Two independent routes of post-translational chemistry in fluorescent protein FusionRed
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Muslinkina, Liya↗
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Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonic
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Henderson, J. Nathan↗
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↗
Identification of the pigment responsible for the blue fluorescence band in the laser induced fluorescence (LIF) spectra of green plants, and the potential use of this band in remotely estimating rates of photosynthesis
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Chappelle, Emmett W.↗
Investigating Fluorescent Amino Acids with Entangled Two-Photon Excited Fluorescence
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Biological and medical sciences, Materials science↗
Hyperspectral Fluorescence and Reflectance Imaging Instrument
The system is a single hyperspectral imaging instrument that has the unique capability to acquire both fluorescence and reflectance high-spatial-resolution data that is inherently spatially and spectrally registered. Potential uses of this instrument include plant stress monitoring, counterfeit document detection, biomedical imaging, forensic imaging, and general materials identification. Until now, reflectance and fluorescence spectral imaging have been performed by separate instruments. Neither a reflectance spectral image nor a fluorescence spectral image alone yields as much information about a target surface as does a combination of the two modalities. Before this system was developed, to benefit from this combination, analysts needed to perform time-consuming post-processing efforts to co-register the reflective and fluorescence information. With this instrument, the inherent spatial and spectral registration of the reflectance and fluorescence images minimizes the need for this post-processing step. The main challenge for this technology is to detect the fluorescence signal in the presence of a much stronger reflectance signal. To meet this challenge, the instrument modulates artificial light sources from ultraviolet through the visible to the near-infrared part of the spectrum; in this way, both the reflective and fluorescence signals can be measured through differencing processes to optimize fluorescence and reflectance spectra as needed. The main functional components of the instrument are a hyperspectral imager, an illumination system, and an image-plane scanner. The hyperspectral imager is a one-dimensional (line) imaging spectrometer that includes a spectrally dispersive element and a two-dimensional focal plane detector array. The spectral range of the current imaging spectrometer is between 400 to 1,000 nm, and the wavelength resolution is approximately 3 nm. The illumination system consists of narrowband blue, ultraviolet, and other discrete wavelength light-emitting-diode (LED) sources and white-light LED sources designed to produce consistently spatially stable light. White LEDs provide illumination for the measurement of reflectance spectra, while narrowband blue and UV LEDs are used to excite fluorescence. Each spectral type of LED can be turned on or off depending on the specific remote-sensing process being performed. Uniformity of illumination is achieved by using an array of LEDs and/or an integrating sphere or other diffusing surface. The image plane scanner uses a fore optic with a field of view large enough to provide an entire scan line on the image plane. It builds up a two-dimensional image in pushbroom fashion as the target is scanned across the image plane either by moving the object or moving the fore optic. For fluorescence detection, spectral filtering of a narrowband light illumination source is sometimes necessary to minimize the interference of the source spectrum wings with the fluorescence signal. Spectral filtering is achieved with optical interference filters and absorption glasses. This dual spectral imaging capability will enable the optimization of reflective, fluorescence, and fused datasets as well as a cost-effective design for multispectral imaging solutions. This system has been used in plant stress detection studies and in currency analysis.
