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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Characterization, comparison, and optimization of lattice light sheets

Lattice light sheet microscopy excels at the noninvasive imaging of three-dimensional (3D) dynamic processes at high spatiotemporal resolution within cells and developing embryos. Recently, several papers have called into question the performance of lattice light sheets relative to the Gaussian sheets most common in light sheet microscopy. Here, we undertake a theoretical and experimental analysis of various forms of light sheet microscopy, which demonstrates and explains why lattice light sheets provide substantial improvements in resolution and photobleaching reduction. The analysis provides a procedure to select the correct light sheet for a desired experiment and specifies the processing that maximizes the use of all fluorescence generated within the light sheet excitation envelope for optimal resolution while minimizing image artifacts and photodamage. We also introduce a new type of “harmonic balanced” lattice light sheet that improves performance at all spatial frequencies within its 3D resolution limits and maintains this performance over lengthened propagation distances allowing for expanded fields of view.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Integrative Quantitative-Phase and Airy Light-Sheet Imaging

Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free organelle recognition and metabolic information that are inaccessible by conventional methods. We report the fusion of these two modalities onto a standard inverted microscope that retains compatibility with microfluidics. We describe the utilization of an accelerating Airy-beam light-sheet yielding identical imaging areas with interferometry, and an application in unmasking the effects of cellular noise on metabolic compartmentalization.

Biological sciences, Biological techniques, Micros↗
DOE OSTI2021

Photon-Sparse, Poisson Light-Sheet Microscopy

Light-sheet microscopy has revolutionized bioimaging by enabling approximately an order of magnitude reduction in specimen irradiance compared to confocal imaging. Here, we introduce a light-sheet imaging system that enables an additional order of magnitude reduction in specimen irradiance by operating at the Poisson limit. To operate at this limit, we integrated classical illumination with single-photon detection and wavelet-based image reconstruction. This integration enabled brightness quantification and object recognition from fewer than one detected photon per image pixel, corresponding to more than 10-fold lower irradiance levels than modern systems. We demonstrate how such photon-sparse imaging can eradicate photobleaching and enable both dim and bright object imaging, thus, further enhancing the related gains of light-sheet microscopy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Image processing tools for petabyte-scale light sheet microscopy data

Light sheet microscopy is a powerful technique for high-speed three-dimensional imaging of subcellular dynamics and large biological specimens. However, it often generates datasets ranging from hundreds of gigabytes to petabytes in size for a single experiment. Conventional computational tools process such images far slower than the time to acquire them and often fail outright due to memory limitations. To address these challenges, we present PetaKit5D, a scalable software solution for efficient petabyte-scale light sheet image processing. This software incorporates a suite of commonly used processing tools that are optimized for memory and performance. Notable advancements include rapid image readers and writers, fast and memory-efficient geometric transformations, high-performance Richardson–Lucy deconvolution and scalable Zarr-based stitching. These features outperform state-of-the-art methods by over one order of magnitude, enabling the processing of petabyte-scale image data at the full teravoxel rates of modern imaging cameras. The software opens new avenues for biological discoveries through large-scale imaging experiments.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2021

Airy light-sheet Raman imaging

Light-sheet fluorescence microscopy has greatly improved the speed and overall photostability of optically sectioning cellular and multi-cellular specimens. Similar gains have also been conferred by light-sheet Raman imaging; these schemes, however, rely on diffraction limited Gaussian beams that hinder the uniformity and size of the imaging field-of-view, and, as such, the resulting throughput rates. Here, we demonstrate that a digitally scanned Airy beam increases the Raman imaging throughput rates by more than an order of magnitude than conventional diffraction-limited beams. Overall, this, spectrometer-less, approach enabled 3D imaging of microparticles with high contrast and 1 µm axial resolution at 300 msec integration times per plane and orders of magnitude lower irradiation density than coherent Raman imaging schemes. We detail the apparatus and its performance, as well as its compatibility with fluorescence light-sheet and quantitative-phase imaging towards rapid and low phototoxicity multimodal imaging.

