Invited Review Article: Pump-probe microscopy Fischer, Martin C; Wilson, Jesse W; Robles, Francisco E ...
Review of scientific instruments,
03/2016, Letnik:
87, Številka:
3
Journal Article
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Multiphoton microscopy has rapidly gained popularity in biomedical imaging and materials science because of its ability to provide three-dimensional images at high spatial and temporal resolution ...even in optically scattering environments. Currently the majority of commercial and home-built devices are based on two-photon fluorescence and harmonic generation contrast. These two contrast mechanisms are relatively easy to measure but can access only a limited range of endogenous targets. Recent developments in fast laser pulse generation, pulse shaping, and detection technology have made accessible a wide range of optical contrasts that utilize multiple pulses of different colors. Molecular excitation with multiple pulses offers a large number of adjustable parameters. For example, in two-pulse pump-probe microscopy, one can vary the wavelength of each excitation pulse, the detection wavelength, the timing between the excitation pulses, and the detection gating window after excitation. Such a large parameter space can provide much greater molecular specificity than existing single-color techniques and allow for structural and functional imaging without the need for exogenous dyes and labels, which might interfere with the system under study. In this review, we provide a tutorial overview, covering principles of pump-probe microscopy and experimental setup, challenges associated with signal detection and data processing, and an overview of applications.
Ion mobility-mass spectrometry (IM-MS) can be a powerful tool for determining structural information about ions in the gas phase, from small covalent analytes to large, native-like or denatured ...proteins and complexes. For large biomolecular ions, which may have a wide variety of possible gas-phase conformations and multiple charge sites, quantitative, physically explicit modeling of collisional cross sections (CCSs) for comparison to IMS data can be challenging and time-consuming. We present a “trajectory method” (TM) based CCS calculator, named “Collidoscope,” which utilizes parallel processing and optimized trajectory sampling, and implements both He and N
2
as collision gas options. Also included is a charge-placement algorithm for determining probable charge site configurations for protonated protein ions given an input geometry in pdb file format. Results from Collidoscope are compared with those from the current state-of-the-art CCS simulation suite, IMoS. Collidoscope CCSs are within 4% of IMoS values for ions with masses from ~18 Da to ~800 kDa. Collidoscope CCSs using X-ray crystal geometries are typically within a few percent of IM-MS experimental values for ions with mass up to ~3.5 kDa (melittin), and discrepancies for larger ions up to ~800 kDa (GroEL) are attributed in large part to changes in ion structure during and after the electrospray process. Due to its physically explicit modeling of scattering, computational efficiency, and accuracy, Collidoscope can be a valuable tool for IM-MS research, especially for large biomolecular ions.
Graphical Abstract
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SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with ...several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 μM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer's therapy, was shown to have antiviral activity between 10 to 66 μM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.
Significance: Ultrafast fiber lasers are an attractive alternative to bulk lasers for nonlinear optical microscopy for their compactness and low cost. The high relative intensity noise (RIN) of these ...lasers poses a challenge for pump-probe measurements such as transient absorption and stimulated Raman scattering, along with modalities that provide label-free contrast from the vibrational and electronic structure of molecules.
Aim: Digital adaptive filtering was applied to determine the applicability for canceling laser RIN in a transient absorption microscope with an ultrafast fiber laser source.
Approach: Digitized signals from the transmitted probe and reference photodetectors were fed to an adaptive filter in MATLAB, running in a noise canceling configuration. This result was then fed to a software lock-in algorithm to demodulate the pump-probe signal. Images were built up one line scan at a time with a 3.5-kHz resonant scanner, with 100 × averaging. The imaging target was Bi4Ge3O12, which exhibits nondegenerate two-photon absorption at the pump/probe wavelengths used (530-nm pump and 490-nm probe).
Results: Without adaptive noise cancellation, the lock-in output primarily passes the laser RIN within its detection bandwidth, resulting in images that closely follow the linear transmissivity and lack sensitivity to pump-probe time delay. With adaptive noise cancellation in front of the lock-in, the RIN rejection is enough to restore the z-sectioning and sensitivity to pump-probe delay, as expected for transient absorption. Results were limited primarily by noise from the photodetector and analog-to-digital converter.
Conclusions: Digital adaptive noise cancellation, even when limited by electronics noise, can recover pump-probe signals by removal of laser RIN, under conditions where averaging alone fails.
