Matthew F. Paige

Fibrous Long Spacing Collagen Ultrastructure Elucidated by Atomic Force Microscopy

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.

Mechanisms of Low-Power Noncoherent Photon Upconversion in Metalloporphyrin−Organic Blue Emitter Systems in Solution

The mechanisms of noncoherent photon upconversion that involve triplet-triplet annihilation (TTA) in solution have been investigated for two model systems. ZnTPP (meso-tetraphenylporphine zinc) is used as the model visible light-absorbing metalloporphyrin because its S(1) fluorescence intensity can be used to monitor the initial rate of porphyrin triplet state production and because its S(2) fluorescence intensity can be used as a direct measure of the rate of porphyrin TTA. When perylene, which has a triplet energy lower than that of ZnTPP, is added as a signaling blue emitter (BE), the mechanism of photon upconversion involves triplet energy transfer from the porphyrin to the BE followed by TTA in the BE to form the fluorescent perylene S(1) state. The kinetics of this process have been characterized and are unremarkable. When coumarin 343 (C343), which has photophysical properties similar to those of perylene except that it has a much higher triplet energy than ZnTPP, is added as the signaling BE, emission from the ZnTPP S(2) state is quenched and fluorescence from the C343 grows in. Contrary to previous suggestions, the mechanism of photon upconversion in this system does not involve singlet energy transfer from the porphyrin S(2) state to the BE. Instead, ground-state C343 complexes with the ZnTPP triplet to form a triplet exciplex, which then undergoes TTA with a second ZnTPP triplet to give the fluorescent state of the BE in a three-center process.

A Comparison of Through-the-Objective Total Internal Reflection Microscopy and Epifluorescence Microscopy for Single-Molecule Fluorescence Imaging

The design and characterization of a wide-field, through-the-objective, Total Internal Reflection (TIR) microscope which has the sensitivity required for characterizing single molecules under ambient conditions is described. A careful comparison between TIR and epifluorescence imaging is made at the single-molecule level with equal incident intensities, using the laser dyes DCM and R6G in a polymer matrix as test samples. With these samples, measurements show that while the signal-to-background ratio (SBR) of single molecules using TIR is comparable with that for epifluorescence imaging over a range of illumination intensities, the signal-to-noise ratio (SNR) for TIR is superior and is the principal source of improved images which have previously been reported with this form of microscopy. The likely source of the improved SNR is simply the reduction in the relative size of photon shot-noise caused by the higher effective pumping intensity inherent in evanescent wave excitation. The techniques general applicability to a wider range of single-molecule studies is discussed. In addition, the effect on detected images produced by variations in the gain of an intensified CCD camera is characterized.

A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils that display a banding with periodicity greater than the 67nm periodicity of native collagen. FLS fibrils can be formed in vitro by addition of alpha(1)-acid glycoprotein to an acidified solution of monomeric collagen, followed by dialysis of the resulting mixture. We have investigated the ultrastructure of FLS fibrils formed in vitro using the atomic force microscope (AFM). The majority of the fibrils imaged showed typical diameters of approximately 150nm and had a distinct banding pattern with a approximately 250nm periodicity. However, we have also observed an additional type of FLS fibril, which is characterized by a secondary banding pattern surrounding the primary bands. These results are compared with those obtained in past investigations of FLS ultrastructure carried out using the transmission electron microscope (TEM). The importance of the fibrils surface topography in TEM staining patterns is discussed. Images of FLS fibrils in various stages of assembly have also been collected, and the implications of these images in determining the mechanism of assembly and the formation of the characteristic banding pattern of the fibrils is discussed.

Fibril formation in collagen

Abstract While simple aggregation in solution typically leads to ramified objects, some polymers and proteins form filamentous structures. The aggregation of collagen, a structural protein, from monomers to fibrils was studied by light scattering and atomic force microscopy. The presence of clear, stable intermediates indicates that the assembly proceeds in a modular or hierarchical fashion. Temperature and concentration dependence studies show that the detailed assembly mechanism may change under different conditions.

A comparison of atomic force microscope friction and phase imaging for the characterization of an immiscible polystyrene/poly(methyl methacrylate) blend film

Abstract Three different forms of atomic force microscope (AFM) measurement, topography, friction force and phase imaging, have been used to investigate the surface morphology and local composition of an immiscible polystyrene (PS)/poly(methyl methacrylate) (PMMA) blend film. This sample forms discrete, micron-size domains in a continuous matrix, which is attributed to the segregation of PMMA in PS. When the samples were imaged in air, contrast in friction and phase images was caused by variations in sample topography only. When the samples were imaged under water, however, both friction and phase imaging yielded non-topographic contrast between domains. We ascribe the contrast in both of these imaging modes to preferential softening of the hydrophilic, PMMA-rich domains and to stronger tip–sample adhesive forces, highlighting the AFMs utility for probing local elastic properties and for compositional mapping of soft polymer samples.

