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Results for “Fluorescence lifetime imaging microscopy (FLIM)”

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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15 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

A General Autofluorescence Method to Characterize Polymerization Progress

An autofluorescence technique to characterize polymerization progress in real time/in line was developed, which functioned in the absence of typical fluorogenic groups on the monomer or polymer. The monomer dicyclopentadiene and polymer polydicyclopentadiene are hydrocarbons that lack traditional functional groups for fluorescence spectroscopy. Here, the autofluorescence of formulations containing this monomer and polymer during ruthenium-catalyzed ring-opening metathesis polymerization (ROMP) was harnessed for reaction monitoring. The methods fluorescence recovery after photobleaching (FRAP) and here-developed fluorescence lifetime recovery after photobleaching (FLRAP) characterized polymerization progress in these native systems—without requiring exogenous fluorophore. (Auto)fluorescence lifetime recovery changes during polymerization correlated linearly to degree of cure, providing a quantitative link with reaction progress. These changing signals also provided relative rates of background polymerization, enabling comparison of 10 different catalyst–inhibitor-stabilized formulations. Multiple-well analysis demonstrated suitability for future high-throughput evaluation of formulations for thermosets. Furthermore, the central concept of the combined autofluorescence and FLRAP/FRAP method may be extendable to monitoring other polymerization reactions previously overlooked for lack of an obvious fluorescence handle.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Self‐Assembly Methods Induce Different Individual‐Polymer‐Block Solvation Responses

An understanding of block-specific responses to stimuli in self-assembled copolymers is essential for the effective design of responsive materials. Here, materials formed from different post-reaction processing methods of the same ROMP block copolymer exhibit different block-specific solvent responses, as characterized in situ by fluorescence lifetime imaging microscopy (FLIM). FLIM data were acquired by tagging the polar or nonpolar block separately with a covalently incorporated viscosity-sensitive fluorescent molecular rotor to measure the changing tightness or looseness of assembly upon changes in solvent composition. Rapid precipitation from an insoluble solvent to form a film results in tighter assembly and less difference in tightness/looseness between the two blocks. Further, it interrupts individual block-solvent response behavior. Both results indicate an apparent disruption of the core–shell assembly. This model from FLIM is further supported by differential scanning calorimetry (DSC) and small-angle X-ray scattering (SAXS) data from isolated solids, which show tighter long-range interactions and more long-range order in these kinetically trapped states. Here, the experiments develop FLIM as a method for pinpointing stimuli-responsive behaviors from different processing methods to individual blocks. Together these outcomes provide a future handle for tailoring solvent-triggered assembly/disassembly behavior.

López, Pía A. [University of California, Irvine, C↗
DOE OSTI2022

NIR fluorescence lifetime macroscopic imaging with a time-gated SPAD camera

The performance of SwissSPAD2 (SS2), a large scale, widefield time-gated CMOS SPAD imager developed for fluorescence lifetime imaging, has recently been described in the context of visible range and fluorescence lifetime imaging microscopy (FLIM) of dyes with lifetimes in the 2.5 – 4 ns range. Here, we explore its capabilities in the NIR regime relevant for small animal imaging, where its sensitivity is lower and typical NIR fluorescent dye lifetimes are much shorter (1 ns or less). We carry out this study in a simple macroscopic imaging setup based on a compact NIR picosecond pulsed laser, an engineered diffuser-based illumination optics, and NIR optimized imaging lens suitable for well-plate or small animal imaging. Because laser repetition rates can vary between models, but the synchronization signal frequency accepted by SS2 is fixed to 20 MHz, we first checked that a simple frequency-division scheme enables data recording for different laser repetition rates. Next, we acquired data using different time gate widths, including gates with duration longer than the laser period, and analyzed the resulting data using both standard nonlinear least-square fit (NLSF) and phasor analysis. We show that the fixed synchronization rate and large gate widths characterizing SS2 (10 ns and over) are not an obstacle to accurately extracting lifetime in the 1 ns range and to distinguishing between close lifetimes. As a result, SS2 and similar very large gated SPAD imagers appear as a versatile alternative to other widefield time-resolved detectors for NIR fluorescence lifetime imaging, including preclinical molecular applications.

FLIM↗
NASA NTRS2004

Biophotonics and Bone Biology

One of the more serious side effects of extended space flight is an accelerated bone loss. Rates of bone loss are highest in the weight-bearing bones of the hip and spine regions, and the average rate of bone loss as measured by bone mineral density measurements is around 1.2% per month for persons in a microgravity environment. It is well known that bone remodeling responds to mechanical forces. We are developing two-photon microscopy techniques to study bone tissue and bone cell cultures to better understand the fundamental response mechanism in bone remodeling. Osteoblast and osteoclast cell cultures are being studied, and the goal is to use molecular biology techniques in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) to study the physiology of in-vitro cell cultures in response to various stimuli, such as fluid flow induced shear stress and mechanical stress. We have constructed a two-photon fluorescence microscope for these studies, and are currently incorporating FLIM detection. Current progress will be reviewed. This work is supported by the NASA John Glenn Biomedical Engineering Consortium.

Zimmerli, Gregory↗
DOE OSTI2024

A red-emitting carborhodamine for monitoring and measuring membrane potential

Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

PLEKHA7 signaling is necessary for the growth of mutant KRAS driven colorectal cancer

Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a “sentry” role preventing PI(3,4)P{sub 2} and PI(3,4,5)P{sub 3} binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.

