Improved Dye Survival in Expansion Microscopy through Stabilizer-Conjugated Linkers

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer-containing organic fluorophores in ExM, as a mitigation strategy for this radical-induced dye degradation. Through direct conjugation of 4-nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore-conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer-modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.     

Live‐Cell Localization Microscopy with a Fluorogenic and Self‐Blinking Tetrazine Probe

Recent developments in fluorescence microscopy call for novel small‐molecule‐based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live‐cell single‐molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live‐cell experiments. At the same time, quality of super‐resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small‐molecule label comprising both fluorogenic and self‐blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single‐molecule localization microscopy on live‐cell samples.     

Quantum dot micropatterning on si

Using InP and PbSe quantum dots, we demonstrate that the Langmuir-Blodgett technique is well-suited to coat nonflat surfaces with quantum dot monolayers. This allows deposition on silicon substrates covered by a developed patterned resist, which results in monolayer patterns with micrometer resolution. Atomic force microscopy and scanning electron microscopy reveal the formation of a densely packed monolayer that replicates predefined structures with high selectivity after photoresist removal. A large variety of shapes can be reproduced and, due to the excellent adhesion of the quantum dots to the substrate, the hybrid approach can be repeated on the same substrate. This final possibility leads to complex, large-area quantum dot monolayer structures with micrometer spatial resolution that may combine different types of quantum dots.     

Classification and dating of black gel pen ink by ion-pairing high-performance liquid chromatography

A novel approach for classification and dating of the black gel pen ink entries on document was developed based on ion-pairing high-performance liquid chromatography (IP-HPLC). Ninety-three black gel pens were collected and divided into two groups, dye-based and pigment-based, by preliminary solubility test. The chromatographic conditions for separation of the dye-based black gel pen inks were optimized and the dye components in inks were satisfactorily separated by using 40 mmol/L tetrabutylammonium bromide as ion-pairing reagent. According to the number and the chromatographic retention times of the main dye components, the 50 dye-based inks were categorized into four classes. The inks within a class can be further identified by the percentage of each dye component. The compositional changes of the dye components in the black gel pen ink entries on paper were investigated in light and natural aging conditions and it has been found that the dye components in the ink entries underwent obvious decomposition, and the decomposing extent of the dye components was related to the aging time. The results can provide scientific evidences for dating of the suspicious black gel pen ink entries on documents.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

 Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers.     

Design,photophysical properties,and applications of fluorene-based fluorophores in two-photon fluorescence bioimaging: A review

Two-photon fluorescence microscopy (2PFM) emerged as a powerful alternative to conventional one-photon microscopy. 2PFM typically uses two near-infrared (NIR) photons to excite fluorescent dyes, which minimizes light scattering in biological samples. Multiphoton absorption also suppresses background signal and autofluorescence from tissues and allows to achieve higher 3D resolution images with low photodamage and photobleaching. Fluorene dyes possess distinct properties that meet the strict criteria of probes used for 2PFM such as enhanced solubility, photostability, and two-photon absorption cross-section. The fluorene molecule also includes many active positions that allow versatile synthesis, selective functionalization, bioconjugation, and tuning solubility. These properties have led to reporting several fluorene probes including monomers, polymers, and dendrimers with important uses in understanding molecular dynamics and bioimaging. The current review presents a compact summary of fluorene-based fluorophores for 2PFM bioimaging applications, shedding light on structure-photophysical property relationships in fluorenes and polyaromatic probe designs.     

Examination of the Spatial Distribution of Dyes and Polymers in Thin Films by Two-Photon Microscopy

Summary.  Two-photon absorption induced fluorescence microscopy was used as a tool for the examination of the spatial distribution of a thin dye film. The two-photon absorption induced fluorescence signal is essentially the same as that produced by excitation with a single photon of equivalent energy. When femtosecond pulses are focused into a sample there is an intrinsic spatial selectivity of the two-photon emission signal, since it is dependent upon the square of the light intensity. This has tremendous implications in fluorescence microscopy. Since two-photon absorption is confined in a small region at the focal waist of an objective lens, photodamage and photobleaching of the sample are significantly reduced. In addition, the two-photon signal has inherent z-axis spatial resolution, which facilitates the construction of 3-D images. In the present work an application of this technique to a thin film of a dye is presented. The method can generally be applied to thin films made from photonic polymers. Received June 23, 2000. Accepted (revised) July 31, 2000     

Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins

Solid‐state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic‐resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long‐distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton–proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin‐labeled 1 mg protein samples using fast magic angle spinning.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

Dye surface coating enables visible light activation of TiO2 nanoparticles leading to degradation of neighboring biological structures

