This PDF file contains the front matter associated with SPIE Proceedings Volume 11246, including the Title Page, Copyright information, Table of Contents, Author and Conference Committee lists.

We developed a dual-beam optical tweezers setup with video-based force detection to precisely determine the mechanic properties of living cells in suspension with superior sensitivity compared to other techniques like optical stretchers and atomic force microscopy (AFM). With high time resolution we are able to determine both the elastic and the viscous properties of the cells. This novel setup is combined with custom-designed microfluidic cartridges to automatically and reliably pattern cells and beads at specific positions. The beads and cells are trapped and coupled to yield bead-cell-bead complexes. First results of the elasticity of HEK293 (human embryonic kidney) cells and skin fibroblasts are presented. The latter contain TMEM43-p.S358L mutation, which is linked to arrhythmogenic right ventricular cardiomyopathy.

Commercial atomic force microscope (AFM) solutions do not typically lend themselves to easy integration into a wide range of optical microscope platforms. Here we present a custom AFM platform which is readily integrated into an FPGA controlled fluorescence microscope and is easily programmed for a variety of unique applications. Our collimated laser design allows for an adjustable sensitivity depending on the range of forces required, and the large range piezo (100μm in all three dimensions) presents the freedom to perform precise measurement and manipulation in a high throughput manner, over a broad range of distances. We demonstrate a high bandwidth of 1MHz with low noise levels of less than 71pN at full bandwidth and below 6pN at a relevant bandwidth of 1kHz.

Image Scanning Microscopy (ISM) enhances the spatial resolution of a confocal microscope by simple means. An extension to realize fluorescence lifetime imaging in combination with ISM seems straight-forward. First realizations have been reported by Israel et al. and Castello et al. [1,2] Here, we present a cost-efficient detector scheme based on a commercial multi-anode PMT that allows to perform fluorescence lifetime imaging microscopy (FLIM) in combination with ISM. We developa dedicated amplification electronics that allows for counting signals from 32 detector pixels using a commercial eight channel TCSPC hardware. [1] Y. Israel, R. Tenne, D. Oron, and Y. Silberberg “Quantum correlation enhanced super-resolution localization microscopy enabled by a fibre bundle camera” Nat. Commun. 8 (2017) 14786. [2] M. Castello et al. “A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM” Nat. Methods 16 (2019) 175-178.

Scanning FCS (sFCS) is a great tool for studying slowly diffusing species as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for simultaneous measurement at multiple locations. The shorter residence times also lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. Here, we show results from sFCS measurements on supported lipid bilayers (SLBs) where fluorescence lifetime information is used to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET).

Time Correlated Single Photon Counting (TCSPC) is a widely diffused technique used in scientific experiments requiring the analysis of optical pulses with high timing precision. One of the major limitations affecting this tool are distortion phenomena at high count rates happening due to pile-up. As a result, experiments must be carried out at a slower operating rate than the laser excitation frequency (1%-5%). It has been recently demonstrated that matching the detector dead time with the duration of the laser excitation period allows to overcome the aforementioned speed limitation, while still keeping distortion low. Theoretical results envision a speed improvement by almost an order of magnitude. In this work we present dedicated integrated electronics to implement the proposed idea. The selected detector for this design is a custom technology SPAD in order to achieve high performance. The SPAD is externally driven by an Active Quenching Circuit (AQC) that senses the avalanche current and provides a prompt quenching and reset of the detector. The AQC features a finely tunable dead time and a low reset time, two key aspects to achieve a very-low distortion regime and high efficiency. The detector electric signal is read out by a fully differential pick-up circuit, delivering a timing differential signal with picosecond precision and rejecting disturbances thanks to a dummy cell. A fast time-to-amplitude converter is used to measure the time of arrival of the photons with picoseconds precision and high linearity.