Hemeproteins are frequent subjects for ultrafast transient absorption spectroscopy (TAS) because of biological importance, strong UV–vis absorption, high photostability, and interesting transient ...dynamics that depend on redox, conformation, and ligand binding. TAS on hemeproteins is usually performed on isolated, purified proteins, though their response is likely to be different in their native molecular environment, which involves the formation of protein complexes and supercomplexes. Recently, we reported a transient absorption microscopy (TAM) experiment which elicited a transient response from hemeproteins in intact biological tissue using a visible-wavelength pump (530 nm) and probe (490 nm). Here, we find that adaptive noise canceling plus resonant galvanometer scanning enables a high-repetition-rate fiber laser source to make redox-sensitive measurements of cytochrome c (Cyt-c). We investigate the origins of the visible-wavelength response of biological tissue through TAS of intact mitochondrial respiratory supercomplexes, separated via gel electrophoresis. We find that each of these high-molecular-weight gel bands yields a TAS response characteristic of cytochrome hemes, implying that the TAS response of intact cells and tissue originates from not just Cyt-c but a mixture of respiratory cytochromes. We also find differences in excited-state lifetime between wild-type (WT) and a tafazzin-deficient (TAZ) mouse model of mitochondrial disease.
The transcription factor Gata6 regulates proliferation and differentiation of epithelial and endocrine cells and cancers. Among hematopoietic cells, Gata6 is expressed selectively in resident ...peritoneal macrophages. We thus examined whether the loss of Gata6 in the macrophage compartment affected peritoneal macrophages, using Lyz2-Cre x Gata6(flox/flox) mice to tackle this issue. In Lyz2-Cre x Gata6(flox/flox) mice, the resident peritoneal macrophage compartment, but not macrophages in other organs, was contracted, with only a third the normal number of macrophages remaining. Heightened rates of death explained the marked decrease in peritoneal macrophage observed. The metabolism of the remaining macrophages was skewed to favor oxidative phosphorylation and alternative activation markers were spontaneously and selectively induced in Gata6-deficient macrophages. Gene expression profiling revealed perturbed metabolic regulators, including aspartoacylase (Aspa), which facilitates generation of acetyl CoA. Mutant mice lacking functional Aspa phenocopied the higher propensity to death and led to a contraction of resident peritoneal macrophages. Thus, Gata6 regulates differentiation, metabolism, and survival of resident peritoneal macrophages.
Many soluble and membrane proteins form symmetrical homooligomeric complexes. However, determining the oligomeric state of protein complexes can be difficult. Alpha-hemolysin (αHL) from ...Staphylococcus aureus is a symmetrical homooligomeric protein toxin that forms transmembrane β-barrel pores in host cell membranes. The stable pore structure of αHL has also been exploited in vitro as a nanopore tool. Early structural experiments suggested αHL forms a hexameric pore, while more recent X-ray crystal structure and solution studies have identified a heptameric pore structure. Here, using native ion mobility-mass spectrometry (IM-MS) we find that αHL simultaneously forms hexameric and heptameric oligomers in both tetraethylene glycol monooctyl ether (C8E4) and tetradecylphosphocholine (FOS-14) detergent solutions. We also analyze intact detergent micelle-embedded αHL porelike complexes by native IM-MS without the need to fully strip the detergent micelle, which can cause significant gas-phase unfolding. The highly congested native mass spectra are deconvolved using Fourier- and Gábor-transform (FT and GT) methods to determine charge states and detergent stoichiometry distributions. The intact αHL micelle complexes are found to contain oligomeric state-proportional numbers of detergent molecules. This evidence, combined with IM data and results from vacuum molecular dynamics simulations, is consistent with both the hexamer and the heptamer forming porelike complexes. The ability of αHL to form both oligomeric states simultaneously has implications for its use as a nanopore tool and its pore formation mechanism in vivo. This study also demonstrates more generally the power of FT and GT to deconvolve the charge state and stoichiometry distributions of polydisperse ions.
Mitochondrial redox is an important indicator of cell metabolism and health, with implications in cancer, diabetes, aging, neurodegenerative diseases, and mitochondrial disease. The most common ...method to observe redox of individual cells and mitochondria is through fluorescence of NADH and FAD+, endogenous cofactors serve as electron transport inputs to the mitochondrial respiratory chain. Yet this leaves out redox within the respiratory chain itself. To a degree, the missing information can be filled in by exogenous fluorophores, but at the risk of disturbed mitochondrial permeability and respiration. Here we show that variations in respiratory chain redox can be detected up by visible-wavelength transient absorption microscopy (TAM). In TAM, the selection of pump and probe wavelengths can provide multiphoton imaging contrast between non-fluorescent molecules. Here, we applied TAM with a pump at 520nm and probe at 450nm, 490nm, and 620nm to elicit redox contrast from mitochondrial respiratory chain hemeproteins. Experiments were performed with reduced and oxidized preparations of isolated mitochondria and whole muscle fibers, using mitochondrial fuels (malate, pyruvate, and succinate) to set up physiologically relevant oxidation levels. TAM images of muscle fibers were analyzed with multivariate curve resolution (MCR), revealing that the response at 620nm probe provides the best redox contrast and the most consistent response between whole cells and isolated mitochondria.
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