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NASA NTRS1981

Selection of fluorescence lidar operating parameters for SNR maximization

Fluorescence lidar when applicable offers one of the most sensitive methods for measuring the concentration of trace constituents of the atmosphere. In the conduct of a fluorescence lidar experiment, a number of parameters which can be used to optimize the SNR can be controlled. In this paper the optimum division of laser pulses centered on and off the fluorescence excitation wavelength is calculated as a function of the ratio of the fluorescence signal strength to the strength of fluorescence from interfering species. For strong interference signals the time should be divided equally on and off the line. For strong fluorescence signals the time on line is proportional to the square root of the on-line off-line signal ratio. The optimization of the integration time for varying values of signal-to-background and signal-to-interference ratios, atmospheric attenuation, laser energy variations, background measurement time, and on-line off-line time division is also considered.

Heaps, W. S.↗

NASA NTRS1987

Fluorescence yields from photodissociative excitation of chloromethanes by vacuum ultraviolet radiation

The photoabsorption and fluorescence cross sections of chloromethanes were measured in the 105-220 nm region using synchrotron radiation as a light source. The fluorescence threshold for CCl4 is at 152 nm with a maximum yield of 3 percent at 113 nm. The fluorescence results from the CCl2(A-X) system. For CHCl3, the fluorescence threshold is at 155 nm with a maximum yield of 0.6 percent at 110 nm. For CH2Cl2, the threshold is at 137 nm with a maximum yield of 0.35 percent at 107 nm. The fluorescence yield of CH3Cl is very small with an upper limit of 0.02 percent. The photodissociation processes are discussed in accord with the fluorescence data observed. Vibrational structures in CHCl3 and CH3Cl2 are observed and classified into progressions.

Lee, L. C.↗

NASA NTRS1989

Red fluorescence and 3-12 micron emission in NGC 2023, HD 44179, M 82, and Lynds 1780

A red excess observed in the Red Rectangle (HD 44179), was attributed to a possible molecular fluorescence mechanism was discovered in NGC 2023 and analyzed in subsequent work in this and other nebulae. An unexpected red light excess was also noticed in a high latitude dark cloud L 1780. The fluorescence was attributed to hydrogenated amorphous carbon by Duley (1985), on the basis of laboratory work. Alternatively, transitions between electronic states of free polycyclic aromatic hydrocarbon molecules, by-passing the cascade along the vibrational states was considered. In L 1780, the red excess was related to the 12 micron emission detected by IRAS. A quantitative comparison of the intensity of the red fluorescence and that of the 3 to 12 micron features is thus warranted in helping assess the physical properties of large interstellar molecules. The red fluorescence radiation, F(R), appears as a bump on the spectra between 0.6 and 0.9 micron. Values were deduced from the spectra for HD 44179, and for the high latitude cloud L 1780. Corrections for the extinction, both interstellar and internal to the nebulae, were included. The 3 to 12 micron brightness, F(IR), was obtained through integration of the spectra for NGC 2023, and for HD 44179 after removal of a smooth continuum due to hot large grains. The values of the ratio of fluorescence flux to the infrared flux, F(R)/F(IR), are summarized. Red fluorescence and infrared radiation are two separate ways to access to the size of the molecules through observation, and it is rewarding that both approaches give similar results. These findings bring a striking coherence into the physical description of the particles, and add further support to the initial attribution of the infrared features to polycyclic aromatic hydrocarbons (PAHs).

Ryter, Charles↗

NASA NTRS1992

Primary production estimates from recordings of solar-stimulated fluorescence in the equatorial Pacific at 150 deg W

Biological, optical, and hydrographical data were collected on the WEC88 cruise along 150 deg W and during a 6-day time-series station on the equator during February/March 1988. This area was characterized by a subsurface chlorophyll maximum (SCM), located at 50-70 m depth at the equator and descending down to 120-125 m at the north and south end of the transect. Highest primary production rates were near-surface and confined to the equatorial region and stations between 7 deg and 11 deg N. To determine the relationship between solar-stimulated fluorescence (centered at 683 nm wavelength) and primary production, a production-fluorescence model based on phytoplankton physiology and marine optics is described. Results of model calculations predict that there is a linear relation between production and fluorescence. A comparison between morning and midday measurements of the production-fluorescence relation showed that there was some difference between the two, whereas evening measurements, on the other hand, were distinctly different from the morning/midday ones. This seems to suggest that diurnal variations contribute significantly to variability in the quantum yield of photochemical processes. The ratio of the quantum yield of photosynthesis to the quantum yield of fluorescence ranged between 0.24 and 0.44 molC/Ein for all stations. The highest value for this ratio occurred at the equatorial stations, indicating that latitudinal variability could have an effect on the production-fluorescence relation.

