Anti-Brownian traps confine single molecules or particles in free solution by closed-loop feedback forces, allowing detailed characterization of photophysical and transport properties. We have recently extended this approach to tracking the particles with interferometric scattering at near infrared wavelengths. This extension allows trapping non-fluorescent particles and performing simultaneous modulated fluorescence measurements in the Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap. Here we use the interferometric scattering signal in the ISABEL trap to measure the scattering cross-sections of single carboxysomes, bacterial nanocompartments involved in carbon fixation. With a core-shell model, we can calculate the total mass and internal loading of single nano-objects.

Two decades ago, we introduced single-molecule FRET measurements to study subunit rotation in individual FoF1-ATP synthases in liposomes. The rotary motors of the enzyme are either driven by ATP hydrolysis, or by internal proton translocation. To counteract diffusive motion of a single enzyme in real time, we built a fast confocal anti-Brownian electrokinetic trap (invented by A. E. Cohen and W. E. Moerner) with laser focus pattern and electrode feedback. We recorded broad distributions of ATP-driven subunit rotation and changing rotor speed in time traces of single enzymes. Now we explore the speed limit by circumventing the biological regulatory controls.

Luminosa, the new single photon counting confocal microscope from PicoQuant addresses the challenges of an expanding single-molecule FRET community. In this talk we present how easily these measurements can be performed with Luminosa single photon-counting confocal microscope and how all necessary correction parameters are automatically determined requiring no interaction from the user by employing methodologies benchmarked by the scientific community. We will also show how the variable PSF feature can be used in such measurements to fine-tune the observation window of freely diffusing biomolecules.

SPAD array sensors support higher-throughput fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser-scanning to wide-field geometries. While a SPAD camera in epi-fluorescence geometry enables wide-field FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus culture medium masks cell autofluorescence and biases lifetime measurements. Here, we address this problem in a proof-of-concept implementation by integrating the SPAD camera in a light-sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling label-free wide-field FLIM of single-cell NAD(P)H autofluorescence.

Super-resolution imaging has become an essential tool used by the bioimaging community. In this study, we present a novel technique to resolve emitters located closer than the diffraction limit, by using the differences in their fluorescence lifetime. By calculating the centre of mass of the point spread function of the emitter on the timescale of the fluorescence decay, protein-protein interactions via FRET can be identified. This technique will also be able to distinguish the number of proximal neighbouring donors without the need to fit the time-resolved fluorescence decays.

Super-resolution fluorescence microscopy for imaging in thick samples would benefit greatly from a reduction in fluorescence background and an increase in acquisition speed. To address these concerns, we present a single-objective tilted light sheet combined with four additional innovations: (i) a 3D-printed microfluidic chip, (ii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iii) deep learning for improved localization precision and imaging speeds, and (iv) engineered point spread functions for 3D imaging. Our approach achieves improved localization precision, imaging speeds, and multi-target accuracy, enabling fast and precise multi-target 3D super-resolution imaging.

Dendritic spines are neuronal structures which play a key role in memory formation by strengthening synaptic pathways through actin cytoskeleton reorganization. The protein CaMKII is hypothesized to perform major structural functions in this rearrangement. Here, we use single-particle tracking and super-resolution imaging to extract information on the bindings dynamic and nanoscale architecture of reconstituted CaMKII-actin assemblies adhered to a coverslip. To improve resolution over the entire field of view, we use a homogeneously illuminated total internal reflection fluorescence scheme. Our method closes an experimental gap present in the field and provides relevant knowledge of actin/CaMKII interactions at the nanoscale.

The potential of micro-and nanofabricated samples as a platform to modulate cell behavior using 3D physical cues has shown tremendous interest in cell differentiation. Investigating cell behavior and organization at the molecular level requires advanced imaging techniques. We produced multiscale 3D substrates with glass fractal pyramids composed of several generations of octahedra of decreasing sizes, which allow direct observation of cells in 3D SMLM. We show how these samples can be fluorescently labeled and used as a self-referenced sample for calibration and resolution measurements. Moreover, we perform quantitative 3D SMLM on cells growing on such fractal substrates, observing spheroid-like behavior.

