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We report the design and synthesis of a bright red cationic molecular probe with impressive optical properties for intracellular RNA imaging.
Ultrahigh dose rate (FLASH) radiation therapy is reported to reduce normal tissue toxicity while maintaining tumor control; however, mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a specialized high-energy electron linear accelerator, High Intensity Gamma Ray Source (HIGS), paired with an organotypic ex vivo brain metastasis model. We varied interpulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR). We characterized dosimetry and targeting accuracy of the FLASH beam with film dosimetry. We combined this FLASH beam with an organotypic rat brain slice/breast carcinoma coculture model of brain metastasis to assess effects on normal and neoplastic tissues. Live-cell and bioluminescence imaging demonstrated cancer cell growth effects, whereas normal tissue responses and immune activation were assessed using live-cell imaging, cytokine profiles, and confocal microscopy. Here, we performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) using conventional dose rates. The highest IDR of the FLASH beam to date was 20.7 ± 0.6 MGy/s, with maximum MDR of 20.7 MGy/s delivered in 1 pulse of 1 µs duration. Beam targeting was accurate to <1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased tumor necrosis factor α and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology slices suggesting microglia activation. Our novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation in the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.
Disclosed herein are systems, methods, systems and devices for measurement and visualization of chemical dynamics in living cells or tissues for diagnostic pathology. Devices can be open- or closed-channel microfluidic membrane devices for long-term IR spectroscopy of live adherent cells and ultimately for rapidly identifying time-dependent spectral features indicative of chemical abnormality in individual cells.
Nonlinear optical microscopy that leverages an objective based total internal reflection (TIR) excitation scheme is an attractive means for rapid, wide-field imaging with enhanced surface sensitivity. Through select combinations of distinct modalities, one can, in principle, access complementary chemical and structural information for various chemical species near interfaces. Here, we report a successful implementation of such a wide-field nonlinear optical microscope system, which combines coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and sum frequency generation (SFG) modalities on the same platform. The intense optical fields needed to drive these high order nonlinear optical processes are achieved through the use of femtosecond pulsed light in combination with the intrinsic field confinement induced by TIR over a large field of view. The performance of our multimodal microscope was first assessed through the experimental determination of its chemical fidelity, intensity and polarization dependences, and spatial resolution using a set of well-defined model systems. Subsequently, its unique capabilities were validated through imaging complex biological systems, including Hydrangea quercifolia pollen grains and Pantoea sp. YR343 bacterial cells. Specifically, the spatial distribution of different molecular groups in the former was visualized via vibrational contrast mechanisms of CARS, whereas co-registered TPF imaging allowed the identification of spatially localized intrinsic fluorophores. We further demonstrate the feasibility of our microscope for wide-field CARS imaging on live cells through independent characterization of cell viability using spatially co-registered TPF imaging. Overall, this approach to TIR enabled wide-field imaging is expected to provide new insights into bacterial strains and their interactions with other species in the rhizosphere in a time-resolved and chemically selective manner.
The multiplexing capability of fluorescence microscopy is severely limited by the broad fluorescence spectral width. Spectral imaging offers potential solutions, yet typical approaches to disperse the local emission spectra notably impede the attainable throughput. Here we show that using a single, fixed fluorescence emission detection band, through frame-synchronized fast scanning of the excitation wavelength from a white lamp via an acousto-optic tunable filter, up to six subcellular targets, labeled by common fluorophores of substantial spectral overlap, can be simultaneously imaged in live cells with low (~1%) crosstalks and high temporal resolutions (down to ~10 ms). The demonstrated capability to quantify the abundances of different fluorophores in the same sample through unmixing the excitation spectra next enables us to devise novel, quantitative imaging schemes for both bi-state and Förster resonance energy transfer fluorescent biosensors in live cells. We thus achieve high sensitivities and spatiotemporal resolutions in quantifying the mitochondrial matrix pH and intracellular macromolecular crowding, and further demonstrate, for the first time, the multiplexing of absolute pH imaging with three additional target organelles/proteins to elucidate the complex, Parkin-mediated mitophagy pathway. Together, excitation spectral microscopy provides exceptional opportunities for highly multiplexed fluorescence imaging. The prospect of acquiring fast spectral images without the need for fluorescence dispersion or care for the spectral response of the detector offers tremendous potential.
The formation of stable protein complexes enables much of biotechnology, but even high-affinity complexes can dissociate, limiting potential applications in biomaterials, bioimaging, nanomedicine, and other protein-based technologies. Here, in this study, we describe lock coupling, a simple and selective one-step reaction between interfacial lysine and glutamate or aspartate side chains to form stable isopeptide bonds and be used for the precise labeling of native antibodies. We identify conditions in which short-lived activated esters formed by the aqueous carbodiimide EDC promote isopeptide bond formation specifically at preassociated amine-acid pairs. Indiscriminate cross-linking is minimized by formation of protein complexes before addition of catalyst, use of acidic pH to suppress exposed Lys reactivity, and limiting the aqueous stability of activated esters. For native antibody (Ab) labeling, we show that the small IgG-binding protein GB1 can be covalently attached to the Ab Fc domain and that introduction of Cys into GB1 loops allows for facile conjugation of fluorophores, micelles, or inorganic nanocrystals for imaging in live cells and animals. By varying Cys substituents and protein stoichiometry, a defined number of probes can be uniformly attached without the need for extensive purification. In live-cell confocal microscopy, labeled GB1 serves as a stable replacement for secondary Abs, enabling simple multicolor immunostaining and imaging. Lock coupling requires just a single reagent in aqueous buffer and leverages both the innate ability of proteins to form high-affinity complexes and the widespread presence of Lys-Glu/Asp pairs at their interfaces, with the potential for precision synthesis of protein-based probes for imaging, biomaterials, biophysics, and medicine.