Stegmann, P. M.↗

NASA NTRS1993

Laser-induced fluorescence detection strategies for sodium atoms and compounds in high-pressure combustors

A variety of laser-induced fluorescence schemes were examined experimentally in atmospheric pressure flames to determine their use for sodium atom and salt detection in high-pressure, optically thick environments. Collisional energy transfer plays a large role in fluorescence detection. Optimum sensitivity, at the parts in 10 exp 9 level for a single laser pulse, was obtained with the excitation of the 4p-3s transition at 330 nm and the detection of the 3d-3p fluorescence at 818 nm. Fluorescence loss processes, such as ionization and amplified spontaneous emission, were examined. A new laser-induced atomization/laser-induced fluorescence detection technique was demonstrated for NaOH and NaCl. A 248-nm excimer laser photodissociates the salt molecules present in the seeded flames prior to atom detection by laser-induced fluorescence.

Weiland, Karen J. R.↗

NASA NTRS1993

Fluorescence observations of LDEF exposed materials as an indicator of induced material reactions

Observations and measurements of induced changes in the fluorescent emission of materials exposed to the space environment on the Long Duration Exposure Facility (LDEF) have revealed systematic patterns of material-dependent behavior. These results have been supplemented by inspection of similar materials exposed on previous Space Shuttle Missions and in laboratory testing. The space environmental factors affecting the fluorescence of exposed materials have been found to include (but are not necessarily limited to) solar ultraviolet (UV) radiation, atomic oxygen (AO), thermal vacuum exposure, and synergistic combinations of these factors. Observed changes in material fluorescent behavior include stimulation, quenching, and spectral band shifts of emission. For example, the intrinsic yellow fluorescence of zinc oxide pigmented thermal control coatings undergoes quenching as a result of exposure, while coloration is stimulated in the fluorescent emission of several polyurethane coating materials. The changes in fluorescent behavior of these materials are shown to be a revealing indicator of induced material reactions as a result of space environmental exposure.

Linton, Roger C.↗

NASA NTRS2000

Preparation and Characterization of Fluorescent Derivatives of Chicken Egg White Lysozyme

Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow, cascade blue, and 5-(2-aminoethyl)aminonapthalene-l-sulfonic acid (EDANS) have been attached to the side chain carboxyl of asp101 using a carbodiimide coupling procedure. asp101 lies within the active site cleft, and it is believed that the probes are at least partially "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to hisl5, located on the "back side" of the molecule relative to the active site. The fluorescently labeled protein is readily purified from the starting material by cation exchange chromatography. All the derivatives fluoresce in both the solution and the crystalline states. Fluorescence characterization has focused on determining the bound probe quantum yields, lifetimes, absorption and emission spectra, and quenching by added solutes in comparison to the free probe. No appreciable changes are found in the lifetimes of any of the probes except for cascade blue, where Tau(sub free) = 3.52 ns vrs Tau(sub bound) = 2.8 ns. Spectral shifts are found in most cases. Particularly strong quenching upon binding is found in the case of the cascade blue derivative, likely due to probe interactions with the active site cleft. While none of the asp101 bound probes are well quenched by commonly employed solutes, such as potassium and sodium iodide, acrylamide, primuline, the chloride salts of manganese, cesium, and cobalt, trifluoroacetamide, trichloroethanol, and thallium iodide, in those cases where quenching is observed the bound probe is less efficiently quenched relative to the free probe. This indicates that the bound probes are less accessible to the bulk solution, an expected finding for attachment within the active site cleft. Attempts have been made to bind other molecules to these sites, with varying success. Interestingly, all three probes contain one or more sulfonate ((Ar-S03)-) groups. We have not been successful in binding analogous probes without sulfate groups such as pyrene, or with derivatized sulfonate groups such as dansyl type probes, analogous to MANS but where the sulfonate group is derivatized, Ar-S02-N2C2H7. None of the probes is rigidly bound to the protein, i.e., they all have a probe motion superimposed on that of the protein.