Biologically and chemically modified nanoparticles are gaining much attention as a new tool in cancer detection and treatment. Herein, we demonstrate that an alizarin red S (ARS) dye coating on TiO2 nanoparticles enables visible light activation of the nanoparticles leading to degradation of neighboring biological structures through localized production of reactive oxygen species. Successful coating of nanoparticles with dye is demonstrated through sedimentation, spectrophotometry, and gel electrophoresis techniques. Using gel electrophoresis, we demonstrate that visible light activation of dye-TiO2 nanoparticles leads to degradation of plasmid DNA in vitro. Alterations in integrity and distribution of nuclear membrane associated proteins were detected via fluorescence confocal microscopy in HeLa cells exposed to perinuclear localized ARS-TiO2 nanoparticles that were photoactivated with visible light. This study expands upon previous studies that indicated dye coatings on TiO2 nanoparticles can serve to enhance imaging, by clearly showing that dye coatings on TiO2 nanoparticles can also enhance the photoreactivity of TiO2 nanoparticles by allowing visible light activation. The findings of our study suggest a therapeutic application of dye-coated TiO2 nanoparticles in cancer research; however, at the same time they may reveal limitations on the use of dye assisted visualization of TiO2 nanoparticles in live-cell imaging.     

Multicolor Caged dSTORM Resolves the Ultrastructure of Synaptic Vesicles in the Brain

The precision of single‐molecule localization‐based super‐resolution microscopy, including dSTORM, critically depends on the number of detected photons per localization. Recently, reductive caging of fluorescent dyes followed by UV‐induced recovery in oxidative buffer systems was used to increase the photon yield and thereby the localization precision in single‐color dSTORM. By screening 39 dyes for their fluorescence caging and recovery kinetics, we identify novel dyes that are suitable for multicolor caged dSTORM. Using a dye pair suited for registration error‐free multicolor dSTORM based on spectral demixing (SD), a multicolor localization precision below 15 nm was achieved. Caged SD‐dSTORM can resolve the ultrastructure of single 40 nm synaptic vesicles in brain sections similar to images obtained by immuno‐electron microscopy, yet with much improved label density in two independent channels.     

Analytical electron microscopy of materials

Analytical electron microscopy enables combined crystallographic and chemical information with a high spatial resolution to be gained from microregions of electron-transparent specimens. This is reached by the combined application of imaging, diffraction and spectroscopic methods, using either a dedicated scanning transmission electron microscope or a conventional high-resolution electron microscope (having a strong objective lens) equipped with suitable X-ray or electron spectrometers. Of the diffraction methods especially the technique of convergent beam diffraction is used, yielding valuable information on crystal structures, lattice parameter changes, symmetry variations and crystal perfection, respectively. For chemical analysis, either energy-dispersive X-ray spectroscopy (EDX) is used or electron energy loss spectroscopy (EELS). Finally, high-resolution electron microscopy in the lateral resolution range of some 0.1 nm allows the reliable geometrical inspection of extreme microregions.     

Flash column chromatograms estimated from thin-layer chromatography data

Given a sample mass and TLC data, a spreadsheet has been developed that provides information on the amount of silica gel needed, the optimal fraction size, and the degree of separation to be expected before flash chromatography is attempted. The spreadsheet is the first utility of its kind to accurately estimate the retention volume and band volume of analytes, as well as the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted; ensuring a successful first separation.     

Optimization of the preparation of glass-coated, dye-tagged metal nanoparticles as SERS substrates

Dye-tagged metal nanoparticles are of significant interest in SERS-based sensitive detection applications. Coating these particles in glass results in an inert spectral tag that can be used in applications such as flow cytometry with significant multiplexing potential. Maximizing the SERS signal obtainable from these particles requires care in partitioning available nanoparticle surface area (binding sites) between the SERS dyes and the functionalized silanes necessary for anchoring the glass coating. In this article, we use the metal-mediated fluorescence quenching of SERS dyes to measure surface areas occupied by both dyes and silanes and thus examine SERS intensities as a function of both dye and silane loading. Notably, we find that increased surface occupation by silane increases the aggregative power of added dye but that decreasing the silane coverage allows a greater surface concentration of dye. Both effects increase the SERS intensity, but obtaining the optimum SERS intensity will require balancing aggregation against surface dye concentration.     

Self-quenched Fluorophore Dimers for DNA-PAINT and STED Microscopy

Super-resolution techniques like single-molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non-covalent, weak affinity-based transient labelling systems. DNA-based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi-target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore-labelled single-stranded oligonucleotides. Here, we introduce short-distance self-quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2-fold. We characterise the optical and thermodynamic properties of fluorophore-dimer single-stranded DNA, and show super-resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background.     

