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All sourcesNASA NTRSDOE OSTI
NASA NTRS2007

Mosaic-Detector-Based Fluorescence Spectral Imager

A battery-powered, pen-sized, portable instrument for measuring molecular fluorescence spectra of chemical and biological samples in the field has been proposed. Molecular fluorescence spectroscopy is among the techniques used most frequently in laboratories to analyze compositions of chemical and biological samples. Heretofore, it has been possible to measure fluorescence spectra of molecular species at relative concentrations as low as parts per billion (ppb), with a few nm spectral resolution. The proposed instrument would include a planar array (mosaic) of detectors, onto which a fluorescence spectrum would be spatially mapped. Unlike in the larger laboratory-type molecular fluorescence spectrometers, mapping of wavelengths to spatial positions would be accomplished without use of relatively bulky optical parts. The proposed instrument is expected to be sensitive enough to enable measurement of spectra of chemical species at relative concentrations <1 ppb, with spectral resolution that could be tailored by design to be comparable to a laboratory molecular fluorescence spectrometer. The proposed instrument (see figure) would include a button-cell battery and a laser diode, which would generate the monochromatic ultraviolet light needed to excite fluorescence in a sample. The sample would be held in a cell bounded by far-ultraviolet-transparent quartz or optical glass. The detector array would be, more specifically, a complementary metal oxide/ semiconductor or charge-coupled- device imaging photodetector array, the photodetectors of which would be tailored to respond to light in the wavelength range of the fluorescence spectrum to be measured. The light-input face of the photodetector array would be covered with a matching checkerboard array of multilayer thin film interference filters, such that each pixel in the array would be sensitive only to light in a spectral band narrow enough so as not to overlap significantly with the band of an adjacent pixel. The wavelength interval between adjacent pixels (and, thus, the spectral resolution) would typically be chosen by design to be approximately equal to the width of the total fluorescence wavelength range of interest divided by the number of pixels. The unitary structure comprising the photodetector array overlaid with the matching filter array would be denoted a hyperspectral mosaic detector (HMD) array.

Son, Kyung-Ah↗
NASA NTRS2004

Phytoplankton-Fluorescence-Lifetime Vertical Profiler

A battery-operated optoelectronic instrument is designed to be lowered into the ocean to measure the intensity and lifetime of fluorescence of chlorophyll A in marine phytoplankton as a function of depth from 0 to 300 m. Fluorescence lifetimes are especially useful as robust measures of photosynthetic productivity of phytoplankton and of physical and chemical mechanisms that affect photosynthesis. The knowledge of photosynthesis in phytoplankton gained by use of this and related instruments is expected to contribute to understanding of global processes that control the time-varying fluxes of carbon and associated biogenic elements in the ocean. The concentration of chlorophyll in the ocean presents a major detection challenge because in order to obtain accurate values of photosynthetic parameters, the intensity of light used to excite fluorescence must be kept very low so as not to disturb the photosynthetic system. Several innovations in fluorometric instrumentation were made in order to make it possible to reach the required low detection limit. These innovations include a highly efficient optical assembly with an integrated flow-through sample interface, and a high-gain, low-noise electronic detection subsystem. The instrument also incorporates means for self-calibration during operation, and electronic hardware and software for control, acquisition and analysis of data, and communications. The electronic circuitry is highly miniaturized and designed to minimize power demand. The instrument is housed in a package that can withstand the water pressure at the maximum depth of 300 m. A light-emitting diode excites fluorescence in the sample flow cell, which is placed at one focal point of an ellipsoidal reflector. A photomultiplier tube is placed at the other focal point. This optical arrangement enables highly efficient collection of fluorescence emitted over all polar directions. Fluorescence lifetime is measured indirectly, by use of a technique based on the same principle as the one described in "Fluorometer for Analysis of Photosynthesis in Phytoplankton" (SSC-00110), NASA Tech Briefs, Vol. 24, No. 1 (November 2000), page 79. The excitation is modulated at a frequency of 70 MHz, and the phase shift between the excitation light and the emitted fluorescence is measured by a detection method in which the 70 MHz signal is down-converted to a 400 Hz signal. The fluorescence lifetime can be computed from the known relationship among the fluorescence lifetime, phase shift, and modulation frequency

