RASP: Optimal Single Puncta Detection in Complex Cellular Backgrounds.

Super-resolution and single-molecule microscopies have been increasingly applied to complex biological systems. A major challenge of these approaches is that fluorescent puncta must be detected in the low signal, high noise, heterogeneous background environments of cells and tissue. We present RASP, Radiality Analysis of Single Puncta, a bioimaging-segmentation method that solves this problem.

High-density volumetric super-resolution microscopy.

Volumetric super-resolution microscopy typically encodes the 3D position of single-molecule fluorescence into a 2D image by changing the shape of the point spread function (PSF) as a function of depth. However, the resulting large and complex PSF spatial footprints reduce biological throughput and applicability by requiring lower labeling densities to avoid overlapping fluorescent signals. We quantitatively compare the density dependence of single-molecule light field microscopy (SMLFM) to other 3D PSFs (astigmatism, double helix and tetrapod) showing that SMLFM enables an order-of-magnitude speed improvement compared to the double helix PSF by resolving overlapping emitters through parallax.

Parabolic Potential Surfaces Localize Charge Carriers in Nonblinking Long-Lifetime "Giant" Colloidal Quantum Dots.

Materials for studying biological interactions and for alternative energy applications are continuously under development. Semiconductor quantum dots are a major part of this landscape due to their tunable optoelectronic properties. Size-dependent quantum confinement effects have been utilized to create materials with tunable bandgaps and Auger recombination rates.

The excited-state dynamics of the radical anions of cyanoanthracenes.

The radical anion of 9,10-dicyanoanthracene (DCA) has been suggested to be a promising chromophore for photoredox chemistry, due to its nanosecond excited-state lifetime determined from indirect measurements. Here, we investigate the excited-state dynamics of the radical anion of three cyanoanthracenes, including DCA˙-, produced by photoinduced electron transfer in liquid using both pump-probe and pump-pump probe transient electronic absorption spectroscopy. All three excited radical ions are characterised by a 3-5 ps lifetime, due to efficient non-radiative deactivation to the ground state.

Sub-millisecond Translational and Orientational Dynamics of a Freely Moving Single Nanoprobe.

This paper presents a new experiment with which we are able to measure the 3D translational motion of a single particle at 10 μs time resolution and with ∼10 nm spatial resolution while at the same time determining the 3D orientation of the same single particle with 250 μs time resolution. These high time resolutions are ∼40 times greater than previous simultaneous measurements of 3D position and 3D orientation. Detailed numerical simulations and experiments are used to demonstrate that the technique can measure 3D orientation at the shot-noise limit.

Excited-state Dynamics of Radical Ions in Liquids.

Thomas Bally has acquired international recognition for his work on the photochemistry of reactive intermediates, which include radical ions. Here, we present a brief overview of our investigations of the excited-state dynamics of radical ions in liquids at room temperature, which are still poorly documented. A better understanding of these dynamics is most relevant, as open-shell ions in the excited state are being increasingly used in redox photochemistry and have been proposed to play a key role in highly exergonic photoinduced electron transfer reactions.

Leveraging lifetime information to perform real-time 3D single-particle tracking in noisy environments.

A microscopy platform that leverages the arrival time of individual photons to enable 3D single-particle tracking of fast-moving (translational diffusion coefficient of ≃3.3 µm/s) particles in high-background environments is reported here. It combines a hardware-based time-gating module, which enables the rate of photon processing to be as high as 100 MHz, with a two-photon-excited 3D single-particle tracking confocal microscope to enable high sample penetration depth.

Information bounds in determining the 3D orientation of a single emitter or scatterer using point-detector-based division-of-amplitude polarimetry.

