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All sources NASA NTRS DOE OSTI

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↗

NASA NTRS2006

Advances in Measurement of Skin Friction in Airflow

The surface interferometric skin-friction (SISF) measurement system is an instrument for determining the distribution of surface shear stress (skin friction) on a wind-tunnel model. The SISF system utilizes the established oil-film interference method, along with advanced image-data-processing techniques and mathematical models that express the relationship between interferograms and skin friction, to determine the distribution of skin friction over an observed region of the surface of a model during a single wind-tunnel test. In the oil-film interference method, a wind-tunnel model is coated with a thin film of oil of known viscosity and is illuminated with quasi-monochromatic, collimated light, typically from a mercury lamp. The light reflected from the outer surface of the oil film interferes with the light reflected from the oil-covered surface of the model. In the present version of the oil-film interference method, a camera captures an image of the illuminated model and the image in the camera is modulated by the interference pattern. The interference pattern depends on the oil-thickness distribution on the observed surface, and this distribution can be extracted through analysis of the image acquired by the camera. The oil-film technique is augmented by a tracer technique for observing the streamline pattern. To make the streamlines visible, small dots of fluorescentchalk/oil mixture are placed on the model just before a test. During the test, the chalk particles are embedded in the oil flow and produce chalk streaks that mark the streamlines. The instantaneous rate of thinning of the oil film at a given position on the surface of the model can be expressed as a function of the instantaneous thickness, the skin-friction distribution on the surface, and the streamline pattern on the surface; the functional relationship is expressed by a mathematical model that is nonlinear in the oil-film thickness and is known simply as the thin-oil-film equation. From the image data acquired as described, the time-dependent oil-thickness distribution and streamline pattern are extracted and by inversion of the thin-oil-film equation it is then possible to determine the skin-friction distribution. In addition to a quasi-monochromatic light source, the SISF system includes a beam splitter and two video cameras equipped with filters for observing the same area on a model in different wavelength ranges, plus a frame grabber and a computer for digitizing the video images and processing the image data. One video camera acquires the interference pattern in a narrow wavelength range of the quasi-monochromatic source. The other video camera acquires the streamline image of fluorescence from the chalk in a nearby but wider wavelength range. The interference- pattern and fluorescence images are digitized, and the resulting data are processed by an algorithm that inverts the thin-oil-film equation to find the skin-friction distribution.

Brown, James L.↗

NASA NTRS1999

UV Raman and Fluorescence for Multi-Species Measurement in Hydrocarbon-Fueled High-Speed Propulsion

This report documents work performed through the NASA Graduate Student Researchers Program, Grant No. NGT3-52316. Research performed included investigation of two-line fluorescence imaging of OH for temperature measurement and an investigation of negative flame speeds for modeling of premixed turbulent flames. The laboratory work and initial analysis of the fluorescence imaging was performed at NASA Glen Research Center with follow up analysis at Vanderbilt University. The negative flame speed investigation was performed using an opposed jet flow simulation program at Vanderbilt University. The fluorescence imaging work is presented first followed by the negative flame speed investigation.

Skaggs, Patricia Annette↗

DOE OSTI2025

Spatially resolved charge-transfer kinetics at the quantum dot–microbe interface using fluorescence lifetime imaging microscopy

Integrating the optoelectronic properties of quantum dots (QDs) with biological enzymatic systems to form microbe-semiconductor biohybrids offers promising prospects for both solar-to-chemical conversion and light-modulated biochemical processes. Developing these nano–bio hybrid systems necessitates a deep understanding of charge-transfer dynamics at the nano–bio interface. Photoexcited carrier transfer from QDs to microbes is driven by complex interactions, with emerging insights into the relevant thermodynamic and kinetic factors. The heterogeneities of both microbes and QD ensembles pose significant challenges in mechanistic understanding, which is critical for designing advanced nano–bio hybrids. We used fluorescence lifetime imaging microscopy to analyze charge transfer between a CdSe QD film andShewanella oneidensismicrobes. We correlated the spatiotemporal fluorescence data with an analytical model. Our analysis revealed two distinct distributions of QD de-excitation pathways. The characteristics of these distributions: 1) a faster transfer rate ( k ¯ E T 1 = 1.5 10 9 s - 1 ), with a lower acceptor number ( N ¯ a 1 = 0.03 ) and 2) a slower transfer rate ( k ¯ E T 2 = 4.1 10 8 s - 1 ) with a higher acceptor number ( N ¯ a 2 = 0.18 ). We assign these distributions to the indirect and direct electron transfer mechanisms, respectively. Our findings demonstrate how spectroscopic imaging can uncover fundamental electron transfer mechanisms at complex interfaces, offering valuable design principles for future nano–bio hybrids.

