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At least 37 records · Page 2

DeepLearnMOR: a deep-learning framework for fluorescence image-based classification of organelle morphology

Abstract The proper biogenesis, morphogenesis, and dynamics of subcellular organelles are essential to their metabolic functions. Conventional techniques for identifying, classifying, and quantifying abnormalities in organelle morphology are largely manual and time-consuming, and require specific expertise. Deep learning has the potential to revolutionize image-based screens by greatly improving their scope, speed, and efficiency. Here, we used transfer learning and a convolutional neural network (CNN) to analyze over 47,000 confocal microscopy images from Arabidopsis wild-type and mutant plants with abnormal division of one of three essential energy organelles: chloroplasts, mitochondria, or peroxisomes. We have built a deep-learning framework, DeepLearnMOR (Deep Learning of the Morphology of Organelles), which can rapidly classify image categories and identify abnormalities in organelle morphology with over 97% accuracy. Feature visualization analysis identified important features used by the CNN to predict morphological abnormalities, and visual clues helped to better understand the decision-making process, thereby validating the reliability and interpretability of the neural network. This framework establishes a foundation for future larger-scale research with broader scopes and greater data set diversity and heterogeneity.

Plant Sciences↗

Unraveling Cu Chemical Signature in CdTe by Spectral Fluorescence Mapping

X-ray absorption spectroscopy (XAS) has been shown to be a powerful tool to unravel the chemical environment of a given atom within a matrix. When used in correlative X-ray microscopy approaches, XAS allows one to probe with nanoscale precision regions of particular interest in an absorber. Herein, we use X-ray absorption near edge structure (XANES) to evaluate the chemical environment of Cu atoms within a CdTe solar cell. The reconstruction of XANES spectra from XRF maps have unfolded 2D maps of Cu chemical structures. In this work, we found that most Cu atoms exist in Cu 2 Te and Cu 1.4 Te phase. Moreover, we found traces of CuTe, Cu 2 O, CuO, Cu 2 S, CuS, and metallic Cu phase. Investigating Cu chemical structures at different performing areas, we found no observable correlation between Cu chemical structures and electrical performance. This approach allows tracking of Cu chemical structures along with electrical performance and elemental distribution simultaneously, with high spatial resolution in a statistically practical way.

CdTe↗

Incorporation of sensing modalities into de novo designed fluorescence-activating proteins

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis -planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca 2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

59 BASIC BIOLOGICAL SCIENCES↗

A Chimeric LBT-GFP Biosensor Exhibits Antithetical Fluorescence Responses to Ca 2+ and Dy 3+ Binding

Rare earth elements (REEs) are critical components in emerging technologies, but their mining and refining processes are often laborious, costly, and environmentally damaging. Developing green and efficient separation methods for REEs is crucial. Biomolecular approaches using lanthanide-binding proteins and peptides show promise for selective REE extraction and separation. In this study, we present the design and characterization of a genetically encoded fluorescence indicator (GEFI) construct that combines a superfolder green fluorescent protein (sfGFP) with a dual lanthanide-binding tag (2×dLBT). The 2×dLBT insert induces conformational changes in sfGFP upon lanthanide binding, modulating the fluorescence intensity. The sfGFP-2×dLBT biosensor exhibited distinct fluorescence responses to different lanthanide ions, with the highest dynamic range observed for heavy REEs like dysprosium (Dy 3+ ). Interestingly, the sensor displayed an antithetical response, where low concentrations of lanthanides initially quenched the fluorescence, but higher concentrations led to a significant fluorescence increase (1.5-fold). The Ca 2+ ion on the other hand showed only a dose-dependent quenching of the fluorescence response. Based on these observations, the biphasic response of the biosensor to lanthanides was eliminated by pretreating the sensor with calcium, which further expanded the dynamic range up to 3-fold for Dy 3+ . The lanthanide-selective and concentration-dependent fluorescence changes of the sfGFP-2×dLBT biosensor demonstrate its potential as a platform for developing specific sensors for various REEs. These sensors could enable rapid and cost-effective determination of REE composition in complex mixtures, facilitating the separation and recovery of critical REEs from electronic waste and other REE-containing sources.