Ryan, Robert E.↗
Let's Exploit Available Knowledge on Vegetation Fluorescence
The potential to measure vegetation fluorescence from space (1) and to derive from it direct information on the gross primary productivity (GPP) of terrestrial ecosystems is probably the most thrilling development in remote sensing and global ecology of recent years, as it moves Earth observation techniques from the detection of canopy biophysics (e.g., fraction of absorbed radiation) and biochemistry (chlorophyll and nitrogen content) to the realm of ecosystem function. The existence of a functional relationship between fluorescence and photosynthesis has been elucidated over the last decade by several laboratories, notably as part of the preliminary studies of the European Space Agency Fluorescence Explorer (FLEX) Earth Explorer Mission. The empirical observation presented by Guanter et al. (2) of a linear relationship between fluorescence radiance and GPP, however, provides the first experimental confirmation of the feasibility of the approach— already thoroughly tested at leaf level—at the desired scale, despite the confounding effects associated with the satellite detection of such a faint signal. A word of clarification is needed here. The use of fluorescence as a probe of leaf photochemistry has been a staple of plant ecophysiology for decades, rooted in a sound understanding of photosynthetic energy dissipation. However, most past studies had to rely for the interpretation of results on active (pulse-saturated) techniques, making them unsuitable for remote-sensing applications. Over recent years, however, novel process based models have been developed for the interpretation of steady-state, solar-induced fluorescence at the leaf to canopy scale (3). We are therefore in a position to move beyond the mere empirical observation of an association between GPP and fluorescence radiance. In particular, Guanter et al. (2) base their analysis on the assumption of a constant ratio between photosynthetic and fluorescence light use efficiencies (equation 3 in ref. 2).We know, however, that the ratio is not constant, but changes widely in response to light, CO2, stomatal limitations, and extreme stress (4, 5). What’s more, we can make sense of such changes, thus extracting valuable information from the very scatter that is apparent in their data. However, this process will require the availability of more tailored instruments, such as the one planned for the FLEX mission. As already stressed by Guanter et al. (2), the spatial resolution of the Global Ozone Monitoring Experiment-2 sensor (40 × 80 km) makes it difficult to compare meaningfully the fluorescence signal with ground measurements, when only 60–70% of the footprint consists of the desired land-cover type (table S1 in ref. 2), suggesting that this could be largely responsible for the low signals observed in European grasslands. Moreover, the overpass time of the MetOp-A satellite (9:30 AM) implies that fluorescence is generally measured under light-limiting conditions, when fluorescence is only marginally affected by stomatal closure even under stress conditions. This result could explain the seasonal mismatch with daily GPP observed in natural ecosystems in the absence of irrigation (figure 4 in ref. 2). We hope, therefore, that this welcome contribution to this fast-advancing field will help demonstrate the potential of the new technique, and pave the way for more refined studies under both a technological and scientific point of view.
Space↗
Molecules for Fluorescence Detection of Specific Chemicals
A family of fluorescent dye molecules has been developed for use in on-off fluorescence detection of specific chemicals. By themselves, these molecules do not fluoresce. However, when exposed to certain chemical analytes in liquid or vapor forms, they do fluoresce (see figure). These compounds are amenable to fixation on or in a variety of substrates for use in fluorescence-based detection devices: they can be chemically modified to anchor them to porous or non-porous solid supports or can be incorporated into polymer films. Potential applications for these compounds include detection of chemical warfare agents, sensing of acidity or alkalinity, and fluorescent tagging of proteins in pharmaceutical research and development. These molecules could also be exploited for use as two-photon materials for photodynamic therapy in the treatment of certain cancers and other diseases. A molecule in this family consists of a fluorescent core (such as an anthracene or pyrene) attached to two end groups that, when the dye is excited by absorption of light, transfer an electron to the core, thereby quenching the fluorescence. The end groups can be engineered so that they react chemically with certain analytes. Upon reaction, electrons on the end groups are no longer available for transfer to the core and, consequently, the fluorescence from the core is no longer quenched. The chemoselectivity of these molecules can be changed by changing the end groups. For example, aniline end groups afford a capability for sensing acids or acid halides (including those contained in chemical warfare agents). Pyridine or bipyridyl end groups would enable sensing of metal ions. Other chemicals that can be selectively detected through suitable choice of end groups include glucose and proteins. Moreover, the fluorescent cores can be changed to alter light-absorption and -emission characteristics: anthracene cores fluoresce at wavelengths around 500 nm, whereas perylene cores absorb and emit at wavelengths of about 600 nm.
