Project description:Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.
Project description:PurposeTo psychophysically determine macular pigment optical density (MPOD) employing the heterochromatic modulation photometry (HMP) paradigm by estimating 460 nm absorption at central and peripheral retinal locations.MethodsFor the HMP measurements, two lights (B: 460 nm and R: 660 nm) were presented in a test field and were modulated in counterphase at medium or high frequencies. The contrasts of the two lights were varied in tandem to determine flicker detection thresholds. Detection thresholds were measured for different R:B modulation ratios. The modulation ratio with minimal sensitivity (maximal threshold) is the point of equiluminance. Measurements were performed in 25 normal subjects (11 male, 14 female; age: 30 ± 11 years, mean ± sd) using an eight channel LED stimulator with Maxwellian view optics. The results were compared with those from two published techniques - one based on heterochromatic flicker photometry (Macular Densitometer) and the other on fundus reflectometry (MPR).ResultsWe were able to estimate MPOD with HMP using a modified theoretical model that was fitted to the HMP data. The resultant MPODHMP values correlated significantly with the MPODMPR values and with the MPODHFP values obtained at 0.25° and 0.5° retinal eccentricity.ConclusionsHMP is a flicker-based method with measurements taken at a constant mean chromaticity and luminance. The data can be well fit by a model that allows all data points to contribute to the photometric equality estimate. Therefore, we think that HMP may be a useful method for MPOD measurements, in basic and clinical vision experiments.
Project description:We present axial plane optical microscopy (APOM) that can, in contrast to conventional microscopy, directly image a sample's cross-section parallel to the optical axis of an objective lens without scanning. APOM combined with conventional microscopy simultaneously provides two orthogonal images of a 3D sample. More importantly, APOM uses only a single lens near the sample to achieve selective-plane illumination microscopy, as we demonstrated by three-dimensional (3D) imaging of fluorescent pollens and brain slices. This technique allows fast, high-contrast, and convenient 3D imaging of structures that are hundreds of microns beneath the surfaces of large biological tissues.
Project description:We describe a new method for calibrating optical trapping measurements in which tension is applied in the direction of the laser beam to a molecule tethered between a surface and an optically trapped bead. Specifically, we present a generally-applicable procedure for converting from the measured scattering intensity and the measured stage displacement to applied tension and bead-coverslip separation, using measurements of the light intensity scattered from an untethered, trapped bead. Our calibration accounts for a number of effects, including aberrations and the interference of forward-reflected bead-scattered light with the trapping beam. To demonstrate the accuracy of our method, we show measurements of the DNA force-versus-extension relation using a range of laser intensities, and show that these measurements match the expected extensible wormlike-chain (WLC) behavior. Finally, we also demonstrate a force-clamp, in which the tension in a tether is held fixed while the extension varies as a result of molecular events.
Project description:PURPOSE:To discuss the impact of intraocular lens-(IOL)-power, IOL-thickness, IOL-shape, corneal power and effective lens position (ELP) on the distance between the anterior IOL vertex (ALP) of a thick IOL and the ELP of its thin lens equivalent. METHODS:We calculated the ALP of a thick IOL in a model eye, which results in the same focal plane as a thin IOL placed at the ELP using paraxial approximation. The model eye included IOL-power (P), ELP, IOL-thickness (Th), IOL-shape-factor (X), and corneal power (DC). The initial values were P = 10 D (diopter: 1 D = 1 m-1), 20 D, 30 D, Th = 0.9 mm, ELP = 5 mm, X = 0, DC = 43 D. The difference between ALP and the ELP was illustrated as a function of each of the model parameters. RESULTS:The ALP of a thick lens has to be placed in front of the ELP for P>0 IOLs to achieve the same optical effect as the thin lens equivalent. The difference ALP-ELP for the initial values is -0.57 mm. Minus power IOLs (ALP-ELP = -0.07 mm, for IOL-power = -5 D) and convex-concave IOLs (ALP-ELP = -0.16 mm, for X = 1) have to be placed further posterior. The corneal power and ELP have less influence, but corneal power cannot be neglected. CONCLUSION:The distance between ELP and ALP primarily depends on IOL-power, IOL-thickness, and shape-factor.
