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NASA NTRS1976

Computer synthesis of high resolution electron micrographs

Specimen damage, spherical aberration, low contrast and noisy sensors combine to prevent direct atomic viewing in a conventional electron microscope. The paper describes two methods for obtaining ultra-high resolution in biological specimens under the electron microscope. The first method assumes the physical limits of the electron objective lens and uses a series of dark field images of biological crystals to obtain direct information on the phases of the Fourier diffraction maxima; this information is used in an appropriate computer to synthesize a large aperture lens for a 1-A resolution. The second method assumes there is sufficient amplitude scatter from images recorded in focus which can be utilized with a sensitive densitometer and computer contrast stretching to yield fine structure image details. Cancer virus characterization is discussed as an illustrative example. Numerous photographs supplement the text.

Nathan, R.↗

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 NTRS2005

Use of a Novel Rover-mounted Fluorescence Imager and Fluorescent Probes to Detect Biological Material in the Atacama Desert in Daylight

We have developed an imaging system, the Fluorescence Imager (FI), for detecting fluorescence signals from sparse microorganisms and biofilms during autonomous rover exploration. The fluorescence signals arise both from naturally occurring chromophores, such as chlorophyll of cyanobacteria and lichens, and from fluorescent probes applied to soil and rocks. Daylight imaging has been accomplished by a novel use of a high-powered flashlamp synchronized to a CCD camera. The fluorescent probes are cell permanent stains that have extremely low intrinsic fluorescence (quantum yields less than 0.01) and a large fluorescence enhancement (quantum yields greater than 0.4) when bound to the target. Each probe specifically targets either carbohydrates, proteins, nucleic acids or membrane lipids, the four classes of macromolecules found in terrestrial life. The intent of the probes is to interrogate the environment for surface and endolithic life forms.

Weinstein, S.↗

DOE OSTI2026

Quantum Light Imaging of the Plant Root─Rhizobacteria─System Using iLOV Fluorescence

It has been demonstrated recently that the use of specific quantum states of light for two-photon microscopy can lead to a substantial reduction of the intensity of light needed for the imaging of biological objects. This opens new ways to better understand the delicate mechanisms of the interactions of living organisms in the rhizosphere on an extended time scale without optical damage or alteration of their native behavior. Herein, we report a successful realization of entangled two-photon excited fluorescence microscopy of wheat plant roots and a set of biological samples, including purified iLOV protein, bacterial cells expressing iLOV, and combination of these under an extremely low excitation intensity. The entangled two-photon coupling strength of iLOV was modeled using electronic structure calculations which demonstrated that the FMN chromophore in the iLOV protein has an entangled two-photon absorption (ETPA) cross section that is large enough for imaging using weak quantum light for fluorescence excitation. Successful imaging of plant-associated Pantoea sp. YR343 cells expressing iLOV validates the feasibility of using iLOV as a molecular label for characterizing plant-microbe interactions based on entangled photon imaging. In conclusion, the results demonstrate the advantageous use of entangled photon imaging under noninvasive and nondestructive conditions.

Varnavksi, Oleg [Univ. of Michigan, Ann Arbor, MI ↗

DOE OSTI2024

Superconducting nanowire single-photon detector enhanced near-infrared II portable confocal microscopy for tissue imaging with indocyanine green

In this Letter a novel, to our knowledge, approach for near-infrared (NIR) fluorescence portable confocal microscopy is introduced, aiming to enhance fluorescence imaging of biological samples in the NIR-II window. By integrating a superconducting nanowire single-photon detector (SNSPD) into a confocal microscopy, we have significantly leveraged the detection efficiency of the NIR-II fluorescence signal from indocyanine green (ICG), an FDA-approved dye known for its NIR-II fluorescence capabilities. The SNSPD, characterized by its extremely low dark count rate and optimized NIR system detection efficiency, enables the excitation of ICG with 1 mW and the capture of low-light fluorescence signals from deep regions (up to 512 µm). Consequently, our technique was able to produce high-resolution images of bio samples with a superior signal-to-noise ratio, making a substantial advancement in the field of fluorescence microscopy and offering a promising opportunity for future clinical study.