Fernandez, Salvador M.↗
NASA NTRS1973

Detection of S02 and N02 in Stack Plume by Raman Scattering and Fluorescence

We have been studying laser-Raman radar which can be used as the remote detector of sulfur dioxide concentration in the stack plume of boiler exhaust gas, and some results have been published. In this paper, we report the interference of nitrogen dioxide fluorescence against sulfur dioxide Raman scattering and the measuring method of sulfur dioxide and nitrogen dioxide concentration. In a stack plume, high density dust and high concentration carbon dioxide are included, therefore very strong Mie back-scattering and carbon dioxide Raman scattering are observed. The separation of these scattering signals from sulfur dioxide Raman signal was the first problem for the laser-Raman radar. But, this problem was solved by using the filter which have high resolving power. It is well known that nitrogen dioxide can be excited to emit fluorescence of broad spectrum by blue-green light. The light source of the laser-Raman radar is SH of Nd:YAG laser, and boiler exhaust gas includes several tens ppm of nitrogen dioxide, then the interference of nitrogen dioxide fluorescence brings the error to sulfur dioxide measurement. 'rhe rejection of the interference can be achieved by the subtraction of the nitrogen dioxide fluorescence contribution from detected signal at sulfur dioxide Raman scattering wavelength. The nitrogen dioxide fluorescence contribution can be measured by two methods. The first is to convert the nitrogen dioxide fluorescence intensity which is measured at a different wavelength from sulfur dioxide Raman line into the one at sulfur dioxide Raman line. The second is to convert the detected signal intensity, which is obtained when the range gate is set just behind the plume, into the one obtained at the range of the plume using the difference of the time dependence between sulfur dioxide Raman scattering and nitrogen dioxide fluorescence. Nitrogen dioxide fluorescence has lifetime of about 300nS, while Raman scattering has none. By either of the two methods, the contribution of nitrogen dioxide fluorescence is determined. Then, we can measure the nitrogen dioxide and sulfur dioxide concentration.The minimum detectable concentration and the experimental results of the remote sensing of sulfur dioxide and nitrogen dioxide in stack plume will be discussed in detail.

Nakahara, S.↗
DOE OSTI2026

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↗
DOE OSTI2025

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↗
NASA NTRS1984

Laser-induced fluorescence of green plants. I - A technique for the remote detection of plant stress and species differentiation

The laser-induced fluorescence (LIF) of green plants was evaluated as a means of remotely detecting plant stress and determining plant type. Corn and soybeans were used as representatives of monocots and dicots, respectively, in these studies. The fluorescence spectra of several plant pigments was excited with a nitrogen laser emitting at 337 nm. Intact leaves from corn and soybeans also fluoresced using the nitrogen laser. The two plant species exhibited fluorescence spectra which had three maxima in common at 440, 690, and 740 nm. However, the relative intensities of these maxima were distinctly different for the two species. Soybeans had an additional slight maxima at 525 nm. Potassium deficiency in corn caused an increase in fluorescence at 690 and 740 nm. Simulated water stress in soybeans resulted in increased fluorescence at 440, 525, 690, and 740 nm. The inhibition of photosynthesis in soybeans by 3-(3-4-dichlorophenyl)-1-1-dimethyl urea (DCMU) gave incresed fluorescence primarily at 690 and 740 nm. Chlorosis as occurring in senescent soybean leaves caused a decrease in fluorescence at 690 and 740 nm. These studies indicate that LIF measurements of plants offer the potential for remotely detecting certain types of stress condition and also for differentiating plant species.