Project description:Purpose:This retrospective longitudinal study aimed to examine the relationship between ocular higher-order aberrations (HOA) and axial eye growth in young myopic children undergoing orthokeratology (ortho-k) treatment. Methods:Axial length and ocular HOA, measured under cycloplegia annually over a 2-year period from the right eyes of myopic children, who previously completed ortho-k clinical trials, were retrieved. Linear mixed model analyses were applied to determine the association between ocular HOA, other known confounding variables (age, sex, and refractive error), and axial eye growth. Results:Data from 103 subjects were analyzed. The root-mean square (RMS) values of total ocular HOA (third to sixth orders combined), spherical (\({\rm{Z}}_4^0\) and \({\rm{Z}}_6^0\) combined), and comatic (\({\rm{Z}}_3^{ - 1}\), \({\rm{Z}}_3^1\), \({\rm{Z}}_5^{ - 1}\), and \({\rm{Z}}_5^1\) combined) aberrations increased by approximately 3, 9, and 2 times, respectively, after 2 years of ortho-k treatment. After adjusting for age, sex, and refractive error, higher RMS values of total HOA and spherical aberrations were associated with both longer axial length and slower axial elongation (all P < 0.01). For individual Zernike term coefficients, a higher level of positive spherical aberration (\({\rm{Z}}_4^0\)) was also associated with longer axial length and slower axial elongation (both P < 0.01), after adjusting for baseline HOA. Conclusions:Ortho-k for myopia control significantly increases the Zernike coefficients and therefore the RMS values for a range of total ocular HOA terms or metrics in children. These findings suggest the potential role of HOA, particularly spherical aberration, as the possible mechanism of slowing axial elongation in ortho-k treatment.
Project description:This retrospective longitudinal analysis aimed to investigate the association between ocular higher-order aberrations (HOAs) and axial eye growth in Hong Kong children. Measures of axial length and ocular HOAs under cycloplegia were obtained annually over a two-year period from 137 subjects aged 8.8?±?1.4 years with mean spherical equivalent refraction of -2.04?±?2.38 D. A significant negative association was observed between the RMS of total HOAs and axial eye growth (P?=?0.03), after adjusting for other significant predictors of axial length including age, sex and refractive error. Similar negative associations with axial elongation were found for the RMS of spherical aberrations ([Formula: see text] and [Formula: see text] combined) (P?=?0.037). Another linear mixed model also showed that greater levels of vertical trefoil [Formula: see text], primary spherical aberration [Formula: see text] and negative oblique trefoil [Formula: see text] were associated with slower axial elongation and longer axial length (all P?<?0.05). These findings support the potential role of HOAs, image quality and a vision-dependent mechanism in childhood eye growth.
Project description:We demonstrate a simple method for mapping optical aberrations with 3D resolution within thick samples. The method relies on the local measurement of the variation in image quality with externally applied aberrations. We discuss the accuracy of the method as a function of the signal strength and of the aberration amplitude and we derive the achievable resolution for the resulting measurements. We then report on measured 3D aberration maps in human skin biopsies and mouse brain slices. From these data, we analyse the consequences of tissue structure and refractive index distribution on aberrations and imaging depth in normal and cleared tissue samples. The aberration maps allow the estimation of the typical aplanetism region size over which aberrations can be uniformly corrected. This method and data pave the way towards efficient correction strategies for tissue imaging applications.
Project description:Artificial skyglow is constantly growing on a global scale, with potential ecological consequences ranging up to affecting biodiversity. To understand these consequences, worldwide mapping of skyglow for all weather conditions is urgently required. In particular, the amplification of skyglow by clouds needs to be studied, as clouds can extend the reach of skyglow into remote areas not affected by light pollution on clear nights. Here we use commercial digital single lens reflex cameras with fisheye lenses for all-sky photometry. We track the reach of skyglow from a peri-urban into a remote area on a clear and a partly cloudy night by performing transects from the Spanish town of Balaguer towards Montsec Astronomical Park. From one single all-sky image, we extract zenith luminance, horizontal and scalar illuminance. While zenith luminance reaches near-natural levels at 5?km distance from the town on the clear night, similar levels are only reached at 27?km on the partly cloudy night. Our results show the dramatic increase of the reach of skyglow even for moderate cloud coverage at this site. The powerful and easy-to-use method promises to be widely applicable for studies of ecological light pollution on a global scale also by non-specialists in photometry.
Project description:This study compared the optical axial length (AL) obtained by composite and segmental methods using swept-source optical coherence tomography (SS-OCT) devices, and demonstrated its effects on the post-operative refractive errors (RE) one month after cataract surgery. Conventional AL measured with the composite method used the mean refractive index. The segmented-AL method used individual refractive indices for each ocular medium. The composite AL (24.52 ± 2.03 mm) was significantly longer (P < 0.001) than the segmented AL (24.49 ± 1.97 mm) among a total of 374 eyes of 374 patients. Bland-Altman analysis revealed a negative proportional bias for the differences between composite and segmented ALs. Although there was no significant difference in the RE obtained by the composite and segmental methods (0.42 ± 0.38 D vs 0.41 ± 0.36 D, respectively, P = 0.35), subgroup analysis of extremely long eyes implanted with a low power intraocular lens indicated that predicted RE was significantly smaller with the segmental method (0.45 ± 0.86 D) than that with the composite method (0.80 ± 0.86 D, P < 0.001). Segmented AL with SS-OCT is more accurate than composite AL in eyes with extremely long AL and can improve post-operative hyperopic shifts in such eyes.