This PDF file contains the front matter associated with SPIE Proceedings Volume 12386, including the Title Page, Copyright information, Table of Contents, and Conference Committee information.

Self-interference digital holography (SIDH) can image incoherently emitting objects over large axial ranges with sub-diffraction resolution in all three dimensions from three two-dimensional images. By combining SIDH with single-molecule localization microscopy (SMLM), incoherently emitting objects can be localized with nanometer precision over a wide axial range without mechanical refocusing. Simulations show that SIDH can achieve sub-20 nm precision with only a few thousand photons. However, background light substantially degrades the performance of SIDH due to the relatively large size of the hologram. Therefore, to achieve the best results, the background must be reduced, and the hologram size must be optimized to increase the signal-to-noise ratio (SNR) and maximize the light efficiency of the SIDH optical system. To optimize the performance of SIDH, we performed simulations to study the optimal hologram radius (𝑅𝑅ℎ) for different levels of background photons. The results show that the reduction of the hologram size improves the localization precision of SIDH. For a given hologram size under different background noise levels, a lower background noise level provides a higher localization precision. By reducing the radius of the entry hologram to 1.4 mm and optimizing the SIDH design, we can achieve a localization precision of better than 60 nm laterally and 80 nm axially over a 10 μm axial range under the conditions of low signal level (6000 photons) with ten photons/pixel of background noise.

Semiconductor nanocrystals feature multiply-excited states that display intriguing physics and significantly impact nanocrystal-based technologies. Fluorescence supplies a natural probe to investigate these states. Still, direct observation of multiexciton fluorescence has proved elusive to existing spectroscopy techniques. Heralded Spectroscopy is a new tool based on a breakthrough single-particle, single-photon, sub-nanosecond spectrometer that utilizes temporal photon correlations to isolate multiexciton emission. This proceedings paper introduces Heralded Spectroscopy and reviews some of the novel insights it uncovered into exciton–exciton interactions within single nanocrystals. These include weak exciton–exciton interactions and their correlation with quantum confinement, biexciton spectral diffusion, multiple biexciton species and biexciton emission polarization.

In Total Internal Reflection Fluorescence (TIRF) microscopy, a specimen is illuminated by the evanescent field produced by a beam undergoing total internal reflection, whose characteristic depth is orders of magnitude below the axial diffraction limit. The axial super-resolution and much improved contrast of TIRF gives a substantially improved signalto-background ratio (SBR) than that which can be achieved with widefield illumination, and it is used extensively for imaging of the cell membrane [1] to [3].

Commercial TIRF objectives allow for a simple adaption to existing microscope systems. To attain a super-critical angle at the specimen plane, the illumination must enter the back focal plane of these objectives off-axis, requiring a high numerical aperture. As such, the magnification of these objectives is generally a minimum of 60x, reducing the lateral imaging field to less than 100 µm in diameter. Sub-cellular axial resolution is therefore restricted to a tiny population of cells and statistically significant data sampling may be difficult to achieve.

To address this, we have developed a TIRF illuminator for the Mesolens, a custom giant objective lens with a 4x/0.47NA specification. The Mesolens provides an imaging field of 4.4 mm x 3.0 mm, and in combination with our new TIRF illuminator which we call MesoTIRF we have performed imaging of large cell populations with sub-micron resolution in three-dimensions. With MesoTIRF we demonstrate more than a five-fold improvement in SBR and significantly reduced photobleaching rate compared to widefield epifluorescence illumination. We will present details of the MesoTIRF system together with current and emerging applications in cell imaging.

Active-feedback 3D single-particle tracking in complex environments is limited to bright slow-moving subjects. Extending this technique to include complex background environments requires the development of particle localization strategies that can account for changing particle and background intensities. In previously reported simulations, 2D tracking utilizing combined online Bayesian with estimation of background and signal (COBWEBS) position estimation yielded improved stability in complex background environments for a variety of particle intensities, diffusive speeds, and patterns. Here, COBWEBS’ improved stability in spatially dependent backgrounds is demonstrated to extend to 3D tracking in numerical simulations. In even background tracking, 3D implementations including COBWEBS estimation show slightly reduced position estimation errors and similar tracking accuracy to traditional Kalman estimation. Uneven background tracking shows improved tracking stability for COBWEBS estimation along XY paired with conventional Kalman estimation. Pairing COBWEBSXY with COBWEBS adapted to the Z axis further improves performance in limited cases. In 3D, the windowed signal and background estimation approach remains proportionally responsive to changing particle and background intensities but systematically underestimates true signal values. Overall, COBWEBS’ improved stability in 3D complex environments should expand the application scope of active-feedback single-particle tracking approaches.