Abstract X-ray computed tomography (XCT) and X-ray fluorescence (XRF) imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray computed tomography and fluorescence imaging for the same cell have yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative XCT and XRF measurement, and computational reconstruction of the correlative and multi-modality images. With XCT, cellular structures including the overall structure and intracellular organelles are visualized, while XRF imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of chemical elements and related proteins in signaling and other biological processes.
Sucrose is the main saccharide used for longdistance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real-time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small ( 50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne-tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real-time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.
Here, we report some new observations on the luminescence of carbon dots (CDs) that are passivated with polyethylene glycol, and explored them, for the first time, as highly sensitive H2S detection probe. The as-prepared CDs have an ultraviolet (UV) emission at 350 nm and a green emission at 540 nm when excited at 270 nm, but they have only a green emission at 523 nm when excited at 365 nm. As Na2S is added to the CDs, the UV emission decreases gradually, while the green emission increases slightly, and interestingly a new emission appears at 455 nm is increased linearly in intensity with the increase of Na2S concentration. The blue emission at 455 nm is from the intrinsic core of CDs, and the green emissions at 540 or 523 nm are from their surface states, while the UV emission at 350 nm is from the anchored PEG. Both the emission intensity ratios of F-450/F-350 and F-455/F-523 show an excellent linear relationship with the Na2S concentration in the range of 0-800 mu M, and the detection limit estimated from both of them is approximately 7.0 nM, indicating our method is the most sensitive H2S CDs based detection method reported thus far. Furthermore, the detection was tested successfully for H2S imaging in live cells. We propose that the interactions of H2S with the CDs and the anchored ligands, as well as the energy transfer between the ligands and the CDs, are responsible for the luminescence responses for H2S detection. Our studies enrich the world of carbon dots with plenty of potential applications. (C) 2020 Elsevier Ltd. All rights reserved.
Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90–99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.
The microscopy research at the Bionanoprobe (currently at beamline 9-ID and later 2-ID after APS-U) of Argonne National Laboratory focuses on applying synchrotron X-ray fluorescence (XRF) techniques to obtain trace elemental mappings of cryogenic biological samples to gain insights about their role in critical biological activities. The elemental mappings and the morphological aspects of the biological samples, in this instance, the bacterium Escherichia coli ( E. Coli ), also serve as label-free biological fingerprints to identify E. coli cells that have been treated differently. The key limitations of achieving good identification performance are the extraction of cells from raw XRF measurements via binary conversion, definition of features, noise floor and proportion of cells treated differently in the measurement. Automating cell extraction from raw XRF measurements across different types of chemical treatment and the implementation of machine-learning models to distinguish cells from the background and their differing treatments are described. Principal components are calculated from domain knowledge specific features and clustered to distinguish healthy and poisoned cells from the background without manual annotation. The cells are ranked via fuzzy clustering to recommend regions of interest for automated experimentation. The effects of dwell time and the amount of data required on the usability of the software are also discussed.
A class of POSS-containing 2D nanocrystals was developed to offer high stability, biocompatibility, and programmability.
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.
Abstract Confocal imaging has shown that CELLULOSE SYNTHASE (CESA) particles move through the plasma membrane as they synthesize cellulose. However, the resolution limit of confocal microscopy circumscribes what can be discovered about these tiny biosynthetic machines. Here, we applied Structured Illumination Microscopy (SIM), which improves resolution two-fold over confocal or widefield imaging, to explore the dynamic behaviors of CESA particles in living plant cells. SIM imaging reveals that Arabidopsis thaliana CESA particles are more than twice as dense in the plasma membrane as previously estimated, helping explain the dense arrangement of cellulose observed in new wall layers. CESA particles tracked by SIM display minimal variation in velocity, suggesting coordinated control of CESA catalytic activity within single complexes and that CESA complexes might move steadily in tandem to generate larger cellulose fibrils or bundles. SIM data also reveal that CESA particles vary in their overlaps with microtubule tracks and can complete U-turns without changing speed. CESA track patterns can vary widely between neighboring cells of similar shape, implying that cellulose patterning is not the sole determinant of cellular growth anisotropy. Together, these findings highlight SIM as a powerful tool to advance CESA imaging beyond the resolution limit of conventional light microscopy.
Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.
Abstract Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM‐polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM‐based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type‐A CBMs from families 3a and 64 with crystalline cellulose‐I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose‐I using equilibrium binding assays. To compute the adsorption ( nk on ) and desorption ( k off ) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide‐field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.
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.
Signal-mediated regulation of protein trafficking is an elegant mechanism for controlling the delivery of molecules to a precise location for critical signaling events that occur over short time frames. During plant reproduction, the FERONIA receptor complex is critical for intercellular communication that leads to gamete delivery; however, the impact of the FERONIA signal transduction cascade on protein trafficking in synergid cells remains unknown. Live imaging of pollen tube reception has revealed that a key outcome of FERONIA signaling is polar accumulation of the MLO protein NORTIA at the filiform apparatus in response to signals from an arriving pollen tube. Artificial delivery of NORTIA to the filiform apparatus is sufficient to bypass the FERONIA signaling pathway and to promote interspecific pollen tube reception. We propose that polar accumulation of NORTIA leads to the production of a secondary booster signal to ensure that pollen tubes burst to deliver the sperm cells for double fertilization.