Liu, Yifan (ORCID:0000000328877704)↗

DOE OSTI2025

Enhanced Monte Carlo Simulations for Electron Energy Loss Mitigation in Real-Space Nanoimaging of Thick Biological Samples and Microchips

High-resolution imaging using Transmission Electron Microscopy (TEM) is essential for applications such as grain boundary analysis, microchip defect characterization, and biological imaging. However, TEM images are often compromised by electron energy spread and other factors. In TEM mode, where the objective and projector lenses are positioned downstream of the sample, electron–sample interactions cause energy loss, which adversely impacts image quality and resolution. This study introduces a simulation tool to estimate the electron energy loss spectrum (EELS) as a function of sample thickness, covering electron beam energies from 300 keV to 3 MeV. Leveraging recent advances in MeV-TEM/STEM technology, which includes a state-of-the-art electron source with 2-picometer emittance, an energy spread of 3 × 10 -5 , and optimized beam characteristics, we aim to minimize energy spread. By integrating EELS capabilities into the BNL Monte Carlo (MC) simulation code for thicker samples, we evaluate electron beam parameters to mitigate energy spread resulting from electron–sample interactions. Based on our simulations, we propose an experimental procedure for quantitively distinguishing between elastic and inelastic scattering. The findings will guide the selection of optimal beam settings, thereby enhancing resolution for nanoimaging of thick biological samples and microchips.

36 MATERIALS SCIENCE↗

NASA NTRS1993

High resolution and image processing of otoconia matrix

This study was designed to investigate patterns of fibrils organization in histochemically stained otoconia. Transmission electron microscope and video imaging were used. These data indicate that otoconia of the chick (Gallus domesticus) inner ear may have central cores in vivo. The data also show that the ultrastructural organization of fibrils fixed with aldehydes and histochemical stains follows trajectories that conform to the hexagonal shape of otoconia. These changes in direction may contribute to the formation of a central core. The existence of central cores is important for the in vivo buoyancy of otoconia. Packing of fibrils is tighter after phosphotungstic acid (PTA) stained otoconia than with other histochemical stains, which usually produce looser packing of fibrils and seemingly larger central core. TEM of tilted and untilted material showed that turning of fibrils occurs at the points where the face angles of otoconia form and where central cores exist. Video image processing of the images allowed reconstructing a template which, if assumed to repeat and change trajectories, would fit the pattern of fibrils seen in fixed otoconia. Since it is highly unlikely that aldehyde primary fixation or PTA stain caused such drastic change in the direction of fibrils, the template derived from these results may closely approximate patterns of otoconia fibrils packing in vivo. However, if the above is correct, the perfect crystallographic diffraction pattern of unfixed otoconia do not correspond to patterns of fixed fibrils.

NASA Discipline Neuroscience↗

DOE OSTI2024

Calibrating cryogenic temperature of TEM specimens using EELS

Cryogenic Scanning/Transmission Electron Microscopy has been established as a leading method to image sensitive biological samples and is now becoming a powerful tool to understand materials' behavior at low temperatures. However, achieving precise local temperature calibration at low temperatures remains a challenge, which is especially crucial for studying phase transitions and emergent physical properties in quantum materials. In this study, we employ electron energy loss spectroscopy (EELS) to measure local cryogenic specimen temperatures. Here we use the temperature-dependent characteristics of aluminum's bulk plasmon peak in EEL spectra, which shifts due to changes in electron density caused by thermal expansion and contraction. We successfully demonstrate the versatility of this method by calibrating different liquid nitrogen cooling holders in various microscopes, regardless of whether a monochromated or non-monochromated electron beam is used. Temperature discrepancies between the actual temperature and the setpoint temperatures are identified across a range from room temperature to 100 K. This work demonstrates the importance of temperature calibrations at intermediate temperatures and presents a straightforward, robust method for calibrating local temperatures of cryogenically-cooled specimens in electron microscopes.

36 MATERIALS SCIENCE↗

DOE OSTI2025

Prolonging All-Optical Molecular Electron Spin Coherence in the Tissue Transparency Window

Coherent electron spin states within paramagnetic molecules hold significant potential for microscopic quantum sensing. However, all-optical coherence measurements amenable to high spatial and temporal resolution under ambient conditions remain a significant challenge. Here we conduct room-temperature, picosecond time-resolved Faraday ellipticity/rotation (TRFE/R) measurements of the electron spin decoherence time T 2 * in [IrBr 6 ] 2- . Decoherence is strongly sensitive to solution phase viscosity, pointing to molecular tumbling as an important decoherence mechanism. Accordingly, immobilization of [IrBr 6 ] 2- molecules in thin polymer films results in an order-of-magnitude increase in coherence lifetime and significantly greater magnetic field sensitivity. Here, by tuning energies of ligand-to-metal charge transfer (LMCT) states, TRFE/R enables spin initialization and readout in the tissue transparency window, paving the way toward all-optical, ultrafast molecular electron spin coherence imaging in biological systems.

