysphotopsia2, sometimes leading to a patient requesting the IOL explantation. In addition to dysphotopsia, scattered light reduces the amount of ballistic light, resulting in lower overall image quality3. Developing a device that can non-clinically evaluate the IOL potential to scatter the incoming light (and later correlated with clinically reported outcomes) can be useful.
Evaluating optical properties of IOLs (the lens used to replace the human crystalline lens after cataract surgery) is of particular interest as it is the most commonly implanted medical device in the world (almost 20 million per year)4 and the United States (over 3 million per year)5. As a result, even a small percentage of patients reporting dysphotopsia may have a large impact. In addition, rapidly improving technologies (e.g. new IOL designs, materials, and optical capabilities) have the potential to increase concerns related to light scattering. For example, multifocal IOLs have been designed to improve near and far visual acuity by designing lenses that utilize refraction and diffraction optical principles. Although highly successful, these lenses have also been found to increase the amount of reported halos and glare, largely associated with scattering of light6.
A few non-clinical laboratory studies attempt to predict dysphotopsia from scattered light as it passes through IOLs7. For example, research has identified that IOL haptics (the arms of the IOL used to set it in place) and the edge of the IOLs are prone to induce a large amount of the observed glare scattered light8. One method, a ballistic-photon removing integrating-sphere method (BRIM), was introduced to quantitatively measure the amount of total non-ballistic light after passing through an IOL9. However, this highly sensitive technique is designed to measure the total intensity of scattered light and is unable to identify directionality of the scattered light. Computer simulation software can be used with model eyes to help predict intensity and directionality of light scatter from various IOL designs and materials. For example, the propensity for the IOL edge to induce the light scattering was simulated to identify designs that would limit the amount of scattered light10. Furthermore, computer simulations that incorporated the Mie scattering theory verified that increased light scatter can reduce the modulation transfer function (MTF) of the IOL (a direct correlation to image quality)3. Although helpful, real bench tests would be necessary to verify these predictive simulations.
To verify predictive simulations a bench test is necessary that is capable of detecting and quantitatively evaluating two distinct forms of scattered light, forward scattered and backward scattered light. Although not a source of dysphotopsia, backward scattered light (light scattering away from the eye) is a cause for reduced image quality, as less light passes through the IOL to ultimately reach the retina. Forward scattered light (light scattering towards the retina) is a concern for ophthalmologists as it may result in complaints of dysphotopsia (e.g. glare, halo, and glistening). One common example is patients reporting additional unwanted glare from passing oncoming cars during night driving; this issue is particularly common with multifocal IOLs11. However, current practice to identify potential forward scattered light is for ophthalmologists to shine light onto the patient&#39;s eye and qualitatively observe how much light is reflected back (backward scattered light) and assuming that the backward scattered light will be approximately the same as the forward scattered light (which is not always the case)12.
Here, we describe a simple test methodology using goniophotometry principles to quantitatively measure the magnitude and direction of scattered light at it passes through an intraocular lens. The SLSP operates by rotating a photodiode sensor 360 degrees around an IOL that is exposed to a light source, see Figure 1a. We chose a green laser source (543 nm) to best represent the known photopic maximum and to agree with the international standard specifications13. Here, an IOL is adapted onto a rotational and translational holder where a photodiode sensor can circle around and observe light scattering off of the lens. As a result, the SLSP has the unique capability to quantitatively measurement the magnitude and directionality of scattered light. However, although not described here, for better predictive capabilities, experiments should be conducted within a controlled environment using an appropriate eye model. The distance between the IOL and the optical sensor (as well as the size of the sensor element) will determine the resolution capabilities of the device; however, there will be a tradeoff between resolution and signal strength that will need to be adjusted, as needed.
To accurately describe the principles of the SLSP platform we define three types of rotational angles, see Figures 1b and 1c. Specifically, the rotation angle (&#730;R) represents the rotation of a photodiode sensor as it rotates around an IOL. Here, 0&#730;R would represent when the sensor is behind the lens (backward scattered light) and 180&#730;R represents when the sensor is in front of the lens (forward scattered light). Angles of 90&#730; and 270&#730; represent the transition points between forward and backward scattered light. The sensing angle (&#730;S) represents degrees that the sensor is pivoted (in the up and down direction) so that it can detect more than one plane of scattered light. Here, 0&#730;S means the sensor surface is parallel to the IOL (and light source). Finally, the angle of incidence (&#730;I) represents the angle that the light source is approaching the IOL from. Here, 0&#730;I corresponds to when the incident light is on the optical axis of the IOL and 90&#730; would represent when the light source is perpendicular to the Meridional plane.