59 BASIC BIOLOGICAL SCIENCES↗

Fluorescent CQD-Doped Styrene Acrylic Emulsion Coating Film with Enhanced Optical Properties

Styrene acrylic emulsions (SAEs) have emerged as a promising material for water-based coatings. However, they are still limited by their own defects in practical applications, poor weatherability, and degradation of performance at lower or higher temperatures. Here, we introduce a facile approach to producing fluorescent carbon quantum dots (CQDs) from wood processing residues and fabricating fluorescent CQD/SAE coating films via emulsion-casting. The addition of the fluorescent CQDs enhanced the optical performance of the CQD/SAE coating films. The fluorescent CQDs were prepared via a hydrothermal approach and were obtained after heating at 180 °C for 6 h at a reaction concentration of 50 mg/mL. The synthesized CQDs resulted in a high fluorescence, and the CQDs had an average size of 1.63 nm. Various concentrations of the fluorescent CQDs were doped into the SAE coating film, which improved its optical properties. We also characterized and discussed the products and then explored their optical properties. This study presents the potential of fluorescent CQD/SAE coating films for applications in anti-counterfeiting coatings, fluorescent adhesives, and papermaking.

36 MATERIALS SCIENCE↗

Ultra-high Information-content Chemical Imaging with Broadband Coherent Anti-Stokes Raman and Two-photon Fluorescence Lifetime Microscopy

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence(TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. Furthermore, this protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Xu, Haoyu [Georgia Institute of Technology, Atlant↗

Infrared fluorescent coatings

The present invention provides for a composition comprising a pigment, wherein the composition is suitable for coating a surface that is, or is expected to be, exposed to the sun. The pigment comprises particles that fluoresce in sunlight, thereby remaining cooler in the sun than coatings pigmented with non-fluorescent particles. The particles comprise solids that fluoresce or glow in the visible or near infrared (NIR) spectra, or that fluoresce when doped. Suitable dopants include, but are not limited to, ions of rare earths and transition metals. A coating composition includes: (i) a film-forming resin; (ii) an infrared reflective pigment; and (iii) an infrared fluorescent pigment different from the infrared reflective pigment. When the coating composition is cured to form a coating and exposed to radiation comprising fluorescence-exciting radiation, the coating has a greater effective solar reflectance (ESR) compared to the same coating exposed to the radiation comprising fluorescence-exciting radiation except without the infrared fluorescent pigment. A multi-layer coating including the coating composition, and a substrate at least partially coated with the coating composition is also disclosed. A method of reducing temperature of an article includes applying the coating composition to at least a portion of the article.

Zalich, Michael Andrew↗

Infrared fluorescent coatings

The present invention provides for a composition comprising a pigment, wherein the composition is suitable for coating a surface that is, or is expected to be, exposed to the sun. The pigment comprises particles that fluoresce in sunlight, thereby remaining cooler in the sun than coatings pigmented with non-fluorescent particles. The particles comprise solids that fluoresce or glow in the visible or near infrared (NIR) spectra, or that fluoresce when doped. Suitable dopants include, but are not limited to, ions of rare earths and transition metals. A coating composition includes: (i) a film-forming resin; (ii) an infrared reflective pigment; and (iii) an infrared fluorescent pigment different from the infrared reflective pigment. When the coating composition is cured to form a coating and exposed to radiation comprising fluorescence-exciting radiation, the coating has a greater effective solar reflectance (ESR) compared to the same coating exposed to the radiation comprising fluorescence-exciting radiation except without the infrared fluorescent pigment. A multi-layer coating including the coating composition, and a substrate at least partially coated with the coating composition is also disclosed. A method of reducing temperature of an article includes applying the coating composition to at least a portion of the article.