60 APPLIED LIFE SCIENCES↗
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 OSTI2024

A modular chemigenetic calcium indicator for multiplexed in vivo functional imaging

Abstract Genetically encoded fluorescent calcium indicators allow cellular-resolution recording of physiology. However, bright, genetically targetable indicators that can be multiplexed with existing tools in vivo are needed for simultaneous imaging of multiple signals. Here we describe WHaloCaMP, a modular chemigenetic calcium indicator built from bright dye-ligands and protein sensor domains. Fluorescence change in WHaloCaMP results from reversible quenching of the bound dye via a strategically placed tryptophan. WHaloCaMP is compatible with rhodamine dye-ligands that fluoresce from green to near-infrared, including several that efficiently label the brain in animals. When bound to a near-infrared dye-ligand, WHaloCaMP shows a 7× increase in fluorescence intensity and a 2.1-ns increase in fluorescence lifetime upon calcium binding. We use WHaloCaMP1a to image Ca 2+ responses in vivo in flies and mice, to perform three-color multiplexed functional imaging of hundreds of neurons and astrocytes in zebrafish larvae and to quantify Ca 2+ concentration using fluorescence lifetime imaging microscopy (FLIM).

Biochemistry & Molecular Biology↗
NASA NTRS2003

Cellular Oxygen and Nutrient Sensing in Microgravity Using Time-Resolved Fluorescence Microscopy

Oxygen and nutrient sensing is fundamental to the understanding of cell growth and metabolism. This requires identification of optical probes and suitable detection technology without complex calibration procedures. Under this project Microcosm developed an experimental technique that allows for simultaneous imaging of intra- and inter-cellular events. The technique consists of frequency-domain Fluorescence Lifetime Imaging Microscopy (FLIM), a set of identified oxygen and pH probes, and methods for fabrication of microsensors. Specifications for electronic and optical components of FLIM instrumentation are provided. Hardware and software were developed for data acquisition and analysis. Principles, procedures, and representative images are demonstrated. Suitable lifetime sensitive oxygen, pH, and glucose probes for intra- and extra-cellular measurements of analyte concentrations have been identified and tested. Lifetime sensing and imaging have been performed using PBS buffer, culture media, and yeast cells as a model systems. Spectral specifications, calibration curves, and probes availability are also provided in the report.

Szmacinski, Henryk↗
DOE OSTI2025

Ultra-high Information-content Chemical Imaging with Broadband Coherent Anti-Stokes Raman and Two-photon Fluorescence Lifetime Microscopy

Raman fingerprint spectroscopy and fluorescence lifetime imaging are emerging tools for studying metabolic profiles of biological specimens. While Raman fingerprint spectroscopy detects intrinsic molecular vibrations that reflect the molecular composition and chemical environment of a sample, fluorescence lifetime imaging measures changes in the excited-state lifetime of fluorophores that are sensitive to their microenvironments. Here, we present a multimodal imaging platform combining broadband coherent anti-Stokes Raman scattering (BCARS) and two-photon fluorescence lifetime imaging (2p-FLIM) microscopy that can acquire biologically relevant Raman fingerprint spectra and fluorescence lifetime signals in vivo and simultaneously. The tremendous chemical information obtained from spatially co-registered BCARS and 2p-FLIM images allows us to characterize the subtle differences between sub-cellular compartments and verify the potential false-positive results generated by fluorescence imaging alone. This is demonstrated by directly comparing the BCARS, 2p-FLIM, and two-photon excitation fluorescence(TPEF) signals simultaneously obtained from the same dye-stained organelle in the live, intact C. elegans expressing a green fluorescent protein (GFP) marker. In this work, we introduce the BCARS/2p-FLIM/TPEF setup scheme, the image acquisition steps, data processing, and representative results showing that the cross-modality imaging method enables rigorous characterization and in vivo detection at sub-cellular resolution. Furthermore, this protocol provides a framework for simultaneous chemical and fluorescence lifetime imaging to improve the accuracy of biological interpretation in complex living systems.

Xu, Haoyu [Georgia Institute of Technology, Atlant↗
DOE OSTI2022

Excited state lifetime modulation in semiconductor nanocrystals for super-resolution imaging

Abstract We report on proof of principle measurements of a concept for a super-resolution imaging method that is based on excitation field density-dependent lifetime modulation of semiconductor nanocrystals. The prerequisite of the technique is access to semiconductor nanocrystals with emission lifetimes that depend on the excitation intensity. Experimentally, the method requires a confocal microscope with fluorescence-lifetime measurement capability that makes it easily accessible to a broad optical imaging community. We demonstrate with single particle imaging that the method allows one to achieve a spatial resolution of the order of several tens of nanometers at moderate fluorescence excitation intensity.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Data for "In vitro and in vivo NIR Fluorescence Lifetime Imaging with a time-gated SPAD camera"

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo. Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

2DG↗
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↗
DOE OSTI2022

In vitro and in vivo NIR fluorescence lifetime imaging with a time-gated SPAD camera

Near-infrared (NIR) fluorescence lifetime imaging (FLI) provides a unique contrast mechanism to monitor biological parameters and molecular events in vivo . Single-photon avalanche diode (SPAD) cameras have been recently demonstrated in FLI microscopy (FLIM) applications, but their suitability for in vivo macroscopic FLI (MFLI) in deep tissues remains to be demonstrated. Herein, we report in vivo NIR MFLI measurement with SwissSPAD2, a large time-gated SPAD camera. We first benchmark its performance in well-controlled in vitro experiments, ranging from monitoring environmental effects on fluorescence lifetime, to quantifying Förster resonant energy transfer (FRET) between dyes. Next, we use it for in vivo studies of target-drug engagement in live and intact tumor xenografts using FRET. Information obtained with SwissSPAD2 was successfully compared to that obtained with a gated intensified charge-coupled device (ICCD) camera, using two different approaches. Our results demonstrate that SPAD cameras offer a powerful technology for in vivo preclinical applications in the NIR window.

47 OTHER INSTRUMENTATION↗