Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level can reveal the stoichiometry, dynamics and interactions of multiple molecular species in mixtures and other complex samples. However, fluorescence-based sensing is typically limited to the detection of just 3-4 colours at a time due to low signal-to-noise ratio, high spectral overlap and the need to maintain the chemical compatibility of dyes. Here we engineered a palette of several dozen composite fluorescent labels, called FRETfluors, for multiplexed spectroscopic measurements at the single-molecule level. FRETfluors are compact nanostructures constructed from three chemical components (DNA, Cy3 and Cy5) with tunable spectroscopic properties due to variations in geometry, fluorophore attachment chemistry and DNA sequence. We demonstrate FRETfluor labelling and detection for low-concentration (<100 fM) mixtures of mRNA, dsDNA and proteins using an anti-Brownian electrokinetic trap. In addition to identifying the unique spectroscopic signature of each FRETfluor, this trap differentiates FRETfluors attached to a target from unbound FRETfluors, enabling wash-free sensing. Although usually considered an undesirable complication of fluorescence, here the inherent sensitivity of fluorophores to the local physicochemical environment provides a new design axis complementary to changing the FRET efficiency. As a result, the number of distinguishable FRETfluor labels can be combinatorically increased while chemical compatibility is maintained, expanding prospects for spectroscopic multiplexing at the single-molecule level using a minimal set of chemical building blocks.

Multi-scale datasets for monitoring Mediterranean oak forests from optical remote sensing during the SENTHYMED/MEDOAK experiment in the north of Montpellier (France).

Mediterranean forests represent critical areas that are increasingly affected by the frequency of droughts and fires, anthropic activities and land use changes. Optical remote sensing data give access to several essential biodiversity variables, such as species traits (related to vegetation biophysical and biochemical composition), which can help to better understand the structure and functioning of these forests. However, their reliability highly depends on the scale of observation and the spectral configuration of the sensor. Thus, the objective of the SENTHYMED/MEDOAK experiment is to provide datasets from leaf to canopy scale in synchronization with remote sensing acquisitions obtained from multi-platform sensors having different spectral characteristics and spatial resolutions. Seven monthly data collections were performed between April and October 2021 (with a complementary one in June 2023) over two forests in the north of Montpellier, France, comprised of two oak endemic species with different phenological dynamics (evergreen: Quercus ilex and deciduous: Quercus pubescens) and a variability of canopy cover fractions (from dense to open canopy). These collections were coincident with satellite multispectral Sentinel-2 data and one with airborne hyperspectral AVIRIS-Next Generation data. In addition, satellite hyperspectral PRISMA and DESIS were also available for some dates. All these airborne and satellite data are provided from free online download websites. Eight datasets are presented in this paper from thirteen studied forest plots: (1) overstory and understory inventory, (2) 687 canopy plant area index from Li-COR plant canopy analyzers, (3) 1475 in situ spectral reflectances (oak canopy, trunk, grass, limestone, etc.) from ASD spectroradiometers, (4) 92 soil moistures and temperatures from IMKO and Campbell probes, (5) 747 leaf-clip optical data from SPAD and DUALEX sensors, (6) 2594 in-lab leaf directional-hemispherical reflectances and transmittances from ASD spectroradiometer coupled with an integrating sphere, (7) 747 in-lab measured leaf water and dry matter content, and additional leaf traits by inversion of the PROSPECT model and (8) UAV-borne LiDAR 3-D point clouds. These datasets can be useful for multi-scale and multi-temporal calibration/validation of high level satellite vegetation products such as species traits, for current and future imaging spectroscopic missions, and by fusing or comparing both multispectral and hyperspectral data. Other targeted applications can be forest 3-D modelling, biodiversity assessment, fire risk prevention and globally vegetation monitoring.

Single-molecule fluorescence multiplexing by multi-parameter spectroscopic detection of nanostructured FRET labels.