Sumida, John↗

NASA NTRS1996

Red Fluorescent Line Emission from Hydrogen Molecules in Diffuse Molecular Clouds

We have modeled the fluorescent pumping of electronic and vibrational emissions of molecular hydrogen (H2) within diffuse molecular clouds that are illuminated by ultraviolet continuum radiation. Fluorescent line intensities are predicted for transitions at ultraviolet, infrared, and red visible wavelengths as functions of the gas density, the visual extinction through the cloud, and the intensity of the incident UV continuum radiation. The observed intensity in each fluorescent transition is roughly proportional to the integrated rate of H2 photodissociation along the line of sight. Although the most luminous fluorescent emissions detectable from ground-based observatories lie at near-infrared wavelengths, we argue that the lower sky brightness at visible wavelengths makes the red fluorescent transitions a particularly sensitive probe. Fabry-Perot spectrographs of the type that have been designed to observe very faint diffuse Ha emissions are soon expected to yield sensitivities that will be adequate to detect H2 vibrational emissions from molecular clouds that are exposed to ultraviolet radiation no stronger than the mean radiation field within the Galaxy. Observations of red H2 fluorescent emission together with cospatial 21 cm H I observations could serve as a valuable probe of the gas density in diffuse molecular clouds.

Neufeld, David A.↗

NASA NTRS2003

Foliar Reflectance and Fluorescence Responses for Corn and Soybean Plants Under Nitrogen Stress

We are investigating the use of spectral indices derived from actively induced fluorescence spectra and passive optical spectra. We examined the influence of photosynthetic pigment, carbon (C) and nitrogen (N) content on the spectral fluorescence and passive optical property characteristics of mature, upper leaves from plants provided different N fertilizer application rates: 20%, 50%, 100% and 150% of recommended 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. Steady state laser-induced fluorescence emission spectra (5 nm resolution) were obtained from adaxial and abaxial surfaces resulting from excitation at single wavelengths (280, 380 or 360, and 532 nm). For emission spectra produced by each of these excitation wavelengths, ratios of emission peaks were calculated, including the red far-red chlorophyll fluorescence (ChlF) ratio (F685/F740) and the far-red/green (F740/F525) ratio. High resolution (< 3 nm) optical spectra (350-2500 nm) of reflectance, transmittance, and absorptance were also acquired for both adaxial and abaxial leaf surfaces. Species differences were demonstrated for several optical parameters. A 'red edge' derivative ratio determined from transmittance spectra [as the maximum first deivative, between 650-750 nm, normalized to the value at 744 nm, or Dmax/D744], was strongly associated with the C/N ratio (r(exp 2) = 0.90, P +/- 0.001). This ratio, calculated from reflectance spectra, was inversely related to chlorophyll b content (r(exp 2) = 0.91, P +/- 0.001) as was the ChlF (F685/F740) ratio obtained with 532 nm excitation (r(exp 2) = 0.76, P +/- 0.01). Discrimination of N treatment groups was possible with specific fluorescence band ratios (e.g., F740/F525 obtained with 380 nm excitation). Higher ChlF and blue-green emissions were measured from the abaxial leaf surfaces. Abaxial surfaces also produced higher reflectances, in general, in the 400-800 nm spectrum.