Chappelle, E. W.↗
NASA NTRS1987

Quantitative photoabsorption and fluorescence spectroscopy of H2S and D2S at 49-240 nm

Photoabsorption and fluorescence cross sections of H2S and D2S were measured in the 49-240 nm region using synchrotron radiation as a light source. Fluorescence from photoexcitation of H2S appears at 49-97 nm, but not in the long wavelength region. Fluorescence spectra were dispersed, and used to identify the emitters to be H2S(+) (A), SH(+)(A), and H(n greater than 2). The fluorescence quantum yield is about 6 percent. Photoexcitation of D2S at 49-96 nm produces fluorescence with a quantum yield of about 5 percent. The emitters are identified from the fluorescence spectra to be D2S(+)(A), SD(+)(A), and D(n greater than 2). The Franck-Condon factors for the SH(+) and SD(+) (A-X) transitions were determined. The SD(A-X) fluorescence was observed from photoexcitation of D2S at 100-151 nm, for which the fluorescence cross section and quantum yield were measured.

Lee, L. C.↗
NASA NTRS1998

Fluorescence Studies of Lysozyme Nucleation

Fluorescence is one of the most powerful tools available for the study of macromolecules. For example, fluorescence can be used to study self association through methods such as anisotropy (the rotational rate of the molecule in solution), quenching (the accessibility of a bound probe to the bulk solution), and resonance energy transfer (measurement of the distance between two species). Fluorescence can also be used to study the local environment of the probe molecules, and the changes in that environment which accompany crystal nucleation and growth. However fluorescent techniques have been very much underutilized in macromolecular growth studies. One major advantage is that the fluorescent species generally must be at low concentration, typically ca 10-5 to 10-6 M. Thus one can study a very wide range of solution conditions, ranging from very high to very low protein concentration, he latter of which are not readily accessible to scattering techniques. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme (CEWL). Fluorescent probes have been attached to two different sites, ASP 101 and the N-terrninal amine, with a sought for use in different lines of study. Preliminary resonance energy transfer studies have been -carried out using pyrene acetic acid (Ex 340 mn, Em 376 nm) lysozyme as a donor and cascade blue (Ex 377 run, Em 423 nm) labeled lysozyme as an acceptor. The emission of both the pyrene and cascade blue probes was followed as a function of the salt protein concentrations. The data show an increase in cascade blue and a concomitant decrease in the pyrene fluorescence as either the salt or protein concentrations are increased, suggesting that the two species are approaching each other close enough for resonance energy transfer to occur. This data can be analyzed to measure the distance between the probe molecules and, knowing their locations on the protein molecule their distances from and orientations with respect to each other. The results of these and other studies will be discussed.

Pusey, Marc L.↗
NASA NTRS2000

Fluorescence Studies of Protein Crystal Nucleation

One of the most powerful and versatile methods for studying molecules in solution is fluorescence. Crystallization typically takes place in a concentrated solution environment, whereas fluorescence typically has an upper concentration limit of approximately 1 x 10(exp -5)M, thus intrinsic fluorescence cannot be employed, but a fluorescent probe must be added to a sub population of the molecules. However the fluorescent species cannot interfere with the self-assembly process. This can be achieved with macromolecules, where fluorescent probes can be covalently attached to a sub population of molecules that are subsequently used to track the system as a whole. We are using fluorescence resonance energy transfer (FRET) to study the initial solution phase self-assembly process of tetragonal lysozyme crystal nucleation, using covalent fluorescent derivatives which crystallize in the characteristic P432121 space group. FRET studies are being carried out between cascade blue (CB-lys, donor, Ex 376 nm, Em 420 nm) and lucifer yellow (LY-lys, acceptor, Ex 425 nm, Em 520 nm) asp101 derivatives. The estimated R0 for this probe pair, the distance where 50% of the donor energy is transferred to the acceptor, is approximately 1.2 nm, compared to 2.2 nm between the side chain carboxyls of adjacent asp101's in the crystalline 43 helix. The short CB-lys lifetime (approximately 5 ns), coupled with the large average distances between the molecules ((sup 3) 50 nm) in solution, ensure that any energy transfer observed is not due to random diffusive interactions. Addition of LY-lys to CB-lys results in the appearance of a second, shorter lifetime (approximately 0.2 ns). Results from these and other ongoing studies will be discussed in conjunction with a model for how tetragonal lysozyme crystals nucleate and grow, and the relevance of that model to microgravity protein crystal growth

Pusey, Marc L.↗
NASA NTRS2001

Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating the filaments. Below 8.5 mm the filamentous morphology of the upper layers gives way to punctate 1-2 micron particles evidencing fluorescent activity following excitation at both deep ultraviolet wavelengths.