Multiplexed, real-time fluorescence detection at the single-molecule level is highly desirable to reveal the stoichiometry, dynamics, and interactions of individual molecular species within complex systems. However, traditionally fluorescence sensing is limited to 3-4 concurrently detected labels, due to low signal-to-noise, high spectral overlap between labels, and the need to avoid dissimilar dye chemistries. We have engineered a palette of several dozen fluorescent labels, called FRETfluors, for spectroscopic multiplexing at the single-molecule level. Each FRETfluor is a compact nanostructure formed from the same three chemical building blocks (DNA, Cy3, and Cy5). The composition and dye-dye geometries create a characteristic F\"orster Resonance Energy Transfer (FRET) efficiency for each construct. In addition, we varied the local DNA sequence and attachment chemistry to alter the Cy3 and Cy5 emission properties and thereby shift the emission signatures of an entire series of FRET constructs to new sectors of the multi-parameter detection space. Unique spectroscopic emission of each FRETfluor is therefore conferred by a combination of FRET and this site-specific tuning of individual fluorophore photophysics. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs statistically selected to minimize classification errors, measured using an Anti-Brownian ELectrokinetic (ABEL) trap which provides precise multi-parameter spectroscopic measurements. The ABEL trap also enables discrimination between FRETfluors attached to a target (here: mRNA) and unbound FRETfluors, eliminating the need for washes or removal of excess label by purification. We show single-molecule identification of a set of 27 FRETfluors in a sample mixture using a subset of constructs selected to minimize classification errors.

Integrated Behavioral Health Prevention for Infants in Pediatric Primary Care: A Mixed-Methods Pilot Study.

OBJECTIVE: Pediatric primary care is a promising setting in which to deliver preventive behavioral health services to young children and their families. Integrated behavioral health care models typically emphasize treatment rather than prevention. This pilot study examined the efficacy of an integrated behavioral health preventive (IBH-P) intervention delivered by psychologists and focused on supporting parenting in low-income mothers of infants as part of well-child visits in the first 6 months of life. METHODS: Using a mixed-methods approach that included a pilot randomized clinical trial and post-intervention qualitative interviews, 137 mothers were randomly assigned to receive IBH-P or usual care. Self-report measures of parenting, child behavior, and stress were obtained at pre- and/or post-intervention. Direct observation of mother-infant interactions was conducted at post-intervention. RESULTS: No differences between groups were found on maternal attunement, knowledge of child development, nurturing parenting, or infant behavior. A secondary analysis on a subsample with no prior exposure to IBH-P with older siblings found that mothers in IBH-P reported increased self-efficacy relative to controls. In the qualitative interviews, mothers stated that they valued IBH-P, learning about their baby, liked the integration in primary care, and felt respected and comfortable with their provider. CONCLUSIONS: Findings are discussed in terms of the next steps in refining IBH-P approaches to prevention in primary care.

NIH Toolbox executive function measures with developmental extensions: Reliability and validity with preschoolers in emergency housing.

Research has shown that executive function (EF) skills are associated with resilience in preschoolers experiencing risk and adversity, but these studies have typically relied on large batteries of tasks to measure children's EF skills. There is a need for brief, reliable EF assessments that can be used in the field with diverse young children. The current study assessed the validity and test-retest reliability of two tablet-based EF tasks from the NIH Toolbox: The Dimensional Change Card Sort (DCCS) and the Flanker Inhibitory Control and Attention Test, each with a developmental extension (Dext) that is triggered when a child struggles with the standardized versions. Dext versions include easier levels intended to improve task accessibility for younger and disadvantaged children. Eighty-six preschoolers residing in emergency housing participated in two study sessions about one week apart, completing tablet-based DCCS-Dext and Flanker-Dext tasks, along with a table-top EF task (Peg-Tapping) and measures of vocabulary and numeracy. The majority of participants triggered the Dext portion of the DCCS and almost half triggered the Dext portion of the Flanker, underscoring the need for extensions of the Toolbox EF tasks to lower the floor of these measures. The Dext EF measures were positively associated with Peg-Tapping, after controlling for age and vocabulary, indicating construct validity. They were also correlated with math achievement, suggesting criterion validity. DCCS-Dext and Flanker-Dext showed moderate test-retest reliability after one week. Together, these findings demonstrate the value of developmental extensions for assessing EF skills among children experiencing risk and adversity.