Neural network approach for correction of multiple scattering errors in the LISST-VSF instrument.

The LISST-VSF is a commercially developed instrument used to measure the volume scattering function (VSF) and attenuation coefficient in natural waters, which are important for remote sensing, environmental monitoring and underwater optical wireless communication. While the instrument has been shown to work well at relatively low particle concentration, previous studies have shown that the VSF obtained from the LISST-VSF instrument is heavily influenced by multiple scattering in turbid waters. High particle concentrations result in errors in the measured VSF, as well as the derived properties, such as the scattering coefficient and phase function, limiting the range at which the instrument can be used reliably. Here, we present a feedforward neural network approach for correcting this error, using only the measured VSF as input. The neural network is trained with a large dataset generated using Monte Carlo simulations of the LISST-VSF with scattering coefficients b=0.05-50m-1, and tested on VSFs from measurements with natural water samples. The results show that the neural network estimated VSF is very similar to the expected VSF without multiple scattering errors, both in angular shape and magnitude. One example showed that the error in the scattering coefficient was reduced from 103% to 5% for a benchtop measurement of natural water sample with expected b=10.6m-1. Hence, the neural network drastically reduces uncertainties in the VSF and derived properties resulting from measurements with the LISST-VSF in turbid waters.

GLORIA - A globally representative hyperspectral in situ dataset for optical sensing of water quality.

The development of algorithms for remote sensing of water quality (RSWQ) requires a large amount of in situ data to account for the bio-geo-optical diversity of inland and coastal waters. The GLObal Reflectance community dataset for Imaging and optical sensing of Aquatic environments (GLORIA) includes 7,572 curated hyperspectral remote sensing reflectance measurements at 1 nm intervals within the 350 to 900 nm wavelength range. In addition, at least one co-located water quality measurement of chlorophyll a, total suspended solids, absorption by dissolved substances, and Secchi depth, is provided. The data were contributed by researchers affiliated with 59 institutions worldwide and come from 450 different water bodies, making GLORIA the de-facto state of knowledge of in situ coastal and inland aquatic optical diversity. Each measurement is documented with comprehensive methodological details, allowing users to evaluate fitness-for-purpose, and providing a reference for practitioners planning similar measurements. We provide open and free access to this dataset with the goal of enabling scientific and technological advancement towards operational regional and global RSWQ monitoring.

Polycrystalline Diamond Coating on Orthopedic Implants: Realization and Role of Surface Topology and Chemistry in Adsorption of Proteins and Cell Proliferation.

Polycrystalline diamond has the potential to improve the osseointegration of orthopedic implants compared to conventional materials such as titanium. However, despite the excellent biocompatibility and superior mechanical properties, the major challenge of using diamond for implants, such as those used for hip arthroplasty, is the limitation of microwave plasma chemical vapor deposition (CVD) techniques to synthesize diamond on complex-shaped objects. Here, for the first time, we demonstrate diamond growth on titanium acetabular shells using the surface wave plasma CVD method. Polycrystalline diamond coatings were synthesized at low temperatures (∼400 °C) on three types of acetabular shells with different surface structures and porosities. We achieved the growth of diamond on highly porous surfaces designed to mimic the structure of the trabecular bone and improve osseointegration. Biocompatibility was investigated on nanocrystalline diamond (NCD) and ultrananocrystalline diamond (UNCD) coatings terminated either with hydrogen or oxygen. To understand the role of diamond surface topology and chemistry in the attachment and proliferation of mammalian cells, we investigated the adsorption of extracellular matrix proteins and monitored the metabolic activity of fibroblasts, osteoblasts, and bone-marrow-derived mesenchymal stem cells (MSCs). The interaction of bovine serum albumin and type I collagen with the diamond surfaces was investigated by confocal fluorescence lifetime imaging microscopy (FLIM). We found that the proliferation of osteogenic cells was better on hydrogen-terminated UNCD than on the oxygen-terminated counterpart. These findings correlated with the behavior of collagen on diamond substrates observed by FLIM. Hydrogen-terminated UNCD provided better adhesion and proliferation of osteogenic cells, compared to titanium, while the growth of fibroblasts was poorest on hydrogen-terminated NCD and MSCs behaved similarly on all tested surfaces. These results open new opportunities for application of diamond coatings on orthopedic implants to further improve bone fixation and osseointegration.

Efficient Monte Carlo simulation reveals significant multiple scattering errors in underwater angular scattering measurements.

Multiple scattering can severely affect the accuracy of optical instrumentation. Variance reduction methods have been implemented to improve a Monte Carlo model developed to simulate volume scattering functions measured by LISST-VSF instruments. The implemented methods can result in more than a tenfold increase in efficiency. The simulation is used to analyze multiple scattering errors for a range of Fournier-Forand (FF) phase functions. Our results demonstrate significant errors in the scattering coefficient, backscattering coefficient and phase function, where multiple scattering errors may only be considered negligible (<10%) for scattering coefficients <1 m-1. The errors depend strongly on the scattering coefficient but also increase when phase functions become more forward-peaked.

Versatile compressive microscope for hyperspectral transmission and fluorescence lifetime imaging.

Increasing demand for multimodal characterization and imaging of new materials entails the combination of various methods in a single microscopic setup. Hyperspectral imaging of transmission spectra or photoluminescence (PL) decay imaging count among the most used methods. Nevertheless, these methods require very different working conditions and instrumentation. Therefore, combining the methods into a single microscopic system is seldom implemented. Here we demonstrate a novel versatile microscope based on single-pixel imaging, where we use a simple optical configuration to measure the hyperspectral information, as well as fluorescence lifetime imaging (FLIM). The maps are inherently spatially matched and can be taken with spectral resolution limited by the resolution of the used spectrometer (3 nm) or temporal resolution set by PL decay measurement (120 ps). We verify the system's performance by its comparison to the standard FLIM and non-imaging transmission spectroscopy. Our approach enabled us to switch between a broad field-of-view and micrometer resolution without changing the optical configuration. At the same time, the used design opens the possibility to add a variety of other characterization methods. This article demonstrates a simple, affordable way of complex material studies with huge versatility for the imaging parameters.

Analysis of multiple scattering errors in LISST-VSF volume scattering function measurements using Monte Carlo simulations and experimental data.

A Monte Carlo algorithm has been developed to investigate the effects of multiple scattering on the volume scattering function measured by the LISST-VSF instrument. The developed algorithm is compared to experimental results obtained from bench-top measurements using 508nm spherical polystyrene beads and Arizona test dust as scattering agents. The Monte Carlo simulation predicts measured volume scattering functions at all concentrations. We demonstrate that multiple scattered light can be a major contributor to the detected signal, resulting in errors in the measured volume scattering function and its derived inherent optical properties. We find a relative error of 10% in the scattering coefficient for optical depths ∼0.4, and it can reach 100% at optical depths ∼2.

In situ measurements of the volume scattering function with LISST-VSF and LISST-200X in extreme environments: evaluation of instrument calibration and validity.

The LISST-VSF and LISST-200X are commercial instruments made available in recent years, enabling underwater measurements of the volume scattering function, which has not been routinely measured in situ due to lack of instrumentation and difficulty of measurement. Bench-top and in situ measurements have enabled absolute calibration of the instruments and evaluation of instrument validity ranges, even at environmental extremes such as the clear waters at the North Pole and turbid glacial meltwaters. Key considerations for instrument validity ranges are ring detector noise levels and multiple scattering. In addition, Schlieren effects can be significant in stratified waters.

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein, Quinine Sulfate and Green Fluorescent Protein.

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10- 3 - 10- 5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.

Chlorophyll a fluorescence lifetime reveals reversible UV-induced photosynthetic activity in the green algae Tetraselmis.

The fluorescence lifetime is a very useful parameter for investigating biological materials on the molecular level as it is mostly independent of the fluorophore concentration. The green alga Tetraselmis blooms in summer, and therefore its response to UV irradiation is of particular interest. In vivo fluorescence lifetimes of chlorophyll a were measured under both normal and UV-stressed conditions of Tetraselmis. Fluorescence was induced by two-photon excitation using a femtosecond laser and laser scanning microscope. The lifetimes were measured in the time domain by time-correlated single-photon counting. Under normal conditions, the fluorescence lifetime was 262 ps, while after 2 h of exposure to UV radiation the lifetime increased to 389 ps, indicating decreased photochemical quenching, likely caused by a damaged and down-regulated photosynthetic apparatus. This was supported by a similar increase in the lifetime to 425 ps when inhibiting photosynthesis chemically using DCMU. Furthermore, the UV-stressed sample was dark-adapted overnight, resulting in a return of the lifetime to 280 ps, revealing that the damage caused by UV radiation is repairable on a relatively short time scale. This reversal of photosynthetic activity was also confirmed by [Formula: see text] measurements.

Testing fluorescence lifetime standards using two-photon excitation and time-domain instrumentation: rhodamine B, coumarin 6 and lucifer yellow.

Having good information about fluorescence lifetime standards is essential for anyone performing lifetime experiments. Using lifetime standards in fluorescence spectroscopy is often regarded as a straightforward process, however, many earlier reports are limited in terms of lifetime concentration dependency, solvents and other technical aspects. We have investigated the suitability of the fluorescent dyes rhodamine B, coumarin 6, and lucifer yellow as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine which wavelengths we should use for the excitation and an appropriate detector range. We also measured lifetimes for different concentrations, ranging from 10(-2)- 10(-6) M, in both water, ethanol and methanol solutions. We observed that rhodamine B lifetimes depend strongly on concentration. Coumarin 6 provided the most stable lifetimes, with a negligible dependency on concentration and solvent. Lucifer yellow lifetimes were also found to depend little with concentration. Finally, we found that a mix of two fluorophores (rhodamine B/coumarin 6, rhodamine B/lucifer yellow, and coumarin 6/lucifer yellow) all yielded very similar lifetimes from a double-exponential decay as the separate lifetimes measured from a single-exponential decay. All lifetime measurements were made using two-photon excitation and obtaining lifetime data in the time-domain using time-correlated single-photon counting.

Chlorophyll a and NADPH fluorescence lifetimes in the microalgae Haematococcus pluvialis (Chlorophyceae) under normal and astaxanthin-accumulating conditions.

In vivo fluorescence lifetimes of chlorophyll-a (chl-a) and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from the green microalgae Haematococcus pluvialis under normal and nutrient-stressed conditions (green stage and red stage, respectively), using two-photon excitation provided by a laser generating pulses in the femtosecond range, and a Leica microscope setup. Analysis of the fluorescence lifetime decay curve revealed two separate lifetime components in all our measurements. A short-lifetime component for chl-a of ~250 ps was completely dominant, contributing more than 90% of overall intensity in both green-stage and red-stage cells. Green-stage cells inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) displayed a significant chl-a lifetime increase for the short component. However, this was not the case for red-stage cells, in which DCMU inhibition did not significantly affect the lifetime. For green-stage cells, we found a short NADPH (free) lifetime component at ~150 ps to be completely dominating, but for red-stage cells, a longer component (protein bound) at ~3 ns contributed as much as 35% of the total intensity. We hypothesize that the long lifetime component of NADPH is connected to photoprotection in the cells and coupled to production of astaxanthin. DCMU does not seem to affect the fluorescence lifetimes of NADPH.