47 OTHER INSTRUMENTATION↗
DOE OSTI2022

Data-driven Mapping of the Mouse Connectome: The utility of transfer learning to improve the performance of deep learning models performing axon segmentation on light-sheet microscopy images

Light sheet microscopy has made possible the high temporal and spatial 3D imaging of both fixed and live biological tissue, with samples as large as the entire mouse brain. However, segmentation and quantification of that data remains a time-consuming manual process. Machine learning methods promise the possibility of automating this process. This study seeks to advance the performance of prior models through the application of refinements such as transfer learning.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Scalable Thin Light-Emitting Diode (LED) Light Sheet Platform

The goal of the Scalable Thin Light-Emitting Diode (LED) Light Sheet Platform project is to derisk the manufacturing scalability for a high chip count heterogeneous smart lighting platform, with the potential to enable significant energy savings through dynamic directional light control and custom. Specifically, the project derisks the scalability of a disruptive new computer controlled microassembly fabrication process SRI International is developing, to address fundamental cost barriers to mass production of high chip count systems. This is key for enabling mass adoption and thus maximum societal energy savings impact. The LED light sheet technology is a smart illumination platform that can be applied to lighting, signage, and display. It has features and a form factor similar to bendable or conformal OLED light sheets but uses more efficient LEDs with a remote phosphor layer that is very close to the LED to deliver a luminous efficacy that exceeds 125 lumen/Watt and enable > 50% Lighting Application Efficiency (LAE) energy savings. During Budget Period 1 (BP1), all of the BP1 milestones were successfully completed: (M2.0.1) Design demonstrator details that will meet the final project goals, (M6.1.1) Show lighting output model can predict illuminance, spatial, spectral distribution for specific light-sheet designs and use case, (M3.2.1) Automated loading supports 200 chip arrays, and (M3.3.1) Finish first interconnect process run for 200 chip array. The BP1 Go/No-Go Decision Point (G/NG 1) 200 chip demonstrator sample was in the process of final assembly and test at the end of BP1 on December 31, 2023. The objective was successfully achieved on January 16, 2024, when a wired 200-chip sample underwent confirmation testing that demonstrated a 93.75% first pass electrical yield that successfully exceeded the 75% first-pass electrical yield BP1 G/NG requirement. In addition, all 93.75% of the sample devices lit up, further demonstrating the ability to assemble and transfer small LED chips without damaging their functionality.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2024

Fine-tuning TrailMap: The utility of transfer learning to improve the performance of deep learning in axon segmentation of light-sheet microscopy images

Light-sheet microscopy has made possible the 3D imaging of both fixed and live biological tissue, with samples as large as the entire mouse brain. However, segmentation and quantification of that data remains a time-consuming manual undertaking. Machine learning methods promise the possibility of automating this process. This study seeks to advance the performance of prior models through optimizing transfer learning. We fine-tuned the existing TrailMap model using expert-labeled data from noradrenergic axonal structures in the mouse brain. By changing the cross-entropy weights and using augmentation, we demonstrate a generally improved adjusted F1-score over using the originally trained TrailMap model within our test datasets.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2023

Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array

Significance: Fluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples. Aim: We aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM. Approach: A single-photon avalanche diode camera was integrated into a light sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells. Results: An NAD(P)H light-sheet FLIM system was built and validated with fluores cence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30× to 6× acquisition speed advantage for the light sheet compared to the laser scanning geometry. Conclusions: FLIM of NAD(P)H is feasible in a light-sheet geometry and is attrac tive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.

47 OTHER INSTRUMENTATION↗
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↗
DOE OSTI2023

Scattered–light–sheet microscopy with sub–cellular resolving power

Since its first demonstration over 100 years ago, scattering-based light-sheet microscopy has recently re-emerged as a key modality in label-free tissue imaging and cellular morphometry; however, scattering-based light-sheet imaging with subcellular resolution remains an unmet target to date. This is because related approaches inevitably superimpose speckle or granular intensity modulation on to the native subcellular features. Here, we addressed this challenge by deploying a time-averaged pseudo-thermalized light-sheet illumination. While pseudo-thermalizing the illumination sheet increased its lateral dimensions, we achieved subcellular resolving power at ultralow irradiance levels after image deconvolution. We validated this approach by imaging cytosolic carbon depots in yeast and bacteria with increased specificity and no staining. Altogether, we expect this scattering-based light-sheet microscopy approach will advance live single-cell imaging investigations by conferring low-irradiance and label-free operation towards eradicating phototoxicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Note: Experimental observation of nano-channel pattern in light sheet laser interference nanolithography system

We experimentally observed nano-channel-like pattern in a light-sheet based interference nanolithography system. The optical system created nano-channel-like patterned illumination. Coherent counter-propagating light sheets are made to interfere at and near geometrical focus along the propagation z-axis. This results in the formation of nano-channel-like pattern (of size ≈ 300 nm and inter-channel periodicity of ≈337.5 nm) inside the sample due to constructive and destructive interference. In addition, the technique has the ability to generate large area patterning using larger light-sheets. Exciting applications are in the broad field of nanotechnology (nano-electronics and nano-fluidics).