Zalich, Michael Andrew↗

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii. We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii. Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Here, Clostridium and other anaerobes that will lead to better understanding of these unique organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Specific features of fluorescence transfer in multiply scattering randomly inhomogeneous layers under intense laser pumping

Based on the analysis of experimental data on the effect of the pulsed laser pump intensity on the spectral properties and the size of the fluorescent response zone in randomly inhomogeneous fluorescent layers, we found that the amplification of spontaneous and stimulated emission significantly affects the statistical properties of the propagation lengths of the fluorescent field partial components in the layers. The experiments are performed with layers of SiO{sub 2} and TiO{sub 2} nanoparticles saturated with rhodamine 6G, pumped by 532-nm laser radiation in the intensity range corresponding to the transient regime from excitation of spontaneous fluorescence to random lasing in the layer. The experimental data are compared with the results of statistical modelling of fluorescence transfer. It is shown that, even at a pump intensity below the random lasing threshold, the spontaneous emission amplification in a layer leads to a significant increase in the contributions to the fluorescence response from partial components with propagation lengths much larger than the layer thickness. This can be interpreted as a manifestation of the quasi-waveguide effect, in which the probability of propagation of diffuse fluorescence components along the layer over distances many times greater than its thickness and the size of the pumped region increases significantly with a decrease in the characteristic radiation amplification length in the layer. (paper)

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Editorial special issue: Advancing foundational sun-induced chlorophyll fluorescence science

The first research on the possibilities offered by chlorophyll-a fluorescence to track the daily course of CO 2 assimilation by leaves dates back to the last century (Kautsky and Hirsch, 1931). In the second half of the XXth Century, the development of the field of active fluorescence, which relies mainly on the pulse amplitude modulation (PAM) fluorimetry technique, allowed the unraveling of the relationship between the yield of chlorophyll-a fluorescence and photochemistry (linear electron transport), which is modulated by a third process known as non-photochemical quenching (Genty et al., 1989). Since then, active fluorescence has been regularly used in ecophysiology, forestry, and crop sciences to understand the plant response to stress (Schreiber, 2004). Since the beginning of the 2000s, the development of portable field spectrometers allows for measuring passive sun-induced fluorescence (SIF) at strong solar or telluric absorption features, which do not rely on the use of artificial excitation light (e.g., Meroni and Colombo, 2006; Meroni et al., 2009; Perez-Priego et al., 2005). Since then, the field has rapidly evolved, and now fluorescence is measurable with automated field spectrometers in the field (Grossmann et al., 2018; Gu et al., 2019b; Rossini et al., 2010), airborne platforms (Rascher et al., 2015; Zarco-Tejada et al., 2000), and satellites (Guanter et al., 2012; Sun et al., 2018). These advances offer the potential to couple ecosystem-scale measurements of CO 2 fluxes and SIF to probe new aspects related to ecosystem structural impacts on SIF andphotosynthesis processes at different time-scales and on different ecosystems (e.g., Damm et al., 2010; Magney et al., 2019; Porcar-Castell et al., 2021). While SIF has been proven as a good proxy of gross primary productivity (GPP), mainly across large spatial and temporal gradients, a series of studies showed that different factors could confound this relationship, namely different canopy structures (e.g., Dechant et al., 2020; Migliavacca et al., 2017), stress conditions (e.g., Martini et al., 2022; Wieneke et al., 2018; Wohlfahrt et al., 2018), nutritional conditions (Cendrero-Mateo et al., 2015; Martini et al., 2019), species-specific differences (e.g., Van Wittenberghe et al., 2013) and light regimes (e.g., Liu and Liu, 2018). At the same time, the modeling of SIF developed rapidly (Gu et al., 2019a; Han et al., 2022; Han et al., 2021; van der Tol et al., 2014; van der Tol et al., 2009) and with an increasing degree of realism, offering the possibility to retrieve important vegetation parameters from remote sensing (Pacheco-Labrador et al., 2019; Verrelst et al., 2015; Verrelst et al., 2016; Zhang et al., 2014). Excellent and comprehensive reviews of the field are by (Mohammed et al., 2019; Porcar-Castell et al., 2021; Porcar-Castell et al., 2014; Sun et al., 2023a; Sun et al., 2023b). Despite the exponential increase in the number of publications in the field, significant progress is still needed. Here, this special issue aims to report foundational SIF science, including theoretical modeling and measurement-based research that is urgently needed to unleash the full potential of SIF for physiological and ecological applications at scales spanning from leaf to globe. The 14 articles collected in this special issue reported on the following specific aspects: first, technical capabilities enabling spectrally resolved SIF observations and their inter-comparability in space and time; second, theoretical developments in SIF-photosynthesis relationships to correctly interpret the signal and extract mechanistic information on vegetation structure and function; third, evaluation of the potential of SIF to track process beyond photosynthesis, such as transpiration; fourth, large scale-applications of SIF observations; and finally, upscaling fluorescence from leaf to canopy scales.