Science & Technology - Other Topics↗

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 NTRS2000

Advances in Fluorescence Sensing Systems for the Remote Assessment of Nitrogen Supply in Field Corn

The studies described herein were conducted to better define changes in fluorescence properties of leaves from field grown corn (Zea mays L.) as they relate to varying levels of nitrogen (N) fertilization. This research was directed toward: 1) providing a remote non-destructive sensing technique to aid in the determination of optimal rates of N fertilization in corn crops and, 2) defining parameters for further development of fluorescence instrumentation to be operated remotely at field canopy levels. Fluorescence imaging bands centered in the blue (450 nm), green (525 nm), red (680 nm), and far-red (740 nm) and ratios of these bands were compared with the following plant parameters: rates of photosynthesis, N:C ratio, pigment concentrations, and grain yields. Both the fluorescence and physiological measures exhibited similar curvilinear responses to N fertilization level while significant linear correlations were obtained among fluorescence bands and band ratios to certain physiological measures of plant productivity. The red / blue, red / green, far-red / blue, far-red /green fluorescence ratios are well suited for remote observation and provided high correlations to grain yield, LAI, N:C, and chlorophyll contents. The results from this investigation indicate that fluorescence technology could aid in the determination of N fertilization requirements for corn. This discussion will also address design concepts and preliminary field trials of a mobile field-based Laser Induced Fluorescence Imaging System (LIFIS) capable of simultaneously acquiring images of four fluorescence emission bands from areas of plant canopies equaling 1 sq m and greater without interference of ambient solar radiation.

Corp, L. A.↗

NASA NTRS2021

Recent Improvements in the Dart Model for Atmosphere, Topography, Large Landscape, Chlorophyll Fluorescence, Satellite Image Inversion

Physical models simulating the radiative budget (RB) and remote sensing (RS) observation of three-dimensional (3D) landscapes are critical to better understand human and natural components of the Earth system and further develop RS technology. DART is one of the most comprehensive 3D models of Earth-atmosphere optical radiative transfer (RT), from ultraviolet (UV) to thermal infrared (TIR). It simulates the optical signal of proximal, aerial and satellite imaging spectrometers and laser scanners, the 3D RB and solar induced chlorophyll fluorescence (SIF) signal, for any urban or natural landscape and any experimental or instrument configuration. It is freely available for research and teaching activities (https://dart.omp.eu). Here, five recent advances are presented. 1) Atmosphere RT. 2) RT in non repetitive topography. 3) Monte Carlo modelling for fast RS image simulation of large landscapes. 4) SIF modelling for vegetation simulated as facets and turbid cells. 5) RS image inversion for mapping the optical properties of urban material and the urban radiative budget.

DART↗

DOE OSTI2025

Characterization of Field-Exposed Photovoltaic Modules Featuring Signs of Contact Degradation

Here, this work investigates several photovoltaic (PV) modules that have shown signs of metal contact corrosion due to field exposure in a hot and humid climate. This includes two multicrystalline silicon aluminum back surface field systems with 10 and 14 years of exposure and one monocrystalline silicon passivated emitter and rear cell system with four years of exposure. A comprehensive, multiscale characterization process is used to evaluate these PV modules in great detail. Current–voltage (I−V), Suns-V OC measurements, electroluminescence imaging, infrared imaging, and ultraviolet fluorescence imaging were performed, and locations of interest were cored and analyzed using cross-sectional scanning electron microscopy (SEM). A rigorous, quantitative analysis procedure for the cross-sectional SEM images is proposed and implemented. Careful characterization does reveal that some of these PV modules do indeed exhibit the same classic signs of acetic-acid-based corrosion of the glass frit that is present at the silver/silicon interface, which have been observed previously in PV modules exposed to damp heat in an environmental chamber.