We report a nanohybrid based on an atomically thin, two-dimensional (2D) van Der Waals semiconductor, colloidal quantum dots and a light harvesting protein where step-wise energy transfer takes place. We connect nanocomponents of the nanohybrid via electrostatic self-assembly and layer-by-layer polyelectrolyte deposition and utilize bandgap engineering to created conditions for efficient directional stepwise energy transfer, from quantum dots, to proteins and to the 2D van Der Waals semiconductor, molybdenum diselenide (MoSe2). The biotic/abiotic nanohybrid exhibits enhanced absorption cross section and enhanced light harvesting through the addition of quantum dots and proteins next to MoSe2, which in turn leads to enhanced exciton generation in MoSe2 via energy transfer from quantum dots to proteins and finally to MoSe2.

Semiconductor quantum dots (QDs) in small clusters can exchange excited state energy via various transfer mechanisms such as F¨orster resonant energy transfer (FRET). Such energy transfer enables excitons to move from larger bandgap donors to smaller bandgap acceptors. Clusters of mixed donor/acceptor QD species consequently have a spectral signature that is dependent on which QDs in the clusters are responsible for the emission. Using a dual-color super-resolution imaging approach, we report on the spectral characteristics of interacting QDs in clusters with nanometer spatial resolution. Higher emission intensities from clusters are shown to emanate from sub-regions of the clusters and have spectral signatures that indicate the emission is dominated by the acceptor region of the spectrum. Thus, energy transferring interactions among QDs in clusters funnel excitons primarily to acceptor particles. Acceptor particles are responsible for the majority of the emission from the clusters with an emission spectra corresponding to the spectral profiles of the acceptor species.

We demonstrate ultrafast fluorescence lifetime imaging microscopy (FLIM) based on frequency-division multiplexing. As a proof-of-concept demonstration, we obtained images with fluorescence intensity and lifetime contrasts of MCF-7 breast cancer cells stained by SYTO16 at a record high frame rate of 16,000 fps, which is 100 times higher than that of previously reported FLIM techniques. Our method is expected to expand the utility of FLIM to quantitative analysis of rapid intracellular dynamics and high-throughput cell screening based on fluorescence lifetime images.

We introduce an innovative design of planar plasmonic nanogap antenna arrays and demonstrate its potential to study the spatiotemporal organization of mimetic biological membranes at the nanoscale. We exploit our novel nanogap antenna platform with different nanogap sizes (10-45 nm) combined with fluorescence correlation spectroscopy to reveal the existence of nanoscopic domains in mimetic biological membranes. Our approach takes advantage of the highly enhanced and confined excitation light provided by the antennas together with their extreme planarity to investigate membrane regions as small as 10 nm in size with microsecond temporal resolution. We first demonstrate the ultra-high confinement of photonic antennas on biological membranes. Moreover, we show that cholesterol slows down the diffusion of individual fluorescent molecules embedded in the lipid bilayer, consistent with the formation of nanoscopic domains enriched by cholesterol. Incorporation of hyaluronic acid (HA) to the ternary lipid mixture further slows down molecular diffusion, suggesting a synergistic effect of cholesterol and HA on the dynamic partitioning of mimetic biological membranes.

Single-molecule superresolution methods enable imaging of specifically-labeled biological samples with structures on length scales below the diffraction limit of visible light. Imaging samples at cryogenic temperatures (77 K) significantly reduces photobleaching, allowing more photons to be collected per emitter and thus improving the localization precision. Cryogenic single-molecule imaging also facilitates correlative imaging with cryogenic electron tomography (cryoET), which provides images of whole biological cells with high-resolution cellular contrast. Combining these two techniques by performing optical imaging under conditions that do not damage the sample for cryoET allows the combination of the high sensitivity and specificity from single-molecule fluorescence with the cellular context from cryoET. In this work, we use PAmKate, a red photoactivatable fluorescent protein, to perform cryogenic single-molecule imaging of proteins in the model organism Caulobacter crescentus at 77 K with sufficiently low illumination powers to prevent damage of the cryogenic sample. The enhanced number of photons detected allows localization precision to be improved to values below 10 nm.

