Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology.
On the nanoscale, almost all light sources blink. Surprisingly, such blinking occurs on time scales much larger than predicted by quantum mechanics and has statistics governed by nonergodicity.
We exploit the strong excitonic coupling in a superradiant trimer molecule to distinguish between longlived collective dark states and photobleaching events. The population and depopulation kinetics of the dark states in a single molecule follow power-law statistics over 5 orders of magnitude in time. This result is consistent with the formation of a radical unit via electron tunneling to a time-varying distribution of trapping sites in the surrounding polymer matrix. We furthermore demonstrate that this radicalization process forms the dominant pathway for molecular photobleaching.
Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC.
The blinking behavior of perylene diïmide molecules is investigated at the single-molecule level. We observe long-time scale blinking of individual multi-chromophoric complexes embedded in a poly(methylmethacrylate) matrix, as well as for the monomeric dye absorbed on a glass substrate at ambient conditions. In both these different systems, the blinking of single molecules is found to obey analogous power-law statistics for both the on and off periods. The observed range for single-molecular power-law blinking extends over the full experimental time window, covering four orders of magnitude in time and six orders of magnitude in probability density. From molecule to molecule, we observe a large spread in off-time power-law exponents. The distributions of off-exponents in both systems are markedly different whereas both on-exponent distributions appear similar. Our results are consistent with models that ascribe the power-law behavior to charge separation and (environment-dependent) recombination by electron tunneling to a dynamic distribution of charge acceptors. As a consequence of power-law statistics, single molecule properties like the total number of emitted photons display non-ergodicity.
Template-induced colloidal deposition during solvent evaporation is a promising technique for extending the possibilities of nanosphere lithography and the creation of photonic band gap materials. We investigated the influence of the parameters that determine the surface topography of templates on colloidal crystal structure. On pillar-shaped templates, large defect-free square symmetric monolayers, ordered vacancy arrays, and body-centered cubic (bcc) and simple cubic (sc) colloidal crystals could be grown. Close-packed crystals displayed defects and large defect grains. Our results indicate that this may be avoided when the direction of gravity with respect to the substrate is changed.The ability of colloids to self-assemble into 2D and 3D crystalline structures lies at the heart of many studies in nanoand micrometer-scale materials science. 1 Especially in the field of photonic band gap materials, colloidal crystals are being routinely used.2 Furthermore, 2D and thin 3D colloidal crystals can be used as a mask for creating regular arrays of nanometer-sized features with a technique called nanosphere lithography.3-5 One of the simplest and most frequently used techniques for assembling 2D and 3D colloidal crystals is colloidal crystallization during solvent evaporation. [6][7][8][9] Apart from its simplicity and low cost, the main advantages of the technique are (i) the possibility to grow millimeter-sized single crystals with a hexagonal (111) orientation of the top surface and (ii) the possibility to control the thickness of the deposited crystal by varying the initial particle volume fraction. 7 A major limitation is that the crystal symmetry and 3D orientation of the crystal cannot be influenced. The use of a patterned substrate, or template, provides the possibility to direct colloidal crystallization epitaxially. 10 Recent experiments on colloidal epitaxy under equilibrium conditions have shown the growth of crystal structures that are metastable in bulk crystallization and thus would not grow without the use of the template. 11,12 Several papers have reported results on template-induced colloidal assembly during solvent evaporation, but the systems consisted of (repeated 13 ) 2D deposits of micrometer-scale particles 14 or showed the frequent occurrence of defects and a lack of long-range order. 15In this letter, we demonstrate the templated growth of 100-nm-radius particles in colloidal crystals with close-packed and non-close-packed symmetry. The crystals were grown by convective assembly on a substrate placed in a vertical setup (i.e., parallel to gravity). We will address the influence of various parameters that determine the template topography on 2D and 3D colloidal crystal structure. Our results indicate that the direction of the gravitational field plays a crucial role in template-directed convective assembly.Silica particles with diameters of d ) 202 and 220 nm (polydispersity σ ≈ 0.005) were deposited onto a substrate that was vertically placed in a slowly evaporating dispersion in eth...
A real-space study is presented on the occurrence of stacking faults in crystals of silica colloids with diameters of about 1 and 1.4 m formed through sedimentation. The softness of the interaction potential is varied from slightly repulsive to hard-sphere like, both intrinsically by variation of the diameter, as well as through the addition of salt, which screens the surface charges. Our results indicate that the equilibrium crystal structure for these colloids is an fcc-crystal, with the number of stacking faults determined by the interplay between sedimentation and crystallization kinetics, irrespective of the softness of the interaction potential. For spheres with a certain diameter the number of stacking faults decreases with decreasing initial volume fractions. These results provide a way to grow fcc-crystals of hard-sphere particles by slow sedimentation. The relative number of stacking faults in the first few layers above the bottom wall can be as much as a factor of 10 higher than deeper into the crystal. This effect is due to the crystallization kinetics on a plain wall in a gravitational field. A patterned bottom wall that favors a specific hexagonal orientation was found to drastically reduce the number of stacking faults in the crystal.
Three compounds bearing multiple adamantyl guest moieties and a fluorescent dye have been synthesized for the supramolecular patterning of beta-cyclodextrin (CD) host monolayers on silicon oxide using microcontact printing and dip-pen nanolithography. Patterns created on monolayers on glass were viewed by laser scanning confocal microscopy. Semi-quantitative analysis of the patterns showed that with microcontact printing approximately a single monolayer of guest molecules is transferred. Exposure to different rinsing procedures showed the stability of the patterns to be governed by specific supramolecular multivalent interactions. Patterns of the guest molecules created at CD monolayers were stable towards thorough rinsing with water, whereas similar patterns created on poly(ethylene glycol) (PEG) reference monolayers were instantly removed. The patterns on CD monolayers displayed long-term stability when stored under N(2), whereas patterns at PEG monolayers faded within a few weeks due to the diffusion of fluorescent molecules across the surface. Assemblies at CD monolayers could be mostly removed by rinsing with a concentrated CD solution, demonstrating the reversibility of the methodology. Patterns consisting of different guest molecules were produced by microcontact printing of one guest molecule and specific adsorption of a second guest molecule from solution to non-contacted areas, giving well-defined alternating assemblies. Fluorescent features of sub-micrometer dimensions were written using supramolecular dip-pen nanolithography.
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