Storrie-Lombardi, M. C.↗
NASA NTRS2001

Hyperspectral Imaging and Spectroscopy of Fluorescently Coupled Acyl-CoA: Cholesterol Acyltransferase in Insect Cells

A high-performance hyperspectral imaging module with high throughput of light suitable for low-intensity fluorescence microscopic imaging and subsequent analysis, including single-pixel-defined emission spectroscopy, was tested on Sf21 insect cells expressing green fluorescence associated with recombinant green fluorescent protein linked or not with the membrane protein acyl-CoA:cholesterol acyltransferase. The imager utilized the phenomenon of optical activity as a new technique providing information over a spectral range of 220-1400 nm, and was inserted between the microscope and an 8-bit CCD video-rate camera. The resulting fluorescence image did not introduce observable image aberrations. The images provided parallel acquisition of well resolved concurrent spatial and spectral information such that fluorescence associated with green fluorescent protein alone was demonstrated to be diffuse within the Sf21 insect cell, and that green fluorescence associated with the membrane protein was shown to be specifically concentrated within regions of the cell cytoplasm. Emission spectra analyzed from different regions of the fluorescence image showed blue shift specific for the regions of concentration associated with the membrane protein.

Malak, H.↗
NASA NTRS2000

A Passive Method for Detecting Vegetation Stress from Orbit: Chlorophyll Fluorescence Spectra from Fraunhofer Lines

Solar-stimulated chlorophyll fluorescence measured with the Fraunhofer line depth method has correlated well with vegetation stress in previous studies. However, the instruments used in those studies were limited to a single solar absorption line (e.g. 656.3 nm), obviating the red/far-red ratio (R/FR) method. Optics and detector technology have reached the level whereby multiple, very narrow Fraunhofer lines are resolvable. Thirteen such lines span the visible spectrum in the red to far-red region where chlorophyll fluorescence occurs. Fluorescence intensities at the 13 Fraunhofer line wavelengths were used to model emission spectra. The source data were collected for summer and fall bean crops (Phaseolus vulgaris L.) subjected to various levels of nitrogen fertilization. The intensities were adjusted to account for Fraunhofer line depth and atmospheric transmittance. Multiple R/FR fluorescence ratios, calculated from the modeled fluorescence spectra, correlated strongly with leaf chlorophyll concentration and well with applied nitrogen. The ratio yielding the best correlation with chlorophyll utilized red fluorescence at the 694.5 nm Fraunhofer line and farred fluorescence at the 755.6 nm Fraunhofer line. Twenty R/FR ratios, each evaluated for the maximum differential between low and high (optimal) nitrogen treatments, ranked higher in some cases and lower in others, possibly related to the time of year the crops were grown and the stage of growth of the crops. Ratios with 728.9 nm and 738.9 nm in the denominator consistently ranked in the lowest and next lowest quartile, respectively. Ratios of the 656.3 nm Fraunhofer line and the 755.6 nm line consistently ranked highest for the summer crop. Ratios with 755.6 nm in the denominator ranked in the upper quartile for 10 out of 12 measurement dates. Differences in ratio ranking indicate that physiological conditions may be estimated using selected ratios of Fraunhofer lines within the context of R/FR analysis. A passive instrument designed to monitor R/FR chlorophyll fluorescence (i.e. vegetation stress) from orbit could be built today.