14 SOLAR ENERGY↗

DOE OSTI2024

LITE-SM: A Light Sheet Illuminator Compatible with Super Resolution and Single Molecule Imaging

The LITE-SM (hereinafter referred to as the Tilt-SM) is a light sheet illumination system purpose-built for single molecule (SM) imaging. SM imaging is a fluorescence imaging technique where individual molecules can be tracked in living cells/tissues in real time allowing for unprecedented insights into the intracellular dynamics of these molecules. The innovative all-mirror optical design greatly reduces/eliminates the aberrations that plague lens-based designs. This iteration of the Tilt-SM has significantly better optical performance then its predecessor with over 10x increase in optical power at the sample, allowing for the use of much smaller and more cost-effective lasers then the original Phase 1 system. Excellent STORM imaging as well as SM tracking has been achieved, satisfying the key objectives of this study.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2005

Radiation Dosimetry via Automated Fluorescence Microscopy

A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern-recognition software developed specifically for this instrument, the images in the various colors are processed to recognize the metaphases and count the chromosome fragments of each color within the metaphases. The intermediate results are then further processed to estimate the proportion of cells that have suffered genetic damage. The prototype instrument scans at an average areal rate of 4.7 mm2/h in unattended operation, finding about 14 metaphases per hour. The false-alarm rate is typically less than 3 percent, and the metaphase-miss rate has been estimated to be less than 5 percent. The counts of chromosomes and fragments thereof are 50 to 70 percent accurate.

Castleman, Kenneth R.↗

NASA NTRS2018

Characterization of Firefly, an Imaging Spectrometer Designed for Airborne Measurements of Solar-Induced Fluorescence

FIREFLY (Fluorescence Imaging of REd and Far-red Light Yield) is a compact, fine-resolution imaging spectrometer that was designed and assembled by Headwall Photonics (Fitchburg, MA, USA) in collaboration with NASA scientists for airborne measurements of Solar-Induced Fluorescence (SIF). FIREFLY is integrated into the next generation of NASA Goddard’s Lidar, Hyperspectral and Thermal airborne imager (G-LiHT; www.gliht.nasa.gov), providing a complete system for measuring, interpreting and scaling SIF emissions. Characterization of FIREFLY was performed here to evaluate its performance and suitability for retrieving SIF.

Imaging Spectroscopy↗

NASA NTRS1996

Stable Lobed Mixer With Combustion Demonstrated and Measured

The NASA Lewis Research Center collaborated with the Massachusetts Institute of Technology (MIT) on an experiment to study the use of lobed mixers to improve the fuel-air mixing process and increase combustion intensity in combustors with minimal pressure loss. This experiment is the first known stable combusting flow studied for this device, and the data show a much faster and much more uniform combustion process than for flat-plate mixers. Several potential benefits may be realized from this study in future combustors, including a reduction in NO_x emissions because of the more uniform temperature distribution. The experiment was done in Lewis' Planar Reacting Shear Layer facility, which was adapted to accept a lobed mixer in addition to the original planar tip. A graduate student at MIT provided the mixer design concept, and Lewis provided the engineering, operations, and research expertise. The experiment used hydrogen-nitrogen mixtures to react with vitiated hot air at 920 K. A flow speed of about 120 m/sec and a speed ratio of 0.5 were used. Flow diagnostics consisted of traversing fine-wire thermocouples and pitot probes for flow mapping. Supplementary fluorescence images were taken with a charged coupled device (CCD) camera to show the location and temporal behavior of the reaction zone. The data showed that the lobed mixer consumed the reactants between 3 to 10 times faster than a corresponding planar shear layer. The figure shows the dramatic difference in the measured temperature distribution with and without the lobed mixer. The increased mixing rate was due to a larger interfacial area as well as to the secondary flow from the streamwise vortices off the tips of the lobes. In addition, the fluorescence images showed that the lobes acted as flame stabilizers.

Source record↗

NASA NTRS1990

Spectral characterization of forest damage occurring on Whiteface Mountain, NY - Studies with the Fluorescence Line Imager (FLI) and ground-based spectrometers

Ground-based spectral characteristics of fir wave damage and an analysis of calibrated FLI data acquired along the same fir wave utilized for the in situ measurements are presented. Derivative curve data were produced from both in situ and FLI reflectance measurements for the red edge spectral region for birch and for various portions of a fir wave. The results suggested that with proper atmospheric correction of airborne imaging spectrometer data sets, the derivative curve approach will provide an accurate means of assessing red edge parameters, and that such data will permit identification of specific types of forest damage on the basis of spectral fine features.