Phase separation of palmitic acid and perfluorooctadecanoic acid in mixed Langmuir-Blodgett monolayer films.

Deposition of mixtures of palmitic acid (C15H31COOH) and perfluorooctadecanoic acid (C17F35COOH) onto solid substrates gives rise to irregularly shaped, phase-separated domains under a variety of deposition conditions. The morphology and chemical composition of these phase-separated domains have been investigated using a combination of surface pressure-area isotherms, atomic force microscopy, X-ray photoemission electron microscopy, and confocal fluorescence microscopy imaging. While domain morphology and composition in 2D phase-separated mixed monolayer systems can typically be rationalized in terms of an interplay between line tension and dipole-dipole repulsion effects, it was found that for this system additional kinetic factors, including domain growth rates and the rate of dissolution of the fatty acid component into the aqueous subphase, also play a major role in controlling film properties. The potential importance of these effects for the controlled patterning of solid substrates is discussed.

Photophysics of untethered ZnTPP-fullerene complexes in solution.

The spectroscopy and dynamic behavior of the self-assembled, Soret-excited zinc tetraphenylporphyrin (ZnTPP) plus fullerene (C(60)) model system in solution has been examined using steady state fluorescence quenching, nanosecond time-correlated single photon counting, picosecond fluorescence upconversion, and picosecond transient absorption methods. Evidence of ground state complexation is presented. Steady-state quenching of the S(2) and S(1) fluorescence of ZnTPP by C(60) reveals that the quenching processes only occur in the excited complexes, are ultrafast, and proceed at different rates in the two states. Only uncomplexed ZnTPP is observed by fluorescence lifetime methods; the locally excited complexes are either dark or, more likely, rapidly relax to products that do not radiate strongly. Both short-range (Dexter) energy transfer and electron transfer relaxation mechanisms are evaluated. Picosecond transient absorption data obtained from the subtle differences between the spectra of Soret-excited ZnTPP with and without a large excess of added C(60) reveal the formation, on a subpicosecond time scale, of relatively long-lived charge-separated species. Soret excitation of ZnTPP···C(60) does not produce a quantitative yield of species in the lower S(1) excited state.

Efficiency of noncoherent photon upconversion by triplet-triplet annihilation: the C60 plus anthanthrene system and the importance of tuning the triplet energies.

As part of a continuing effort to find noncoherent photon upconversion (NCPU) systems with improved energy conversion efficiencies, the photophysics of the blue emitter, anthanthrene (An), and the fullerene absorber-sensitizer, C60, have been examined by both steady-state and pulsed laser techniques. An is a promising candidate for NCPU by homomolecular triplet-triplet annihilation (TTA) because its triplet state lies ∼800 cm(-1) below the triplet energy of the C60 donor (thereby improving efficiency by reducing back triplet energy transfer), and its fluorescent singlet state lies in near resonance with double its triplet energy (thus minimizing thermal energy losses in the annihilation process). In fluid solution, efficient triplet-triplet donor-acceptor energy transfer is observed, and rate constants for homomolecular TTA in the An acceptor are estimated to approach the diffusion limit. NCPU is also observed in An + C60 in poly(methylmethacrylate) thin films.

Rippled Domain Formation in Phase-Separated Mixed Langmuir-Blodgett Films

The morphology and composition of phase-separated Langmuir and Langmuir-Blodgett films of stearic acid (C17H35COOH) (SA) mixed with perfluorotetradecanoic acid (C13F27COOH) (PA) have been investigated using a combination of atomic force microscopy (AFM) measurements and surface pressure-area isotherms. At elevated surface pressures, the mixed film phase-separated to form a distinct series of lines (ripples), as opposed to the hexagons that have previously been observed with mixed films with longer alkyl chain fatty acids. At low surface pressures, phase separation is still observed, though a range of different domain structures was formed. The chemical composition of the phase-separated domains has been investigated by AFM-based compositional mapping, which has allowed unambiguous identification of the chemical composition of the domains. A simple mechanistic model describing how domain formation takes place in this system is presented.