Theisen, Arnold F.↗
NASA NTRS2004

Fluorescent Approaches to High Throughput Crystallography

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution 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 a macromolecules purification. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals will show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "bits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. 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. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Minamitani, Elizabeth Forsythe↗
NASA NTRS2004

Fluorescent Approaches to High Throughput Crystallography

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically can not reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1%, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. 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. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Pusey, Marc L.↗
NASA NTRS2000

Relationship between the Fluorescence Lifetime of Chlorophyll 'a' and Primary Productivity within the Mississippi River Plume and Adjacent Shelf Region

In situ measurements of chlorophyll fluorescence intensity have been widely used to estimate phytoplankton biomass. However, because the fluorescence quantum yield of chlorophyll a in vivo can be highly variable, measurements of chlorophyll fluorescence intensity cannot be directly correlated with phytoplankton biomass and do not provide information on the physiological state of the phytoplankton under study. Conversely, lifetime-based measurements of chlorophyll fluorescence provide a framework in which photosynthetic rates of phytoplankton can be analyzed according to phytoplankton physiology. Along with the measurement of primary production and ambient nutrient concentrations within the Mississippi River plume in the northern Gulf of Mexico, phytoplankton fluorescence lifetimes were measured using a Fluorescence Lifetime Phytoplankton Analyzer (developed under a NASA Small Business Innovative Research contract to Ciencia, Inc.). Variability of fluorescence lifetimes within the plume can be used as a background from which to interpret variations in the maximum quantum yield of photochemistry. The extent to which nutrient and effluent loading in this dynamic coastal area affect the photosynthetic performance of phytoplankton will be presented as a function of phytoplankton fluorescence lifetimes.

Hall, Callie↗
NASA NTRS2005

Fluorescent Approaches to High Throughput Crystallography

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, 51%, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear hits. Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. 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. This presentation will focus on the methodology for fluorescent labeling, the crystallization results, and the effects of the trace labeling on the crystal quality.

Pusey, Marc L.↗
NASA NTRS2005

Fluorescent Approaches to High Throughput Crystallography

X-ray crystallography remains the primary method for determining the structure of macromolecules. The first requirement is to have crystals, and obtaining them is often the rate-limiting step. The numbers of crystallization trials that are set up for any one protein for structural genomics, and the rate at which they are being set up, now overwhelm the ability for strictly human analysis of the results. Automated analysis methods are now being implemented with varying degrees of success, but these typically cannot reliably extract intermediate results. By covalently modifying a subpopulation, less than or = 1 %, of a macromolecule solution 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. The covalently attached probe will concentrate in the crystal relative to the solution, and under fluorescent illumination the crystals show up as bright objects against a dark background. As crystalline packing is more dense than amorphous precipitate, the fluorescence intensity can be used as a guide in distinguishing different types of precipitated phases, even in the absence of obvious crystalline features, widening the available potential lead conditions in the absence of clear "hits." Non-protein structures, such as salt crystals, will not incorporate the probe and will not show up under fluorescent illumination. Also, brightly fluorescent crystals are readily found against less fluorescent precipitated phases, which under white light illumination may serve to obscure the crystals. Automated image analysis to find crystals should be greatly facilitated, without having to first define crystallization drop boundaries and by having the protein or protein structures all that show up. 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. Preliminary experiments show that the presence of the fluorescent probe does not affect the nucleation process or the quality of the X-ray data obtained.

Pusey, Marc L.↗
NASA NTRS2006

Fluorescent Applications to Crystallization

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 tests with model proteins have shown that labeling u to 5 percent of the protein molecules does not affect the X-ray data quality obtained . The presence of the trace fluorescent label gives a number of advantages. Since the label is covalently attached to the protein molecules, it "tracks" the protein s response to the crystallization conditions. 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 darker background. Non-protein structures, such as salt crystals, do not show up under fluorescent illumination. Crystals have the highest protein concentration and are readily observed 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. Preliminary tests, using model proteins, indicates that we can use 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 more rapid amorphous precipitation kinetics may overwhelm and trap more slowly formed ordered assemblies, which subsequently show up as regions of brighter fluorescence intensity. Experiments are now being carried out to 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.↗