54 ENVIRONMENTAL SCIENCES↗

Miniature fluorescence sensor for quantitative detection of brain tumour

Fluorescence-guided surgery has emerged as a vital tool for tumour resection procedures. As well as intraoperative tumour visualisation, 5-ALA-induced PpIX provides an avenue for quantitative tumour identification based on ratiometric fluorescence measurement. To this end, fluorescence imaging and fibre-based probes have enabled more precise demarcation between the cancerous and healthy tissues. These sensing approaches, which rely on collecting the fluorescence light from the tumour resection site and its “remote” spectral sensing, introduce challenges associated with optical losses. In this work, we demonstrate the viability of tumour detection at the resection site using a miniature fluorescence measurement system. Unlike the current bulky systems, which necessitate remote measurement, we have adopted a millimetre-sized spectral sensor chip for quantitative fluorescence measurements. A reliable measurement at the resection site requires a stable optical window between the tissue and the optoelectronic system. This is achieved using an antifouling diamond window, which provides stable optical transparency. The system achieved a sensitivity of 92.3% and specificity of 98.3% in detecting a surrogate tumour at a resolution of 1 × 1 mm 2 . In conclusion, as well as addressing losses associated with collecting and coupling fluorescence light in the current ‘remote’ sensing approaches, the small size of the system introduced in this work paves the way for its direct integration with the tumour resection tools with the aim of more accurate interoperative tumour identification.

59 BASIC BIOLOGICAL SCIENCES↗

Data-driven insights into the fluorescence of asphaltene aggregates using extended Frenkel exciton model

The origin of the fluorescence redshift during asphaltene aggregation remains debated due to the great diversity of asphaltene molecules, while the extended Frenkel exciton model provided a theoretical framework for studying multi-chromophore systems, such as asphaltene aggregates. We investigated the fluorescence energy of hundreds of asphaltene dimers based on 133 experimental revealed asphaltene monomer structures. Our result shows that the dimer's fluorescence energy is always lower than both of its monomers regardless of its intermolecular conformation, with its redshift dominated by superexchange. The dimer oscillator strength predominantly depends on the monomer with the lower fluorescence energy, and the involvement of charge-transfer states and the cancellation between monomer transition dipole moments suppressed the fluorescence especially when two monomers have similar fluorescence energy. The above-mentioned findings hold for all asphaltene dimers despite their diversity, which offers a theoretical interpretation for comprehending the relationship between asphaltene aggregation and its fluorescence variation. Furthermore, this work provides theoretical guidance to other research areas related to organic planar conjugated systems with multiple chromophores.