<table><tbody><tr><td>PD300 series Photodiode Sensor</td><td>Ophir-Spiricon Corp</td><td>7Z02410</td><td>PD300-1W, RoHS</td></tr><tr><td>URS Series Precision Rotation Stage</td><td>Newport Corp.</td><td>URS75BCC</td><td></td></tr><tr><td>ESP301 1-Axis Motion Controller and Driver</td><td>Newport Corp.</td><td>ESP301-1N</td><td></td></tr><tr><td>LabView Software</td><td>National Instruments Corp.</td><td>776671-35</td><td></td></tr><tr><td>Origin</td><td>OriginLab Corp.</td><td>N/A</td><td></td></tr><tr><td>Single Mode FC/APC Fiber Optic Patch Cables</td><td>ThorLabs Inc.</td><td>P3-460B-FC</td><td></td></tr><tr><td>10X Olympus Plan Achromat Objective</td><td>ThorLabs Inc.</td><td>RMS10X</td><td>RMS10X - 10X Olympus Plan Achromat Objective, 0.25 NA, 10.6 mm WD&amp;nbsp;</td></tr></tbody></table>

scanning light scattering profiler (slps) based methodology to quantitatively evaluate forward and backward light scattering from intraocular lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light scattering from intraocular lenses (IOLs) using goniophotometer principles. This protocol describes the SLSP platform and how it employs a 360&#176; rotational photodetector sensor that is scanned around an IOL sample while recording the intensity and location of scattered light as it passes through the IOL medium. The SLSP platform can be used to predict, non-clinically, the propensity for current and novel IOL designs and materials to induce light scatter. Non-clinical evaluation of light scattering properties of IOLs can significantly reduce the number of patient complaints related to unwanted glare, glistening, optical defects, poor image quality, and other phenomena associated with the unintended light scattering. Future studies should be conducted to correlate SLSP data with clinical results to help identify which measured light scatter is most problematic for patients that have undergone cataract surgery subsequent to IOL implantation.

Goniophotometry measurements can produce 360&#730;R of signal when the sensor is not located on the plane of the light source. However, to collect measurements from scattered light on the plane of the light source (0&#730;I) the sensor will need to eclipse the light source, resulting in less than 360&#730;R of signal. In our experiments, it was determined that ~20&#730;R of signal was blocked as the sensor eclipsed the light source.
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Watch this Scientific Journal Video about Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses at JoVE.com

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

scanning-light-scattering-profiler-slps-based-methodology-to

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Engineering

7.5K Views.  U.S. Food and Drug Administration. This protocol describes the scanning light scattering profiler (SLSP) that enables the full-angle quantitative evaluation of forward and backward scattering of light from intraocular lenses (IOLs) using goniophotometer principles.