Middleton, E. M.↗

NASA NTRS2005

Two-Photon Fluorescence Microscope for Microgravity Research

A two-photon fluorescence microscope has been developed for the study of biophysical phenomena. Two-photon microscopy is a novel form of laser-based scanning microscopy that enables three-dimensional imaging without many of the problems inherent in confocal microscopy. Unlike one-photon optical microscopy, two-photon microscopy utilizes the simultaneous nonlinear absorption of two near-infrared photons. However, the efficiency of two-photon absorption is much lower than that of one-photon absorption, so an ultra-fast pulsed laser source is typically employed. On the other hand, the critical energy threshold for two-photon absorption leads to fluorophore excitation that is intrinsically localized to the focal volume. Consequently, two-photon microscopy enables optical sectioning and confocal performance without the need for a signal-limiting pinhole. In addition, there is a reduction (relative to one-photon optical microscopy) in photon-induced damage because of the longer excitation wavelength. This reduction is especially advantageous for in vivo studies. Relative to confocal microscopy, there is also a reduction in background fluorescence, and, because of a reduction in Rayleigh scattering, there is a 4 increase of penetration depth. The prohibitive cost of a commercial two-photon fluorescence-microscope system, as well as a need for modularity, has led to the construction of a custom-built system (see Figure 1). This system includes a coherent mode-locked titanium: sapphire laser emitting 120-fs-duration pulses at a repetition rate of 80 MHz. The pulsed laser has an average output power of 800 mW and a wavelength tuning range of 700 to 980 nm, enabling the excitation of a variety of targeted fluorophores. The output from the laser is attenuated, spatially filtered, and then directed into a confocal scanning head that has been modified to provide for side entry of the laser beam. The laser output coupler has been replaced with a dichroic filter that reflects the longer-wavelength excitation light and passes the shorter-wavelength fluorescence light. Also, the confocal pinhole has been removed to increase the signal strength. The laser beam is scanned by a twoperpendicular- axis pair of galvanometer mirrors through a pupil transfer lens into the side port of an inverted microscope. Finally, the beam is focused by a 63-magnification, 1.3-numerical- aperture oil-immersion objective lens onto a specimen. The pupil transfer lens serves to match the intermediate image planes of the scanning head and the microscope, and its location is critical. In order to maximize the quality of the image, (that is, the point spread function of the objective lens for all scan positions), the entire system was modeled in optical-design software, and the various free design parameters (the parameters of the spatial-filter components as well as the separations of all of the system components) were determined through an iterative optimization process. A modular design was chosen to facilitate access to the optical train for future fluorescence correlation spectroscopy and fluorescence-lifetime experiments.

Fischer, David G.↗

NASA NTRS2015

Spectral and Temporal Laser Fluorescence Analysis Such as for Natural Aquatic Environments

An Advanced Laser Fluorometer (ALF) can combine spectrally and temporally resolved measurements of laser-stimulated emission (LSE) for characterization of dissolved and particulate matter, including fluorescence constituents, in liquids. Spectral deconvolution (SDC) analysis of LSE spectral measurements can accurately retrieve information about individual fluorescent bands, such as can be attributed to chlorophyll-a (Chl-a), phycobiliprotein (PBP) pigments, or chromophoric dissolved organic matter (CDOM), among others. Improved physiological assessments of photosynthesizing organisms can use SDC analysis and temporal LSE measurements to assess variable fluorescence corrected for SDC-retrieved background fluorescence. Fluorescence assessments of Chl-a concentration based on LSE spectral measurements can be improved using photo-physiological information from temporal measurements. Quantitative assessments of PBP pigments, CDOM, and other fluorescent constituents, as well as basic structural characterizations of photosynthesizing populations, can be performed using SDC analysis of LSE spectral measurements.

Chekalyuk, Alexander↗

NASA NTRS2015

Particle Image Velocimetry Applications Using Fluorescent Dye-Doped Particles

Polystyrene latex sphere particles are widely used to seed flows for velocimetry techniques such as Particle Image Velocimetry (PIV) and Laser Doppler Velocimetry (LDV). These particles may be doped with fluorescent dyes such that signals spectrally shifted from the incident laser wavelength may be detected via Laser Induced Fluorescence (LIF). An attractive application of the LIF signal is achieving velocimetry in the presence of strong interference from laser scatter, opening up new research possibilities very near solid surfaces or at liquid/gas interfaces. Additionally, LIF signals can be used to tag different fluid streams to study mixing. While fluorescence-based PIV has been performed by many researchers for particles dispersed in water flows, the current work is among the first in applying the technique to micron-scale particles dispersed in a gas. A key requirement for such an application is addressing potential health hazards from fluorescent dyes; successful doping of Kiton Red 620 (KR620) has enabled the use of this relatively safe dye for fluorescence PIV for the first time. In this paper, basic applications proving the concept of PIV using the LIF signal from KR620-doped particles are exhibited for a free jet and a twophase flow apparatus. Results indicate that while the fluorescence PIV techniques are roughly 2 orders of magnitude weaker than Mie scattering, they provide a viable method for obtaining data in flow regions previously inaccessible via standard PIV. These techniques have the potential to also complement Mie scattering signals, for example in multi-stream and/or multi-phase experiments.