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2021

Fusing Quantitative-Phase Imaging with Airy Light- Sheet Microscopy

We report the integration of quantitative-phase imaging (QPI) with light-sheet (LS) fluorescent microscopy on to a standard inverted microscope that retains compatibility with microfluidics. QPI enables label-free imaging and number-density quantification of single cells and their organelles. Conversely, LS yields considerable speed and phototoxicity gains in quantifying the 4D dynamics of gene-encoded fluorescent biomarkers. We will detail the system design that relied on spatial light interferometry for QPI and an accelerating Airy- beam light-sheet for fluorescence, its performance, as well as results of a representative multivariate imaging analysis of single-cell metabolism.

imaging, lattice light sheet, Yarrowia lipolytica,↗
DOE OSTI2024

Lattice light-sheet microscopy allows for super-resolution imaging of receptors in leaf tissue

Plant leaf tissues are difficult to image via fluorescent microscopy, largely due to the presence of chlorophyll and other pigments that provide large background fluorescence. An advantage of Lattice Light-Sheet microscopy is its use of Bessel beams that illuminate a thin focal region of interest for microscopy, allowing for the excitation of fluorescent molecules within this region without surrounding chlorophyll-like objects outside of the region of interest. Here, we apply STORM Super-resolution techniques to observe Receptor-Like Kinases in Arabidopsis thaliana leaf cells. By applying this technique with the Lattice Light-Sheet, we can localize immune response proteins in sub-100 nm length scales and reconstruct three-dimensional locations of proteins within individual leaf cells. Using this technique, we observed the effect of the elicitors ATP and flg22, where we observed a significant degree of internalization of cognate receptors P2K1 and FLS2. We were also able to similarly observe differences in colocalization due to stimulation with these elicitors, where we observe proteins on the membrane becoming less colocalized as a result of stimulation, suggesting an immune response mechanism involving receptors internalizing via pathways distinct to the receptor. Further, these data show the Lattice Light-Sheet’s capabilities for imaging tissue with problematic background fluorescence that otherwise makes super-resolution fluorescence microscopy difficult.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Video-rate Raman-based metabolic imaging by Airy light-sheet illumination and photon-sparse detection

Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Volumetric imaging of the 3D orientation of cellular structures with a polarized fluorescence light-sheet microscope

Polarized fluorescence microscopy is a valuable tool for measuring molecular orientations in biological samples, but techniques for recovering three-dimensional orientations and positions of fluorescent ensembles are limited. We report a polarized dual-view light-sheet system for determining the diffraction-limited three-dimensional distribution of the orientations and positions of ensembles of fluorescent dipoles that label biological structures. We share a set of visualization, histogram, and profiling tools for interpreting these positions and orientations. We model the distributions based on the polarization-dependent efficiency of excitation and detection of emitted fluorescence, using coarse-grained representations we call orientation distribution functions (ODFs). We apply ODFs to create physics-informed models of image formation with spatio-angular point-spread and transfer functions. We use theory and experiment to conclude that light-sheet tilting is a necessary part of our design for recovering all three-dimensional orientations. We use our system to extend known two-dimensional results to three dimensions in FM1-43-labeled giant unilamellar vesicles, fast-scarlet-labeled cellulose in xylem cells, and phalloidin-labeled actin in U2OS cells. Additionally, we observe phalloidin-labeled actin in mouse fibroblasts grown on grids of labeled nanowires and identify correlations between local actin alignment and global cell-scale orientation, indicating cellular coordination across length scales.

Science & Technology - Other Topics↗
DOE OSTI2016

Light-sheet microscopy by confocal line scanning of dual-Bessel beams

Here, we have developed a light-sheet microscope that uses confocal scanning of dual-Bessel beams for illumination. A digital micromirror device (DMD) is placed in the intermediate image plane of the objective used to collect fluorescence and is programmed with two lines of pixels in the “on” state such that the DMD functions as a spatial filter to reject the out-of-focus background generated by the side-lobes of the Bessel beams. The optical sectioning and out-of-focus background rejection capabilities of this microscope were demonstrated by imaging of fluorescently stained actin in human A431 cells. The dual-Bessel beam system enables twice as many photons to be detected per imaging scan, which is useful for low light applications (e.g., single-molecule localization) or imaging at high speed with a superior signal to noise. While demonstrated for two Bessel beams, this approach is scalable to a larger number of beams.

confocal microscopy↗