Video: Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the milli-scale structure of barbs and barbules largely determines the directional pattern of reflected light; and through the macro-scale spatial structure of overlapping, curved feathers, these directional effects create the visual texture. Milli-scale and macro-scale effects determine where on the organism's body, and from what viewpoints and under what illumination, the iridescent colors are seen. Thus, the highly directional flash of brilliant color from the iridescent throat of a hummingbird is inadequately explained by its nano-scale structure alone and questions remain. From a given observation point, which milli-scale elements of the feather are oriented to reflect strongly? Do some species produce broader "windows" for observation of iridescence than others? These and similar questions may be asked about any organisms that have evolved a particular surface appearance for signaling, camouflage, or other reasons.
In order to study the directional patterns of light scattering from feathers, and their relationship to the bird's milli-scale morphology, we developed a protocol for measuring light scattered from biological materials using many high-resolution photographs taken with varying illumination and viewing directions. Since we measure scattered light as a function of direction, we can observe the characteristic features in the directional distribution of light scattered from that particular feather, and because barbs and barbules are resolved in our images, we can clearly attribute the directional features to these different milli-scale structures. Keeping the specimen intact preserves the gross-scale scattering behavior seen in nature. The method described here presents a generalized protocol for analyzing spatially- and directionally-varying light scattering from complex biological materials at multiple structural scales.

We present a non-destructive method for sampling spatial variation in the direction of light scattered from structurally complex materials. By keeping the material intact, we preserve gross-scale scattering behavior, while concurrently capturing fine-scale directional contributions with high-resolution imaging. Results are visualized in software at biologically-relevant positions and scales.

Light interacts with an organism's integument on a variety of spatial scales. For example in an iridescent bird: nano-scale structures produce color; the ...

Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments

In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. From the measured surface, 3D topographic maps can be subsequently analyzed using a variety of software packages to extract the information that is needed.
In this article we describe how white light interferometry, and optical profilometry (OP) in general, combined with generic surface analysis software, can be used for materials science and engineering tasks. In this article, a number of applications of white light interferometry for investigation of surface modifications in mass spectrometry, and wear phenomena in tribology and lubrication are demonstrated. We characterize the products of the interaction of semiconductors and metals with energetic ions (sputtering), and laser irradiation (ablation), as well as ex situ measurements of wear of tribological test specimens.
Specifically, we will discuss:
<ol class="lroman">
	<li>Aspects of traditional ion sputtering-based mass spectrometry such as sputtering rates/yields measurements on Si and Cu and subsequent time-to-depth conversion.</li>
	<li>Results of quantitative characterization of the interaction of femtosecond laser irradiation with a semiconductor surface. These results are important for applications such as ablation mass spectrometry, where the quantities of evaporated material can be studied and controlled via pulse duration and energy per pulse. Thus, by determining the crater geometry one can define depth and lateral resolution versus experimental setup conditions.</li>
	<li>Measurements of surface roughness parameters in two dimensions, and quantitative measurements of the surface wear that occur as a result of friction and wear tests.</li>
</ol>
Some inherent drawbacks, possible artifacts, and uncertainty assessments of the white light interferometry approach will be discussed and explained.

White light microscope interferometry is an optical, noncontact and quick method for measuring the topography of surfaces. It is shown how the method can be applied toward mechanical wear analysis, where wear scars on tribological test samples are analyzed; and in materials science to determine ion beam sputtering or laser ablation volumes and depths.

In materials science and engineering it is often necessary to obtain quantitative measurements of surface topography with micrometer lateral resolution. ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Chemistry

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as aqueous solutions and biomaterials, with fast acquisition times. Here, we discuss the assembly and operation of a Brillouin spectrometer that uses stimulated Brillouin scattering (SBS) to measure stimulated Brillouin gain (SBG) spectra of water and lipid emulsion-based tissue-like samples in transmission mode with &lt;10 MHz spectral-resolution and &lt;35 MHz Brillouin-shift measurement precision at &lt;100 ms. The spectrometer consists of two nearly counter-propagating continuous-wave (CW) narrow-linewidth lasers at 780 nm whose frequency detuning is scanned through the material Brillouin shift. By using an ultra-narrowband hot rubidium-85 vapor notch filter and a phase-sensitive detector, the signal-to-noise-ratio of the SBG signal is significantly enhanced compared to that obtained with existing CW-SBS spectrometers. This improvement enables measurement of SBG spectra with up to 100-fold faster acquisition times, thereby facilitating high spectral-resolution and high-precision Brillouin analysis of soft materials at high speed.