Petrosky, Brian J.↗

NASA NTRS2021

Scaling photosynthetic function and CO2 dynamics from leaf to canopy level for maize – dataset combining diurnal and seasonal measurements of vegetation fluorescence, reflectance and vegetation indices with canopy gross ecosystem productivity

Recent advances in leaf fluorescence measurements and canopy proximal remote sensing currently enable the non-destructive collection of rich diurnal and seasonal time series, which are required for monitoring vegetation function at the temporal and spatial scales relevant to the natural dynamics of photosynthesis. Remote sensing assessments of vegetation function have traditionally used actively excited foliar chlorophyll fluorescence measurements, canopy optical reflectance data and vegetation indices (VIs), and only recently passive solar induced chlorophyll fluorescence (SIF) measurements. In general, reflectance data are more sensitive to the seasonal variations in canopy chlorophyll content and foliar biomass, while fluorescence observations more closely relate to the dynamic changes in plant photosynthetic function. With this dataset we link leaf level actively excited chlorophyll fluorescence, canopy proximal reflectance and SIF, with eddy covariance measurements of gross ecosystem productivity (GEP). The dataset was collected during the 2017 growing season on maize, using three automated systems (i.e., Monitoring Pulse-Amplitude-Modulation fluorimeter, Moni-PAM; Fluorescence Box, FloX; and from eddy covariance tower). The data were quality checked, filtered and collated to a common 30 minutes timestep. We derived vegetation indices related to canopy functioning (e.g., Photochemical Reflectance Index, PRI; Normalized Difference Vegetation Index, NDVI; Chlorophyll Red-edge, Clre) to investigate how SIF and VIs can be coupled for monitoring vegetation photosynthesis. The raw datasets and the filtered and collated data are provided to enable new processing and analyses.

Petya Campbell↗

DOE OSTI2026

Fluorescent Particle Vertical Profiles (Counts and Fractions) at SAIL

This dataset contains vertically resolved fluorescent aerosol number concentrations and fluorescent fractions of total aerosol number concentrations measured with the ARM Tethered Balloon System during the Surface Atmosphere Integrated Field Laboratory campaign near Gothic, Colorado. Measurements were collected during April and June 2023 as part of the ARM–EMSL FICUS project “Characterization of the Aerobiome during the ARM Surface-Atmosphere Integrated Laboratory Campaign,” which was designed to characterize the vertical distribution of bioaerosol-relevant particles in the Colorado River Basin. The dataset includes profiles of fluorescent particle concentrations measured with a Fluorescent Portable Optical Particle Sizer (FPOPS) in the approximately 0.5–3 µm diameter range. For fluorescent fractions of total aerosol number concentrations, we compare FPOPS measurements to POPS on the same TBS flight.

Fluorescent particle concentration↗

NASA NTRS1994

Rapid in situ assessment of physiological activities in bacterial biofilms using fluorescent probes

Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Rh 123 is incorporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be determined using epifluorescence microscopy. The results obtained with these two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparable and always showed approximately 2-fold higher values than those obtained with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time required for the DVC method and 24 h for colony formation. The results indicate that staining with CTC and Rh 123 provides rapid information regarding cell numbers and physiological activities of bacteria within biofilms.

NASA Discipline Number 04-10↗

DOE OSTI2025

Characterizing leaf-scale fluorescence with spectral invariants

Sun-induced chlorophyll fluorescence (SIF) is increasingly recognized as a non-destructive probe for tracking terrestrial photosynthesis. Emerging developments in spectral invariants theory provide an innovative and efficient approach for representing SIF radiative transfer processes at the canopy scale. However, modeling leaf-scale fluorescence based on the spectral invariants properties (SIP) remains underexplored. In this study, the spectral invariants theory is employed for the first time to model the leaf-scale total, backward and forward fluorescence (leaf-SIP SIF). The leaf-SIP SIF model separates the leaf-scale radiative transfer process into two distinct components: the wavelength-dependent one associated with leaf biochemical properties, and the wavelength-independent component linked to leaf structural characteristics. The leaf structure-related effects are characterized by two spectrally invariant parameters: the photon recollision probability (p) and the scattering asymmetry parameter (q), which are parameterized using the directly measurable leaf dry matter. Evaluation against field measurements shows that the proposed leaf-SIP SIF model has a good performance, with coefficient of determination (R 2 ) of 0.89, 0.89, 0.90 and root mean squared errors (RMSE) of 1.28, 0.69, 0.74 Wm -2 µm -1 sr -1 , respectively for the total, backward, and forward fluorescence (660–800 nm). The leaf-SIP SIF model with a more concise formulation demonstrates comparable performance with the widely used Fluspect model. Further, the leaf-SIP SIF model provides a simple and efficient approach for simulating leaf-scale fluorescence, with the potential to be integrated into a unified SIP-based model framework for simulating the radiative transfer processes across the soil-leaf-canopy-atmosphere continuum.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Atomically Precise Hexanuclear Ce(IV) Clusters as Functional Fluorescent Nanosensors for Rapid One-Step Detection of PFAS

Here, the presence of poly- and perfluoroalkyl substances (PFAS) in the environment is associated with adverse health effects but measuring PFAS is challenging due to the associated high cost and technical complexities of the analysis. Here, the reactivity of atomically precise metal-oxo clusters is reported and the foundation for their use is provided as fluorescent nanosensors for PFAS detection. The material comprises crystalline, water soluble, hexanuclear cerium-oxo clusters [Ce 6 (µ 3 -O) 4 (µ 3 -OH) 4 ] 12+ decorated with glycine molecules (Ce-Gly) characterized by fluorescence emission at 353 nm. The Ce-Gly fluorescence is found sensitive to long chain carboxylated PFAS of CF 3 –(CF 2 ) n –, where n ≥ 6, such as perfluorooctanoic, perfluorononanoic and perfluorodecanoic acids. This unique reactivity leads to a change in the emission spectra in a concentration dependent manner, enabling PFAS detection through ligand exchange and aggregation-induced emission (AIE) enhancement. No significant cross-reactivity from potentially co-existing species, including sulfonated PFAS, octanoic and dodecanoic acids, humic acid, and inorganic ions is observed. With an optimal concentration of 3.3 µg mL -1 Ce-Gly, the method demonstrated detection limits of 0.24 ppb for PFOA and 0.4 ppb for PFNA. These findings highlight the potential of fluorescence-based detection strategies utilizing nanoscale probes such as Ce-Gly as fluorescent probes and nanosensors for PFAS.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

DOE OSTI2026

Development of high throughput light-sheet fluorescence lifetime imaging microscopy for 3D functional imaging of metabolic pathways in plant and microorganisms (Final Technical Report)

This research program will enable new biochemical contrast in the nanosecond lifetime domain through use of the recently demonstrated electro-optic fluorescence lifetime imaging technique (EO-FLIM) for wide-field lifetime imaging. The Stanford/Stanford Linear Accelerator Center multidisciplinary collaboration -- physics, applied physics, and structural biology -- will develop a light-sheet fluorescence lifetime imaging microscope for functional studies of microbial and plant metabolic pathways and dynamic interactions between plants and microorganisms in the rhizosphere. The proposed approach overcomes the imaging time bottleneck associated with existing fluorescence lifetime imaging methods. Initial demonstrations have shown a factor of 100,000 improvement in photon throughput compared to existing methods. High photon efficiency allowed the first wide-field fluorescence lifetime imaging of single molecules. Recent work has improved the technique’s repetition rate to enable compatibility with mode-locked lasers and demonstrated the combination of wide-field fluorescence lifetime imaging with super-resolution localization microscopy, observations of single molecule dynamics, and observation of donor lifetime quenching in single-molecule imaging. These results were achieved on standard camera sensors and would not have been possible with other wide-field approaches. The throughput and photon economy of the EO-FLIM method enables new BER-relevant imaging opportunities. In particular, scanned single- and two-photon light-sheet excitation will be used to achieve volumetric imaging with time-domain contrast.

47 OTHER INSTRUMENTATION↗