Determining the 3D orientation of a single molecule or particle, encoded in its polar and azimuthal angles, is of interest for a variety of fields, being relevant to a range of questions in elementary chemical reactivity, biomolecular motors, and nanorheology. A popular experimental method, known as division-of-amplitude polarimetry, for determining the real-time orientation of a single particle is to split the emitted/scattered light into multiple polarizations and to measure the light intensity using point detectors at these polarizations during a time interval Δt. Here, we derive the Cramér-Rao lower bounds for this method from the perspective of information theory in the cases of utilizing a chromophore or a scattering particle as a 3D orientation probe.

Two Ru Linkage Isomers with Distinctly Different Charge Transfer Photophysics.

The ligand PHEHAT (PHEHAT = 1,10-phenanthrolino[5,6-]1,4,5,8,9,12-hexaazatriphenylene) presents a structural asymmetry that has a dramatic influence on the photophysical properties depending on the chelation site of the metal ion in the linkage isomers. While [Ru(phen)HATPHE] behaves classically, like [Ru(bpy)], [Ru(phen)PHEHAT] exhibits an unusual behavior. It appears that this complex has two MLCT bright states, the lower one being weakly emissive or nonemissive depending on the solvent and temperature.

Ion-Pair Dynamics upon Photoinduced Electron Transfer Monitored by Pump-Pump-Probe Spectroscopy.

The excited-state dynamics of the radical anion of perylene (Pe) generated upon bimolecular photoinduced electron transfer (PET) with a donor was investigated using broadband pump-pump-probe spectroscopy. It was found to depend on the age of the anion, that is, on the time interval between the first pump pulse that triggers PET and the second one that excites the ensuing Pe anion (Pe). These differences, observed in acetonitrile but not in tetrahydrofuran, report on the evolution of the PET product from an ion pair to free ions.

Detecting order and lateral pressure at biomimetic interfaces using a mechanosensitive second-harmonic-generation probe.

A planarizable push-pull molecular probe with mechanosensitive properties was investigated at several biomimetic interfaces, consisting of different phospholipid monolayers located between dodecane and an aqueous buffer solution, using the interface-specific surface-second-harmonic-generation (SSHG) technique. Whereas the SSHG spectra recorded at liquid-disordered interfaces were similar to the absorption spectra in bulk solutions, those measured at liquid-ordered phases exhibited a remarkable shift towards lower energies to an extent depending on the surface pressure of the phospholipid monolayer. On the basis of quantum-chemical calculations, this effect was accounted for by the planarization of the mechanosensitive probe.

Influence of the hydrogen-bond interactions on the excited-state dynamics of a push-pull azobenzene dye: the case of Methyl Orange.

The excited-state dynamics of the push-pull azobenzene Methyl Orange (MO) were investigated in several solvents and water/glycerol mixtures using a combination of ultrafast time-resolved fluorescence and transient absorption in both the UV-visible and the IR regions, as well as quantum chemical calculations. Optical excitation of MO in its trans form results in the population of the S ππ* state and is followed by internal conversion to the S nπ* state in ∼50 fs. The population of this state decays on the sub-picosecond timescale by both internal conversion to the trans ground state and isomerisation to the cis ground state.

Specific Monitoring of Excited-State Symmetry Breaking by Femtosecond Broadband Fluorescence Upconversion Spectroscopy.

Most quadrupolar molecules designed for large two-photon absorption cross section have been shown to undergo symmetry breaking upon excitation to the S state. This was originally deduced from their strong fluorescence solvatochromism and later visualized in real time using transient infrared spectroscopy. For molecules not containing clear IR marker modes, however, a specific real-time observation of the symmetry breaking process remains lacking.

Single-Molecule Imaging of Individual Amyloid Protein Aggregates in Human Biofluids.

The misfolding and aggregation of proteins into amyloid fibrils characterizes many neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. We report here a method, termed SAVE (single aggregate visualization by enhancement) imaging, for the ultrasensitive detection of individual amyloid fibrils and oligomers using single-molecule fluorescence microscopy. We demonstrate that this method is able to detect the presence of amyloid aggregates of α-synuclein, tau, and amyloid-β.