Chemistry↗

Chlorophyll fluorescence imaging captures photochemical efficiency of grain sorghum (Sorghum bicolor) in a field setting

Abstract Background Photosynthesis is one of the most important biological reactions and forms the basis of crop productivity and yield on which a growing global population relies. However, to develop improved plant cultivars that are capable of increased productivity, methods that can accurately and quickly quantify photosynthetic efficiency in large numbers of genotypes under field conditions are needed. Chlorophyll fluorescence imaging is a rapid, non-destructive measurement that can provide insight into the efficiency of the light-dependent reactions of photosynthesis. Results To test and validate a field-deployed fluorescence imaging system on the TERRA-REF field scanalyzer, leaves of potted sorghum plants were treated with a photosystem II inhibitor, DCMU, to reduce photochemical efficiency (F V /F M ). The ability of the fluorescence imaging system to detect changes in fluorescence was determined by comparing the image-derived values with a handheld fluorometer. This study demonstrated that the imaging system was able to accurately measure photochemical efficiency (F V /F M ) and was highly correlated ( r = 0.92) with the handheld fluorometer values. Additionally, the fluorescence imaging system was able to track the decrease in photochemical efficiency due to treatment of DCMU over a 7 day period. Conclusions The system’s ability to capture the temporal dynamics of the plants’ response to this induced stress, which has comparable dynamics to abiotic and biotic stressors found in field environments, indicates the system is operating correctly. With the validation of the fluorescence imaging system, physiological and genetic studies can be undertaken that leverage the fluorescence imaging capabilities and throughput of the field scanalyzer.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Fluorescence lifetime measurements using photon pair correlations generated via spontaneous parametric down conversion (SPDC)

We have used photon pair correlations generated via spontaneous parametric downconversion (SPDC) to measure the fluorescence lifetime of the organic dye rhodamine 6 G, demonstrating that fluorescence lifetime measurements can be achieved using a continuous wave (CW) laser, without pulsed or modulated lasers. Our entangled photon method, quantum fluorescence lifetime (Q-FL) measurements, uses one photon to excite fluorescence and the resulting fluorescence photon is timed and referenced to the arrival time of the other entangled photon. Thus, we can exploit the short timescale of photon pair correlations to conduct experiments that are typically carried out with pulsed lasers and we show that the inherent timing of the photons is fast enough to resolve the nanosecond scale fluorescence lifetime of the sample. This measurement paves the way towards using the time correlations of entangled photons for fluorescence imaging; capitalizing on the presence of fast, sub-100 ps correlations that have not been demonstrated classically.

Eshun, Audrey (ORCID:0000000167697082)↗

Fluorescent compositions

A fluorescent composition includes at least one of a fluorescent sensor compound and organic reporter molecules encapsulated in a microsphere structure. When encapsulated, the fluorescent sensor compound and the organic reporter molecules are distributed in a liquid organic matrix. When non-encapsulated, the remaining one of the fluorescent sensor compound and the organic reporter molecules reside in the matrix. In response to a force applied to the composition sufficient to break at least a portion of the microsphere structure, the fluorescent sensor compound and the organic reporter molecules are transformed into a non-reversible fluorescent state exhibiting a quantum yield greater than 0.2. The fluorescent state is objectively visually verifiable without physically contacting the composition.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Fluorescent compositions

An article that includes a fluorescent composition having at least one of a fluorescent sensor compound and organic reporter molecules encapsulated in a microsphere structure. When encapsulated, the fluorescent sensor compound and the organic reporter molecules are distributed in a liquid organic matrix. When non-encapsulated, the remaining one of the fluorescent sensor compound and the organic reporter molecules reside in the matrix. In response to a force applied to the composition sufficient to break at least a portion of the microsphere structure, the fluorescent sensor compound and the organic reporter molecules are transformed into a non-reversible fluorescent state exhibiting a quantum yield greater than 0.2. The fluorescent state is objectively visually verifiable without physically contacting the composition.

Smartt, Heidi A.↗