Fedor, Steve↗
Improved Charge-Transfer Fluorescent Dyes
Improved charge-transfer fluorescent dyes have been developed for use as molecular probes. These dyes are based on benzofuran nuclei with attached phenyl groups substituted with, variously, electron donors, electron acceptors, or combinations of donors and acceptors. Optionally, these dyes could be incorporated as parts of polymer backbones or as pendant groups or attached to certain surfaces via self-assembly-based methods. These dyes exhibit high fluorescence quantum yields -- ranging from 0.2 to 0.98, depending upon solvents and chemical structures. The wavelengths, quantum yields, intensities, and lifetimes of the fluorescence emitted by these dyes vary with (and, hence, can be used as indicators of) the polarities of solvents in which they are dissolved: In solvents of increasing polarity, fluorescence spectra shift to longer wavelengths, fluorescence quantum yields decrease, and fluorescence lifetimes increase. The wavelengths, quantum yields, intensities, and lifetimes are also expected to be sensitive to viscosities and/or glass-transition temperatures. Some chemical species -- especially amines, amino acids, and metal ions -- quench the fluorescence of these dyes, with consequent reductions in intensities, quantum yields, and lifetimes. As a result, the dyes can be used to detect these species. Another useful characteristic of these dyes is a capability for both two-photon and one-photon absorption. Typically, these dyes absorb single photons in the ultraviolet region of the spectrum (wavelengths < 400 nm) and emit photons in the long-wavelength ultraviolet, visible, and, when dissolved in some solvents, near-infrared regions. In addition, these dyes can be excited by two-photon absorption at near-infrared wavelengths (600 to 800 nm) to produce fluorescence spectra identical to those obtained in response to excitation by single photons at half the corresponding wavelengths (300 to 400 nm). While many prior fluorescent dyes exhibit high quantum yields, solvent-polarity- dependent fluorescence behavior, susceptibility to quenching by certain chemical species, and/or two-photon fluorescence, none of them has the combination of all of these attributes. Because the present dyes do have all of these attributes, they have potential utility as molecular probes in a variety of applications. Examples include (1) monitoring curing and deterioration of polymers; (2) monitoring protein expression; (3) high-throughput screening of drugs; (4) monitoring such chemical species as glucose, amines, amino acids, and metal ions; and (5) photodynamic therapy of cancers and other diseases.
Meador, Michael↗
On the uncertainty in single molecule fluorescent lifetime and energy emission measurements
Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least square methods agree and are optimal when the number of detected photons is large however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67% of those can be noise and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous poisson processes, we derive the exact joint arrival time probably density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. the ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background nose and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.
Brown, Emery N.↗
On the Uncertainty in Single Molecule Fluorescent Lifetime and Energy Emission Measurements
Time-correlated single photon counting has recently been combined with mode-locked picosecond pulsed excitation to measure the fluorescent lifetimes and energy emissions of single molecules in a flow stream. Maximum likelihood (ML) and least squares methods agree and are optimal when the number of detected photons is large, however, in single molecule fluorescence experiments the number of detected photons can be less than 20, 67 percent of those can be noise, and the detection time is restricted to 10 nanoseconds. Under the assumption that the photon signal and background noise are two independent inhomogeneous Poisson processes, we derive the exact joint arrival time probability density of the photons collected in a single counting experiment performed in the presence of background noise. The model obviates the need to bin experimental data for analysis, and makes it possible to analyze formally the effect of background noise on the photon detection experiment using both ML or Bayesian methods. For both methods we derive the joint and marginal probability densities of the fluorescent lifetime and fluorescent emission. The ML and Bayesian methods are compared in an analysis of simulated single molecule fluorescence experiments of Rhodamine 110 using different combinations of expected background noise and expected fluorescence emission. While both the ML or Bayesian procedures perform well for analyzing fluorescence emissions, the Bayesian methods provide more realistic measures of uncertainty in the fluorescent lifetimes. The Bayesian methods would be especially useful for measuring uncertainty in fluorescent lifetime estimates in current single molecule flow stream experiments where the expected fluorescence emission is low. Both the ML and Bayesian algorithms can be automated for applications in molecular biology.
Brown, Emery N.↗
Foliar Reflectance and Fluorescence Responses for Plants Under Nitrogen Stress Determined with Active and Passive Systems
Vegetation productivity is driven by nitrogen (N) availability in soils. Both excessive and low soil N induce physiological changes in plant foliage. In 2001, we examined the use of spectral fluorescence and reflectance measurements to discriminate among plants provided different N fertilizer application rates: 20%, 50%, 100% and 150% of optimal N levels. A suite of optical, fluorescence, and biophysical measurements were collected on leaves from field grown corn (Zea mays L.) and soybean plants (Glycine max L.) grown in pots (greenhouse + ambient sunlight daily). Three types of steady state laser-induced fluorescence measurements were made on adaxial and abaxial surfaces: 1) fluorescence images in four 10 nm bands (blue, green, red, far-red) resulting from broad irradiance excitation; 2) emission spectra (5 nm resolution) produced by excitation at single wavelengths (280,380 or 360, and 532 nm); and 3) excitation spectra (2 nm resolution), with emission wavelengths fixed at wavelengths centered on selected solar Fraunhofer lines (532,607,677 and 745 nm). Two complementary sets of high resolution (less than 2 nm) optical spectra were acquired for both adaxial and abaxial leaf surfaces: 1) optical properties (350-2500 nm) for reflectance, transmittance, and absorptance; and 2) reflectance spectra (500-1000 nm) acquired with and without a short pass filter at 665 nm to determine the fluorescence contribution to apparent reflectance in the 650-750 spectrum, especially at the 685 and 740 nm chlorophyll fluorescence (ChIF) peaks. The strongest relationships between foliar chemistry and optical properties were demonstrated for C/N content and two optical parameters associated with the red edge inflection point. Select optical properties and ChIF parameters were highly correlated for both species. A significant contribution of ChIF to apparent reflectance was observed, averaging 10-25% at 685 nm and 2 - 6% at 740 nm over all N treatments. Discrimination of N treatment groups was possible with specific fluorescence band ratios (e.g., F740/F525 obtained with 380EX). From all measurements assessing fluorescence, higher ChIF and blue/green emissions were measured from the abaxial leaf surfaces; Abaxial surfaces also produced higher reflectances in the 400-800 nm spectrum. Fluorescence information collected in Fraunhofer regions located on the shoulders of ChIF features compared favorably with peak emissions. This supports the potential capability of a future space-born interferometer sensor to capture plant canopy fluorescence.