Rock, B. N.↗

DOE OSTI2025

Multiphoton and Harmonic Imaging of Microarchitected Materials

Microadditive manufacturing has revolutionized the production of complex, nano- to microscale components across various fields. This work investigates two-photon (2P) and three-photon (3P) fluorescence imaging, as well as third-harmonic generation (THG) microscopy, to examine periodic microarchitected lattice structures fabricated using multiphoton lithography (MPL). By immersing the structures in refractive index matching fluids, we demonstrate high-fidelity 3D reconstructions of both fluorescent structures using 2P and 3P microscopy as well as low-fluorescence structures using THG microscopy. These results show that multiphoton fluorescence (MPF) imaging offers reduced signal decay with respect to depth compared to single-photon techniques in the examined structures. We further demonstrate the ability to nondestructively identify intentional internal modifications of the structure that are not immediately visible with scanning electron microscope (SEM) images and compression-induced fractures, highlighting the potential of these techniques for quality control and defect detection in microadditively manufactured components.

36 MATERIALS SCIENCE↗

NASA NTRS2013

Characterization of Fluorescent Polystyrene Microspheres for Advanced Flow Diagnostics

Fluorescent dye-doped polystyrene latex microspheres (PSLs) are being developed for velocimetry and scalar measurements in variable property flows. Two organic dyes, Rhodamine B (RhB) and dichlorofluorescence (DCF), are examined to assess laser-induced fluorescence (LIF) properties for flow imaging applications and single-shot temperature measurements. A major interest in the current research is the application of safe dyes, thus DCF is of particular interest, while RhB is used as a benchmark. Success is demonstrated for single-point laser Doppler velocimetry (LDV) and also imaging fluorescence, excited via a continuous wave 2 W laser beam, for exposures down to 10 ms. In contrast, when exciting with a pulsed Nd:YAG laser at 200 mJ/pulse, no fluorescence was detected, even when integrating tens of pulses. We show that this is due to saturation of the LIF signal at relatively low excitation intensities, 4-5 orders of magnitude lower than the pulsed laser intensity. A two-band LIF technique is applied in a heated jet, indicating that the technique effectively removes interfering inputs such as particle diameter variation. Temperature measurement uncertainties are estimated based upon the variance measured for the two-band LIF intensity ratio and the achievable dye temperature sensitivity, indicating that particles developed to date may provide about +/-12.5 C precision, while future improvements in dye temperature sensitivity and signal quality may enable single-shot temperature measurements with sub-degree precision.

Maisto, Pietro M. F.↗

DOE OSTI2025

Single-Objective Airy Light-Sheet Imaging

Despite its massive potential, standard light-sheet imaging (LSI) faces key challenges, such as the incompatibility with common sample mounting techniques and low-resolution imaging. Single-objective LSI attempts to address these issues but often suffers from limited fields-of-view and throughput rates, or requires multiple optics that increase costs, alignment complexity, and losses. To overcome these challenges of standard single-objective LSI, we introduce single-objective Airy light-sheet imaging (SoALSI). SoALSI leverages the extraordinary self-acceleration properties of the Airy beam, achieving 5× higher imaging rates and enhanced imaging efficiency than standard single-objective LSI. Here, we demonstrate SoALSI’s versatility through rigorous contrast and resolution characterizations and by high-resolution imaging of diverse biological specimens, including malaria parasite-infected red blood cells and plant root tissue. SoALSI seamlessly integrates with any standard inverted microscope frame, enabling broader accessibility for the bioimaging community to explore biological processes in a wide range of specimens with enhanced resolution and imaging contrast.

airy beam↗

NASA NTRS1991

Electron beam dispersion measurements in nitrogen using two-dimensional imaging of N2(+) fluorescence

Experimental results are presented related to the radial spread of fluorescence excited by 10 and 20 KeV electron beams passing through nonflowing rarefied nitrogen at 293 K. An imaging technique for obtaining species distributions from measured beam-excited fluorescence is described, based on a signal inversion scheme mathematically equivalent to the inversion of the Abel integral equation. From fluorescence image data, measurements of beam radius, integrated signal intensity, and spatially resolved distributions of N2(+) first-negative-band fluorescence-emitting species have been made. Data are compared with earlier measurements and with an heuristic beam spread model.

Clapp, L. H.↗

NASA NTRS2008

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.↗