The typical time-scales of photo-physical processes in fluorophores are in the order of nano- to microseconds. A powerful, sensitive yet relatively simple technique is fluorescence correlation spectroscopy (FCS). This method allows for studying any dynamic process that modulates the fluorescence intensity measured within the femtoliter-sized detection volume of a confocal microscope. Mostly, FCS is applied for measuring diffusion properties but it can also be used for determining photophysical transitions, To evaluate such measurements one needs to know the excitation rate throughout the detection volume. Absolute excitation rates of fluorescent molecules can be obtained from fluorescence antibunching measurements Here, we combine fluorescence antibunching with FCS on the same experimental setup to measure intersystem crossing rates and triplet state lifetimes of Rhodamine 110 and ATTO 655 with errors less than 5%.

A dual-color super-resolution microscope with polarization and orientation-resolving capabilities is presented. Combining single-molecule localization methods with simultaneous polarization measurements enables the determination of the orientation of single emitters, such as quantum dot nanocrystals, with sub-10 nm precision. Additional simultaneous spectral characterization of particle emission allows the capture of multiple optical properties that impact energy transfer. We report on the instrumentation development and the results from coupled quantum dot clusters.

The appropriate sample illumination method varies depending on the nature of the sample and the goals of the study, especially for single-molecule localization microscopy. Here, we demonstrate a flexible microscopy system which combines the whole-cell sectioning capability of light sheet with conventional epi-illumination and total internal reflection fluorescence (TIRF), both with a homogeneous flat-field profile to significantly improve the resolution across the entire field of view. In conjunction with point spread function engineering capabilities and fast switching of illumination modes, our system enables 3D single-molecule super-resolution imaging optimized for the sample and region of interest.

Abnormal accumulation of amyloid-β (Aβ) plaques in the brain is one of the major characteristics of Alzheimer’s disease and there is a possibility that the degree of plaque toxicity is related to the distribution of nanoscale oligomeric aggregates in plaques. Here, by super-resolution fluorescence imaging, we visualized that anti-Aβ oligomer antibodies localized differently to plaques compared to anti-Aβ monomer antibodies and characteristic plaque shapes in brain tissues were classified. These differences in nanoscale distribution were hard to be discerned by conventional fluorescence imaging, implying that super-resolution imaging has the potential to reveal the detailed features of oligomeric aggregates in plaques.

A temporal correlation superresolution image is based on the variance of the recorded photon time trace, while its resolution is higher than that of the complementary intensity image, it is noisier. Both images, the intensity and correlation based, are fed into a deep convolutional neural network (CNN), which produces an image that is optimized to have higher resolution than the intensity image and less noise than the correlation image. The image then passes through separate linear networks that mimic the physical blurring of the imaging setup. Preliminary experimental results show similar resolution to the experimental superresolution image with less noise.

In this contribution, we demonstrate the feasibility of fluorescence single-molecule localization microscopy (SMLM) imaging with an event-based (or neuromorphic vision) sensor. Event-based sensors are matrices of independent pixels sensitive to intensity variations only, with a microsecond range temporal resolution. We have integrated such an event-based sensor in a typical SMLM set-up and characterized its localization precision and response linearity. We obtain super-resolution images of biological samples of the same resolution than with conventional scientific cameras. Finally, we exploit the unique characteristics of event-based SMLM to perform high-density acquisitions with overlapping PSFs, achieving much better results than with frame-based cameras.

This conference presentation was prepared for the Single Molecule Spectroscopy and Superresolution Imaging XVI conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Single Molecule Spectroscopy and Superresolution Imaging XVI conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Single Molecule Spectroscopy and Superresolution Imaging XVI conference at SPIE BiOS, 2023.

This conference presentation was prepared for the Single Molecule Spectroscopy and Superresolution Imaging XVI conference at SPIE BiOS, 2023.