Middleton, E. M.↗
Fluorescent Approaches to High Throughput Crystallography
We have shown that by covalently modifying a subpopulation, less than or equal to 1%, of a macromolecule with a fluorescent probe, the labeled material will add to a growing crystal as a microheterogeneous growth unit. Labeling procedures can be readily incorporated into the final stages of purification, and the presence of the probe at low concentrations does not affect the X-ray data quality or the crystallization behavior. The presence of the trace fluorescent label gives a number of advantages when used with high throughput crystallizations. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination crystals show up as bright objects against a dark background. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Brightly fluorescent crystals are readily found against less bright precipitated phases, which under white light illumination may obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries as the protein or protein structures is all that shows up. Fluorescence intensity is a faster search parameter, whether visually or by automated methods, than looking for crystalline features. We are now testing the use of high fluorescence intensity regions, in the absence of clear crystalline features or "hits", as a means for determining potential lead conditions. A working hypothesis is that kinetics leading to non-structured phases may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Preliminary experiments with test proteins have resulted in the extraction of a number of crystallization conditions from screening outcomes based solely on the presence of bright fluorescent regions. Subsequent experiments will test this approach using a wider range of proteins. The trace fluorescently labeled crystals will also emit with sufficient intensity to aid in the automation of crystal alignment using relatively low cost optics, further increasing throughput at synchrotrons.
Pusey, Marc L.↗
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↗
Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate
Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.
Evaluation Studies↗
Global Monitoring of Terrestrial Chlorophyll Fluorescence from Moderate-Spectral-Resolution Near-Infrared Satellite Measurements: Methodology, Simulations, and Application to GOME-2
Globally mapped terrestrial chlorophyll fluorescence retrievals are of high interest because they can provide information on the functional status of vegetation including light-use efficiency and global primary productivity that can be used for global carbon cycle modeling and agricultural applications. Previous satellite retrievals of fluorescence have relied solely upon the filling-in of solar Fraunhofer lines that are not significantly affected by atmospheric absorption. Although these measurements provide near-global coverage on a monthly basis, they suffer from relatively low precision and sparse spatial sampling. Here, we describe a new methodology to retrieve global far-red fluorescence information; we use hyperspectral data with a simplified radiative transfer model to disentangle the spectral signatures of three basic components: atmospheric absorption, surface reflectance, and fluorescence radiance. An empirically based principal component analysis approach is employed, primarily using cloudy data over ocean, to model and solve for the atmospheric absorption. Through detailed simulations, we demonstrate the feasibility of the approach and show that moderate-spectral-resolution measurements with a relatively high signal-to-noise ratio can be used to retrieve far-red fluorescence information with good precision and accuracy. The method is then applied to data from the Global Ozone Monitoring Instrument 2 (GOME-2). The GOME-2 fluorescence retrievals display similar spatial structure as compared with those from a simpler technique applied to the Greenhouse gases Observing SATellite (GOSAT). GOME-2 enables global mapping of far-red fluorescence with higher precision over smaller spatial and temporal scales than is possible with GOSAT. Near-global coverage is provided within a few days. We are able to show clearly for the first time physically plausible variations in fluorescence over the course of a single month at a spatial resolution of 0.5 0.5. We also show some significant differences between fluorescence and coincident normalized difference vegetation indices (NDVI) retrievals.
Fluorescence↗