Carbon‐Nanotube‐Based Hybrid Materials: Nanopeapods

This review article focuses on the structures and properties of novel hybrid nanocarbon materials, which are created by incorporating atoms and molecules into the hollow spaces of carbon nanotubes (CNTs); thus they are called nanopeapods. After dealing with synthesis procedures, we discuss the structures and electronic properties of the hybrid materials based on high‐resolution transmission electron microscopy (HRTEM), electron energy‐loss spectroscopy (EELS), X‐ray and electron diffraction, scanning tunneling microscopy (STM), and field‐effect transistor transport measurements. Utilization of the low‐dimensional nanosized spaces of CNTs to produce novel low‐dimensional nanocluster, nanowire, and nanotube materials is also discussed.     

Local concentration of gel phase domains in supported lipid bilayers under light irradiation in binary mixture of phospholipids doped with dyes for photoinduced activation

Recently, lipid bilayers supported on solid substrates are considered to offer potential as biological devices utilizing biological membranes and membrane proteins. In particular, artificially patterned supported bilayers hold great promise for the development of biological devices. In this study, we show control of the formation and location of phase-separated domain structures by light irradiation for gel phase and liquid-crystalline phase separation structures in a DMPC-DOPC binary lipid bilayer tagged with dye molecules on SiO2/Si substrates. Upon light irradiation, the gel phase domain structures disappeared from the phase-separated bilayers. This disappearance indicates that the light irradiation causes a local increase in the temperature of the lipid bilayer. In this disappearance phenomenon, the photoinduced activation of dye lipids, e.g. fluorescent lipids, is considered to play an important role, since the same phenomenon does not occur in lipid bilayers that have a low concentration of dye lipids. Thus, the local increase in temperature is propagated by light absorption of the dye lipid and subsequent photoinduced activation of nonradiative molecular vibrations. Subsequent interruption of the photoinduced activation for molecular motion allowed the gel phase domain structures to precipitate and grow again. Moreover, the domain area fraction remaining after the photoinduced activation was higher than that before the photoinduced activation. This result indicates that the local increase in temperature propagated by dye-excitation enhances formation of the gel phase domains. By utilizing this phenomenon, we could preferentially induce formation of domain structures within the light-irradiated regions. This technique could be the basis for a new patterning technique based on domain structures. Moreover, these domain structure patterns can be eliminated by increasing the temperature, allowing rewritable patterning.     

Live-Cell Localization Microscopy with a Fluorogenic and Self-Blinking Tetrazine Probe

Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell experiments. At the same time, quality of super-resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small-molecule label comprising both fluorogenic and self-blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single-molecule localization microscopy on live-cell samples.     

Label-free fluorescent probing of G-quadruplex formation and real-time monitoring of DNA folding by a quaternized tetraphenylethene salt with aggregation-induced emission characteristics

Biosensing processes such as molecular beacons require non-trivial effort to covalently label or mark biomolecules. We report here a label-free DNA assay system with a simple dye with aggregation-induced emission (AIE) characteristics as the fluorescent bioprobe. 1,1,2,2-Tetrakis[4-(2-bromoethoxy)phenyl]ethene is nonemissive in solution but becomes highly emissive when aggregated. This AIE effect is caused by restriction of intramolecular rotation, as verified by a large increase in the emission intensity by increasing viscosity and decreasing temperature of the aqueous buffer solution of 1,1,2,2-tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide (TTAPE). When TTAPE is bound to a guanine-rich DNA strand (G1) via electrostatic attraction, its intramolecular rotation is restricted and its emission is turned on. When a competitive cation is added to the G1 solution, TTAPE is detached and its emission is turned off. TTAPE works as a sensitive poststaining agent for poly(acrylamide) gel electrophoresis (PAGE) visualization of G1. The dye is highly affinitive to a secondary structure of G1 called the G-quadruplex. The bathochromic shift involved in the G1 folding process allows spectral discrimination of the G-quadruplex from other DNA structures. The strong affinity of TTAPE dye to the G-quadruplex structure is associated with a geometric fit aided by the electrostatic attraction. The distinct AIE feature of TTAPE enables real-time monitoring of folding process of G1 in the absence of any pre-attached fluorogenic labels on the DNA strand. TTAPE can be used as a K+ ion biosensor because of its specificity to K+-induced and -stabilized quadruplex structure.     

Quantitative Single-Molecule Electrochemiluminescence Bioassay

A single-molecule electrochemiluminescence bioassay is developed here which allows imaging and direct quantification of single biomolecules. Imaging single biomolecules is realized by localizing the electrochemiluminescence events of the labeled molecules. Such an imaging system allows mapping the spatial distribution of biomolecules with electrochemiluminescence and contains quantitative single-molecule insights. We further quantify biomolecules by spatiotemporally merging the repeated reactions at one molecule site and then counting the clustered molecules. The proposed single-molecule electrochemiluminescence bioassay is used to detect carcinoembryonic antigen, showing a limit of detection of 67 attomole concentration which is 10 000 times better than conventional electrochemiluminescence bioassays. This spatial resolution and sensitivity enable single-molecule electrochemiluminescence bioassay a new toolbox for both specific bioimaging and ultrasensitive quantitative analysis.