Biofilms are omnipresent in the natural environment. They consist of microbial communities that encapsulate themselves in extracellular polymeric substances. The increasing micro- and nanoplastic pollution is of immense concern. The transport and fate of nanoplastics (NPs) in the environment is strongly affected by biofilm induced aggregation. We apply fluorescence correlation spectroscopy (FCS) to investigate the pH-dependent aggregation tendency of polystyrene (PS) nanoparticles due to interactions with model extracellular biofilm substances, such as alginate and bovine serum albumin (BSA). We show that certain alginate-BSA mixtures convey a lower tendency to nanoparticle aggregation, as compared to alginate or BSA alone. The positively charged BSA promotes nanoparticle aggregation through bridging due to attractive electrostatic nanoparticle-BSA interactions. In a mixture of BSA and negatively charged alginate this interaction is hampered. In the BSA-alginates mixture and in alginate alone other weaker attractive forces are causing aggregation, possibly due to hydrophobic interactions, van der Waals interactions or depletion forces that are not electrostatic in nature and thus are less influenced by the pH-value. Thus it is crucial to consider correlative effects between multiple biofilm components to better understand the aggregation tendency of NPs. A single component model system based on total organic carbon content of extracellular biofilm substances, would have led to an underestimation of the stability towards aggregation. Nevertheless a simple model that only depends on the polysaccharide concentration might be feasible if the protein content is not exceeding a critical value.

FCCS (fluorescence cross correlation spectroscopy) is a powerful tool to characterize drug target interaction in physiological environments. Over the last decade this technology was used to understand the binding and kinetics of small molecules and antibodies to challenging target proteins in great details and accuracy. Over the last decade Intana Bioscience expanded the application of FCCS from affinity measurements to comprehensive interaction studies including kinetics, multi component complex formation, assessing stoichiometry and quantification of aggregation, PK studies and target engagement. This presentation will highlight examples of results obtained with FCCS in projects ranging from early stages in the drug discovery up to the support of clinical trials to quantify target occupancies in patient samples.

We investigated starvation-induced changes in the relative nanoviscosity of a cancer cell. Fluorescence correlation spectroscopy was used to determine the movement of GFP particles in the cell cytoplasm and calculate diffusion coefficients during starvation for 96 hrs. Water efflux from cells caused changes in cell volume throughout starvation. A few folds increase in this physical quantity was observed during cell death. Morphological changes during starvation were recorded using confocal microscopy.

From multi-photon to single molecule, the past several decades have witnessed a revolution in fluorescent microscopy. These techniques have revealed the inner working of cells and tissue and have relied on symbiotic advances in advanced molecular probes, light emitting molecules and particles, and novel instrumentation. Following on these developments, researchers began to develop functional nanomaterials or materials that can response to their environment. One of the first such molecules reported electric fields, allowing neuron signaling to be observed. However, the optical signal generated by voltage reporters is often low, placing limitations on the measurements that can be performed. Thus, material scientists and chemists began to pursue the development of alternative systems. In parallel, the fields of organic solar cells and integrated photonics were actively pursuing the design of materials with similar active properties, thus forming a foundation for improved functional organic imaging agents. In this talk, I will discuss some of our recent work in developing functional imaging agents for multi-wavelength and multi-photon live-cell imaging, focusing on recent molecular designs performed using density functional theory as well as in vitro studies.

Irreversible adsorption of biomolecules onto imaging substrates is an impediment to expand the applications of single molecule techniques. Traditional polyethylene glycol (PEG) surfaces are only effective at low concentrations of analytes and their structure prevents their use for interferometric scattering (iSCAT) microscopy. We propose a new platform that virtually eliminates non-specific binding thanks to the omniphobicity of perfluorinated compounds, also known as the fluorous effect. Here, we showcase the anti-fouling properties of these substrates at a single molecule level through iSCAT measurements of a protein mixture. We believe these novel engineered substrates show great promise to study biomachinery processes requiring large analyte concentrations, where other passivation methods are not effective, through iSCAT microscopy and other single molecule techniques.

Single-molecule localization microscopy (SMLM) in combination with DNA barcoding (DNA-PAINT) enables easy-to-implement multi-target super-resolution imaging. However, image acquisition is slow because of the need to spatio-temporally isolate single emitters and to collect sufficient statistical data to generate a super-resolved image. Here, we bypass this limitation by utilizing a neural network, DeepSTORM, that can predict super-resolved SMLM images from high-emitter density data. This reduces the acquisition time 10- to 20-fold, enabling image acquisition as short as one minute. Integrating weak-affinity DNA labels allows precise control of single-molecule emitter densities, which enables recording of training, ground truth, and testing data from the same sample. Sequential imaging of multiple targets using different DNA barcodes with the same fluorophore enables aberration-free multi-target imaging (Exchange-PAINT). The constant exchange of fluorophore labels at target sites minimizes signal loss for long acquisition times, which allows imaging large samples in a matter of minutes. The concept is transferable to other weak-affinity, non-covalent fluorophore labels.

A time-dependent likelihood distribution for analyzing time correlated single photon counting data from a four-pixel time-resolved single molecule localization microscopy experiment is discussed. It is generated by accounting for the probabilities to record photons from two emitters, background counts, and dark counts during two different time channels relative to each incident laser pulse in the experiment. Maximizing the distribution enables localization of each emitter in a dual emitting nanostructure based on the disparate photoluminescence lifetimes of the emitters, even when both emitters are simultaneously in an emissive state. The technique is demonstrated using simulated photon counting data from a hypothetical non-blinking dual-emitter nanostructure in which the distance between the two emitters is less than 10-nm.