We describe the construction of a rapid continuous-wave-stimulated-Brillouin-scattering (CW-SBS) spectrometer. The spectrometer employs single-frequency diode-lasers and an atomic vapor notch-filter to acquire transmission spectra of turbid/non-turbid samples with high spectral-resolution at speeds up to 100-fold faster than those of existing CW-SBS spectrometers. This improvement enables high-speed Brillouin material analysis.

Recent years have witnessed a significant increase in the use of spontaneous Brillouin spectrometers for non-contact analysis of soft matter, such as ...

Scattering And Absorption of Light in Planetary Regoliths

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed microscopic particles. The theoretical and numerical methods constitute a framework of Radiative Transfer with Reciprocal Transactions (R2T2). The R2T2 framework entails Monte Carlo order-of-scattering tracing of interactions in the frequency space, assuming that the fundamental scatterers and absorbers are wavelength-scale volume elements composed of large numbers of randomly distributed particles. The discrete random media are fully packed with the volume elements. For spherical and nonspherical particles, the interactions within the volume elements are computed exactly using the Superposition T-Matrix Method (STMM) and the Volume Integral Equation Method (VIEM), respectively. For both particle types, the interactions between different volume elements are computed exactly using the STMM. As the tracing takes place within the discrete random media, incoherent electromagnetic fields are utilized, that is, the coherent field of the volume elements is removed from the interactions. The experimental methods are based on acoustic levitation of the samples for non-contact, non-destructive scattering measurements. The levitation entails full ultrasonic control of the sample position and orientation, that is, six degrees of freedom. The light source is a laser-driven white-light source with a monochromator and polarizer. The detector is a mini-photomultiplier tube on a rotating wheel, equipped with polarizers. The R2T2 is validated using measurements for a mm-scale spherical sample of densely-packed spherical silica particles. After validation, the methods are applied to interpret astronomical observations for asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko (Figure 1) recently visited by the NASA Dawn mission and the ESA Rosetta mission, respectively.

Numerical and experimental methods are presented for multiple scattering of light in discrete random media of densely-packed particles. The methods are utilized to interpret the observations of asteroid (4) Vesta and comet 67P/Churyumov-Gerasimenko.

Theoretical, numerical, and experimental methods are presented for multiple scattering of light in macroscopic discrete random media of densely-packed ...

Measuring the Behavioral Effects of Intraocular Scatter

Intraocular scatter, with its associated functional manifestations, is a leading cause of automotive accidents and a significant biomarker of covert and overt ocular disease (e.g., diseases of the cornea and lens). Nearly all current methods of measuring the behavioral consequences of light scatter, however, suffer from various limitations mostly reflecting a lack of construct and content validity: to wit, the measures do not adequately reflect real world conditions (e.g., artificial light vs. sunlight) or everyday tasks (e.g., recognition under visually demanding conditions).
This protocol describes two novel, ecologically valid methods of measuring the behavioral effects of intraocular scatter by quantifying scatter geometry and visual recognition under glare conditions. The former was measured by assessing the diameter of halos and spokes that resulted from a bright point source. Light spread (essentially, the point spread function determined using Rayleigh criteria) was quantified by determining the minimum perceivable distance between two small points of broad-band light. The latter was done based on the identification of letters formed using apertures through which bright light was shone.

In this protocol, we outline the conceptual design elements and structural development of a glare acuity apparatus. Additionally, the design of a device for measuring positive dysphotopsia (halos, spokes) and two-point light thresholds is described.

Intraocular scatter, with its associated functional manifestations, is a leading cause of automotive accidents and a significant biomarker of covert and ...

Behavior

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Bioengineering

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

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Chapters in this video

Measuring Spatially- and Directionally-varying Light Scattering from Biological Material

Characterization of Surface Modifications by White Light Interferometry: Applications in Ion Sputtering, Laser Ablation, and Tribology Experiments

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

High-speed Continuous-wave Stimulated Brillouin Scattering Spectrometer for Material Analysis

Scattering And Absorption of Light in Planetary Regoliths

Measuring the Behavioral Effects of Intraocular Scatter

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy