Biochemical Imaging of Human Atherosclerotic Plaques with Fluorescence Lifetime Angioscopy

A prototype angioscopy system with fluorescence lifetime imaging microscopy (FLIM) capabilities was built and applied for biochemical imaging of human coronary atherosclerotic plaques. The FLIM angioscopy prototype consisted of a thin flexible angioscope suitable for UV-excited autofluorescence imaging, and a FLIM detection system based on a pulse sampling approach. The angioscope was composed of an imaging bundle attached to a gradient index objective lens and surrounded by a ring of illumination fibers (2 mm outer diameter, 50 μm spatial resolution). For FLIM detection based on the pulse sampling approach, a gated-intensified charge-couple device camera (200 ps temporal resolution) was used. Autofluorescence was excited with a pulsed UV laser (337 nm) and FLIM images were acquired at three emission bands (390/40 nm, 450/40 nm, 550/88 nm). The system was characterized on standard fluorophores and then used to image postmortem human coronary arteries. The FLIM angioscope allowed us to distinguish elastin-dominant plaques (peak emission at 450 nm, ∼1.5 ns lifetimes) from collagen-dominant plaques (peak emission at 390 n, ∼2–3 ns lifetimes) based on their intrinsic fluorescence spectral and lifetime differences. This study demonstrates the potential of FLIM angioscopy for biochemical imaging of human coronary atherosclerotic plaques.     

Ruthenium(II) Complex Enantiomers as Cellular Probes for Diastereomeric Interactions in Confocal and Fluorescence Lifetime Imaging Microscopy

Ruthenium dipyridophenazine (dppz) complexes are sensitive luminescent probes for hydrophobic environments. Here, we apply multiple-frequency fluorescence lifetime imaging microscopy (FLIM) to Δ and Λ enantiomers of lipophilic ruthenium dppz complexes in live and fixed cells, and their different lifetime staining patterns are related to conventional intensity-based microscopy. Excited state lifetimes of the enantiomers determined from FLIM measurements correspond well with spectroscopically measured emission decay curves in pure microenvironments of DNA, phospholipid membrane or a model protein. We show that FLIM can be applied to monitor the long-lived excited states of ruthenium complex enantiomers and, combined with confocal microscopy, give new insight into their biomolecular binding and reveal differences in the microenvironment probed by the complexes.     

Application of FLIM-FIDSAM for the in vivo analysis of hormone competence of different cell types

Background fluorescence derived from subcellular compartments is a major drawback in high-resolution live imaging, especially of plant cells. A novel technique for contrast enhancement of fluorescence images of living cells expressing fluorescent fusion proteins termed fluorescence intensity decay shape analysis microscopy (FIDSAM) has been recently published and is applied here to plant cells expressing wild-type levels of a low-abundant membrane protein (BRI1-EGFP), demonstrating the applicability of FIDSAM to samples exhibiting about 80% autofluorescence. Furthermore, the combination of FIDSAM and fluorescence lifetime imaging microscopy enables the simultaneous determination and quantification of different ligand-specific responses in living cells with high spatial and temporal resolution even in samples with high autofluorescence background. Correlation of different responses can be used to determine the hormone ligand competence of different cell types as demonstrated here in BRI1-EGFP-expressing root and hypocotyl cells.     

Metabolic state oscillations in cerebral nuclei detected using two-photon fluorescence lifetime imaging microscopy

The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH), a key endogenous coenzyme and metabolic biomarker, can reflect the metabolic state of cells. To implement metabolic imaging of brain tissue at high resolution, we assembled a two-photon fluorescence lifetime imaging microscopy (FLIM) platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehyde-fixed mouse cerebral slices. Furthermore, NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus (SCN). The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night, and an ultradian oscillation at night was observed. Through the combination of high-resolution imaging and immunostaining data, the metabolic tendency of different cell types was detected after the first two hours of the day and at night. Thus, two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.     

Near-Infrared Fluorescence Lifetime Imaging of Biomolecules with Carbon Nanotubes**

Single-walled carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorescent building blocks for biosensors. Their surface is chemically tailored to respond to analytes by a change in fluorescence. However, intensity-based signals are easily affected by external factors such as sample movements. Here, we demonstrate fluorescence lifetime imaging microscopy (FLIM) of SWCNT-based sensors in the NIR. We tailor a confocal laser scanning microscope (CLSM) for NIR signals (>800 nm) and employ time correlated single photon counting of (GT)₁₀-DNA functionalized SWCNTs. They act as sensors for the important neurotransmitter dopamine. Their fluorescence lifetime (>900 nm) decays biexponentially and the longer lifetime component (370 ps) increases by up to 25 % with dopamine concentration. These sensors serve as paint to cover cells and report extracellular dopamine in 3D via FLIM. Therefore, we demonstrate the potential of fluorescence lifetime as a readout of SWCNT-based NIR sensors.     

Rational Design of Cyclometalated Iridium(III) Complexes for Three-Photon Phosphorescence Bioimaging

Compared to 2PE (two-photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three-photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two Ir^III complexes (3PAIr1 and 3PAIr2) were developed as efficient three-photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three-photon transition probability. Confocal and lifetime imaging of mitochondria using Ir^III complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm ), fast uptake (5 min), and low threshold for three-photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 μm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).     

Rational Design of Cyclometalated Iridium(III) Complexes for Three‐Photon Phosphorescence Bioimaging

Compared to 2PE (two‐photon excitation) microscopy, 3PE microscopy has superior spatial resolution, deeper tissue penetration, and less defocused interference. The design of suitable agents with a large Stokes shift, good three‐photon absorption (3PA), subcellular targeting, and fluorescence lifetime imaging (FLIM) properties, is challenging. Now, two Ir^III complexes (3PAIr1 and 3PAIr2) were developed as efficient three‐photon phosphorescence (3PP) agents. Calculations reveal that the introduction of a new group to the molecular scaffold confers a quadruple promotion in three‐photon transition probability. Confocal and lifetime imaging of mitochondria using Ir^III complexes as 3PP agents is shown. The complexes exhibit low working concentration (50 nm ), fast uptake (5 min), and low threshold for three‐photon excitation power (0.5 mW at 980 nm). The impressive tissue penetration depth (ca. 450 μm) allowed the 3D imaging and reconstruction of brain vasculature from a living specimen.     

Trimethylsilyl Chloride Aids in Solubilization of Oxidative Addition Intermediates from Zinc Metal

Trimethylsilyl chloride (TMSCl) is commonly used to “activate” metal(0) powders toward oxidative addition of organohalides, but knowledge of its mechanism remains limited by the inability to characterize chemical intermediates under reaction conditions. Here, fluorescence lifetime imaging microscopy (FLIM) overcomes these prior limitations and shows that TMSCl aids in solubilization of the organozinc intermediate from zinc(0) metal after oxidative addition, a previously unknown mechanistic role. This mechanistic role is in contrast to previously known roles for TMSCl before the oxidative addition step. To achieve this understanding, FLIM, a tool traditionally used in biology, is developed to characterize intermediates during a chemical reaction—thus revealing mechanistic steps that are unobservable without fluorescence lifetime data. These findings impact organometallic reagent synthesis and catalysis by providing a previously uncharacterized mechanistic role for a widely used activating agent, an understanding of which is suitable for revising activation models and for developing strategies to activate currently unreactive metals.     

Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.     

Flat hydrogel substrate for atomic force microscopy to observe liposomes and lipid membranes

In order to avoid denaturation of biomolecules due to strong adsorption on solid surfaces, a soft substrate has to be used for atomic force microscopy (AFM) observation. We propose a hydrophilic agarose gel surface as a soft substrate for AFM to observe liposomes and lipid membranes. Although our simple method does not require any delicate control at the molecular level, an agarose gel surface can be simply flattened to 0.3 nm in roughness using an atomically flat solid surface during gelation. The AFM images revealed that liposomes were unruptured on the gel surface at low liposome density, whereas an unruptured state was difficult to obtain on a solid surface like mica. This indicates that the weak interaction between the liposome and the soft surface inhibits the liposome from rupturing, and also that the surface rougher than the solid surface prevents lateral diffusion of the liposomes along the surface to be fused. Increasing the liposome density resulted in a lipid membrane at various thicknesses forming on the hydrogel surface by the fusion and rupture of liposomes. Using the soft substrate, it can be expected to promote investigations of structures and functions of biomolecules at the nanometer scale under physiological conditions with AFM.     

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments, we found the depth of the polymer matrix to be 1–2?μm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2?μm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Simultaneous Detection of Carbon Monoxide and Viscosity Changes in Cells

A new family of robust, non-toxic, water-compatible ruthenium(II) vinyl probes allows the rapid, selective and sensitive detection of endogenous carbon monoxide (CO) in live mammalian cells under normoxic and hypoxic conditions. Uniquely, these probes incorporate a viscosity-sensitive BODIPY fluorophore that allows the measurement of microscopic viscosity in live cells via fluorescence lifetime imaging microscopy (FLIM) while also monitoring CO levels. This is the first example of a probe that can simultaneously detect CO alongside small viscosity changes in organelles of live cells.     

Self-organization of a sulfonamido-porphyrin in Langmuir monolayers and Langmuir-Blodgett films

Langmuir monolayers (LM) and Langmuir-Blodgett (LB) films of pure lipophilic meso-tetra(4-dodecylaminosulfophenyl)porphyrin (PC12) and mixed with the anionic surfactant sodium hexadecylsulfate (SHS) were studied. The molecular packing and structure of PC12 and PC12-4SHS with variable surface pressure were investigated by surface pressure-area measurements, steady-state absorption, fluorescence emission and anisotropy, as well as by fluorescence lifetime imaging microscopy (FLIM). At low surface pressure, the porphyrin molecules are organized with the rings tilted on the water surface whereas at high surface pressure the porphyrin rings achieve a more perpendicular arrangement. Using the FLIM images a gradual change of aggregates into large "islands" is observed. Different patterns are observed in the pure PC12 multilayer films (n = 3 and 5) with ordered patches superimposed which are not observed in the PC12-4SHS multilayer LB films.     

In cell measurement of fluorescence lifetime imaging microscopy revealed C-terminal conformation changes of Ferroportin upon addition of Mn2+

The C-terminal conformation changes of Ferroportin upon addition of Mn²⁺ were revealed by fluorescence lifetime imaging microscopy.     

Different interactions between the two sides of purple membrane with atomic force microscope tip

Atomic force microscopy (AFM) is known to be capable of measuring local surface charge density based on the DLVO model. However, it has failed to distinguish charge density difference between the extracellular and cytoplasmic sides of purple membrane (PM) in previous studies. In this paper, tapping-mode AFM with thioglycolate-modified tips was used to image PM in buffers of different salt concentrations. When imaged in 25 mM KCl buffer, the topography of membranes appeared to be of two different types, one flat and the other domelike. Such a difference was not observed in buffers of high salt concentrations. This suggests that the topography variation results from differences in electrostatic interaction between the AFM tip and the different membrane surfaces. With images of papain-digested PM and high-resolution images of membrane surface structure, we proved that the membrane surfaces with flat topography were on the extracellular side while the surfaces with domelike topography were on the cytoplasmic side. Hence, this provides a straightforward method to distinguish the two sides of PM without the requirement of high-resolution imaging. Force-distance curves clearly demonstrated the different tip-sample interactions. The force curves recorded on the extracellular side of PM were consistent with the DLVO model, so its surface charge density can be estimated well. However, the curves recorded on the cytoplasmic side had a much longer decay length, which is supposed to be relevant to the flexibility of the C-terminus of bacteriorhodopsin (bR).     

Optical Characterization of Zinc Pyrithione

Zinc pyrithione is ubiquitous in commercial products particularly antidandruff shampoos. For the efficacy of zinc pyrithione therapeutic cleansers to be assessed accurately, the distribution of particles on the scalp needs to be visualized. Currently, no technique is available which provides the chemical specificity and sensitivity required. Here, we report application of fluorescence‐lifetime imaging microscopy (FLIM) for high‐contrast mapping of zinc pyrithione distribution on the scalp. Characterization of the zinc pyrithione emission by using both one‐photon excitation at five specific wavelengths and two‐photon excitation in the range of 740–820 nm revealed its FLIM fingerprint—a characteristic short average time‐weighted emission lifetime of Τ_ZnPT = 250 ps. Bandpass‐filtering FLIM signals at Τ_ZnPT enabled an efficient discrimination between the zinc pyrithione and major endogenous skin species in comparison with that of the conventional reflectance confocal microscopy. Our findings provide means for in vivo high‐sensitivity assaying and high‐contrast imaging of zinc pyrithione in biological systems.     

Deciphering the nanometer-scale organization and assembly of Lactobacillus rhamnosus GG pili using atomic force microscopy

In living cells, sophisticated functional interfaces are generated through the self-assembly of bioactive building blocks. Prominent examples of such biofunctional surfaces are bacterial nanostructures referred to as pili. Although these proteinaceous filaments exhibit remarkable structure and functions, their potential to design bioinspired self-assembled systems has been overlooked. Here, we used atomic force microscopy (AFM) to explore the supramolecular organization and self-assembly of pili from the Gram-positive probiotic bacterium Lactobacillus rhamnosus GG (LGG). High-resolution AFM imaging of cell preparations adsorbed on mica revealed pili not only all around the cells, but also in the form of remarkable star-like structures assembled on the mica surface. Next, we showed that two-step centrifugation is a simple procedure to separate large amounts of pili, even though through their synthesis they are covalently anchored to the cell wall. We also found that the centrifuged pili assemble as long bundles. We suggest that these bundles originate from a complex interplay of mechanical effects (centrifugal force) and biomolecular interactions involving the SpaC cell adhesion pilin subunit (lectin-glycan bonds, hydrophobic bonds). Supporting this view, we found that pili isolated from an LGG mutant lacking hydrophilic exopolysaccharides show an increased tendency to form tight bundles. These experiments demonstrate that AFM is a powerful platform for visualizing individual pili on bacterial surfaces and for unravelling their two-dimensional assembly on solid surfaces. Our data suggest that bacterial pili may provide a generic approach in nanobiotechnology for elaborating functional supramolecular interfaces assembled from bioactive building blocks.     

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.