Electron paramagnetic resonance spectroscopy↗

DOE OSTI2026

Enabling robust, stable, and accurate nonlinear optical measurements from squeezed light generated in hot rubidium vapor

The feasibility of nonlinear optical (NLO) imaging and spectroscopy using low intensity quantum states of light including entangled photon and squeezed light sources, such as those driven by two-photon absorption (TPA), has been a topic of ongoing debate. An unambiguous identification of the appreciable quantum advantage in such quantum light applications could enable NLO imaging of biological samples without photodegradation and phototoxicity. Recently, we have constructed a two-mode squeezed light source based on four-wave mixing in 85Rb vapor, which is capable of 7.9 dB of intensity-difference squeezing (IDS), corresponding to 8.7 dB upon electronic noise correction. In this talk, we discuss the stability of our system, including implementation details on achieving and maintaining 8.7 dB of IDS for several hours, and ensuring the two spatially multimode beams are properly overlapped in a sample with minimal optical loss.

Allen, Harry [ORNL] (ORCID:0000000190253914)↗

NASA NTRS1977

Large-area soft X-ray imaging system

System consists of large-area focusing collector and position sensitive proportional counter. Device can be used to study plasmas, with X-ray imaging in biological sciences, and crystallography.

Gorenstein, P.↗

NASA NTRS1993

Tin-polyimide and indium-polyimide thin-film composites as soft X-ray bandpass filters

A tin-polyimide and an indium-polyimide soft X-ray bandpass filter were fabricated with thicknesses of 1400 and 1750 A for the metal and polyimide components, respectively. The transmission of each filter was measured at the Stanford Synchrotron Radiation Laboratory. The transmission of the tin-polyimide filter was found to be about 40 percent for radiation with wavelengths between 60 and 80 A. The transmission of the indium-polyimide filter was greater than 40 percent between 70 and 90 A. The indium was about 5 percent more transmissive than the tin and attained a maximum transmission of about 48 percent at 76 A. Such filters have potential applications to soft X-ray telescopes that operate in this region. They might also be of interest to investigators who work with X-ray microscopes that image live biological specimens in the 23-44-A water window.

Powell, Stephen F.↗

NASA NTRS1996

3-D Imaging In Virtual Environment: A Scientific Clinical and Teaching Tool

The advent of powerful graphics workstations and computers has led to the advancement of scientific knowledge through three-dimensional (3-D) reconstruction and imaging of biological cells and tissues. The Biocomputation Center at NASA Ames Research Center pioneered the effort to produce an entirely computerized method for reconstruction of objects from serial sections studied in a transmission electron microscope (TEM). The software developed, ROSS (Reconstruction of Serial Sections), is now being distributed to users across the United States through Space Act Agreements. The software is in widely disparate fields such as geology, botany, biology and medicine. In the Biocomputation Center, ROSS serves as the basis for development of virtual environment technologies for scientific and medical use. This report will describe the Virtual Surgery Workstation Project that is ongoing with clinicians at Stanford University Medical Center, and the role of the Visible Human data in the project.

Ross, Muriel D.↗

NASA NTRS2012

Computing Radiative Transfer in a 3D Medium

A package of software computes the time-dependent propagation of a narrow laser beam in an arbitrary three- dimensional (3D) medium with absorption and scattering, using the transient-discrete-ordinates method and a direct integration method. Unlike prior software that utilizes a Monte Carlo method, this software enables simulation at very small signal-to-noise ratios. The ability to simulate propagation of a narrow laser beam in a 3D medium is an improvement over other discrete-ordinate software. Unlike other direct-integration software, this software is not limited to simulation of propagation of thermal radiation with broad angular spread in three dimensions or of a laser pulse with narrow angular spread in two dimensions. Uses for this software include (1) computing scattering of a pulsed laser beam on a material having given elastic scattering and absorption profiles, and (2) evaluating concepts for laser-based instruments for sensing oceanic turbulence and related measurements of oceanic mixed-layer depths. With suitable augmentation, this software could be used to compute radiative transfer in ultrasound imaging in biological tissues, radiative transfer in the upper Earth crust for oil exploration, and propagation of laser pulses in telecommunication applications.

Von Allmen, Paul↗

DOE OSTI2025

Experimental study of energy-dependent angular broadening of MeV electron beams for high-resolution imaging in thick samples

In scanning transmission electron microscopy (STEM), spatial resolution is primarily influenced by the projected size of the electron probe within the specimen. In thin samples, a large semi-convergence angle enables a tightly focused beam and sub-nanometer resolution. However, in thick specimens, resolution is fundamentally limited by transverse beam broadening from multiple large-angle scattering events—for example, a probe with 10 mrad angular divergence can broaden by ∼100 nm over a 10 μm path. Since this broadening scales inversely with beam energy, MeV-STEM offers a promising route for high-resolution imaging in thick materials. To quantitatively assess this effect, we performed high-precision measurements at UCLA’s PEGASUS beamline, characterizing beam divergence and intensity profiles for 3–8 MeV electrons transmitted through a wedged-silicon sample of varying thickness. Our results reconcile discrepancies among analytical models and validate Monte Carlo simulations. Here, we find that increasing beam energy from 3.0 to 5.8 MeV reduces angular broadening by a factor of 2.6, with diminishing returns observed at 7.6 MeV. These findings provide a quantitative framework for optimizing MeV-STEM parameters in high-resolution imaging of thick biological and microelectronic specimens, and for guiding beam energy selection in other advanced imaging modes beyond STEM.

36 MATERIALS SCIENCE↗

DOE OSTI2026

A Step-by-Step Protocol from METASPACE to Biological Interpretation

Mass spectrometry imaging (MSI) represents an exceptional tool for exploring complex biological systems spatially at the molecular level. However, due to its multidimensional nature and large-scale data output, it presents considerable challenges when it comes to extracting meaningful biological insights. Recent advancements, such as the METASPACE platform, have enabled researchers to efficiently process, annotate, and interpret MSI datasets by leveraging machine learning and cloud-based infrastructure. In this tutorial, we present a detailed and user-friendly R-pipeline designed to help METASPACE users navigate untargeted metabolomic annotations and transform them into practical insights about their biological systems. By combining METASPACE annotations with rapid R-based screening, this workflow not only streamlined the analytical process but also enhanced the understanding of spatial molecular distribution, especially for complex systems. Here, this easy-to-follow approach has the potential for applications in diagnostics, drug discovery, environmental and ecological processes, and more. We envision this pipeline to be particularly useful for newcomers to the field of MSI and

Moreno Pedraza, Abigail↗

DOE OSTI2025

3D quantum ghost imaging microscope

Quantum ghost imaging uses quantum-entangled photons to generate a two-dimensional image with only a bucket detector at the sample. Here we expand on this approach to generate a three-dimensional image without scanning. A quantum-entangled light source directly links information between a pair of 2D sensors, one of which captures a standard image from one perspective and a second sensor which captures a ghost image from a perpendicular perspective. By correlating the spatial information from the two detectors for each photon pair, we obtain three dimensions of spatial information (x, y, and z) for each scattered photon. We demonstrate that this system can study microscopic environments by imaging scattering from metallic nanoparticle clusters. This approach has the potential to greatly reduce the flux of light required to obtain a 3D image of a biological sample and thereby extend the number of images that can be obtained before photodamaging the sample.

Eshun, Audrey [Lawrence Livermore National Laborat↗

DOE OSTI2024

Adaptive optical third-harmonic generation microscopy for in vivo imaging of tissues

Third-harmonic generation microscopy is a powerful label-free nonlinear imaging technique, providing essential information about structural characteristics of cells and tissues without requiring external labelling agents. In this work, we integrated a recently developed compact adaptive optics module into a third-harmonic generation microscope, to measure and correct for optical aberrations in complex tissues. Taking advantage of the high sensitivity of the third-harmonic generation process to material interfaces and thin membranes, along with the 1,300-nm excitation wavelength used here, our adaptive optical third-harmonic generation microscope enabled high-resolution in vivo imaging within highly scattering biological model systems. Examples include imaging of myelinated axons and vascular structures within the mouse spinal cord and deep cortical layers of the mouse brain, along with imaging of key anatomical features in the roots of the model plant Brachypodium distachyon. In all instances, aberration correction led to enhancements in image quality.

60 APPLIED LIFE SCIENCES↗