We achieve spontaneous 3D super-resolution on a standard confocal microscope by exploiting bio-friendly fluorescent markers with super-linear excitation-emission dependence (upconversion nanoparticles of NaYF4: Yb, Tm). We refer to this approach as upconversion super-linear excitation-emission (uSEE) microscopy. To demonstrate the applicability of the method for biological applications, we image sugar-coated upconversion nanoparticles in neuronal cells and we achieve resolution twice better than the diffraction limit both in lateral and axial directions. We envision that due to the application simplicity of the developed methodological toolbox, uSEE microscopy can be widely incorporated as an everyday super-resolution method in biological laboratories.

DAISY combined Supercritical Angle Fluorescence (SAF) detection in order to provide an absolute axial reference to a point spread function engineering approach . The dual view optical setup permits to introduce a strong astigmatism and decouple lateral and axial information, thus extending the axial performances without degrading the lateral precision. This technique provides 3D absolute information over a 1-µm capture range above the glass coverslip and an axial localization precision down to 15 nm with minimal loss of lateral resolution and little sensitivity to field aberrations. We will present sequential and simultaneous up to 3 color 3D imaging in cells.

Confocal microscopy features a good sectioning capability, which makes it essential for high-resolution 3D biological imaging. However, a tightly focused excitation beam inevitably leads to irreversible photodamage to live specimens, such as photobleaching and phototoxicity, and point-by-point scanning mechanism hampers its applications in fast volumetric imaging. As an alternative approach, selective plane illumination microscopy has shown outstanding performance in long-term imaging of embryonic development and neuronal activities attributed to its capabilities of intrinsic sectioning, gentle excitation and fast imaging. One drawback of SPIM is that the arrangement of two closely placed objectives greatly restricts the geometry of sample holders and the available numerical aperture (NA) for effective fluorescence collection. Here, we propose a highly-inclined plane illumination with a single high NA objective using for both excitation and detection. Unlike the requirement of two relay objective units in remote focusing imaging, an adaptive optical device serving as a flexible wavefront modulator compensates the systematical aberrations induced by optical components and effectively extended the imaging depth by generating an elongated point spread function (PSF). Our technique is applicable to single-molecule tracking and super-resolution imaging in live cells. Moreover, the adaptive optics can be replaced with a transmitted phase mask to enhance the effective fluorescent collection and simplify the system alignment effort.

Correlating DNA-PAINT (point accumulation for imaging in nanoscale topography) and single-molecule FRET (Förster resonance energy transfer) enables the multiplexed detection with sub-diffraction optical resolution. We designed pairs of short oligonucleotides, labeled with donor and acceptor fluorophores with various distances generating different FRET efficiencies. The strands can transiently bind to a target docking strand, simultaneous binding of both strands results in FRET signals which yield a super-resolved image via DNA-PAINT imaging. We demonstrate FRET-PAINT by designing and imaging DNA origami, which is a useful tool to establish super-resolution methods. The DNA origami structures were equipped with three target binding sites spaced by 55 nm, a sub-diffraction limited distance, however ensuring that no FRET between the target sites occurs. We resolved the individual binding sites in the donor and acceptor channels, and in addition extracted the FRET efficiency for each site in single and mixed populations. The combination of FRET and DNA-PAINT allows for multiplexed super-resolution imaging in conjunction with distance-sensitive readout in the 1 to 10 nm range.

Self-interference digital holography (SIDH) is a promising approach for three-dimensional imaging as it offers the ability to view a complete three-dimensional volume from a single image. SIDH has so far, largely been limited to image samples that emit a large number of photons. We report the use of a Michelson interferometer based SIDH setup which provides higher light throughput compared to previous systems that employed spatial light modulators (SLM). SIDH microscopy incorporating a Michelson interferometer in the proposed optical configuration and high-numerical-aperture oil immersion objectives can be used to perform super-resolution single-molecule localization microscopy (SMLM) and single-particle tracking (SPT) over large axial ranges. We demonstrate this by localizing a single 0.1 µm diameter fluorescent nanosphere using a custom built wide-field microscope. With 49,000 photons detected, the proposed system achieves a localization precision of 4.5 nm in x, 5 nm in y and 39.8 nm in z over a 20 µm axial range. Further, we also discuss the SNR requirements to image photon-limited light sources such as a single-molecules using SIDH.

Knowledge of how proteins organize into functional complexes is essential to understand their biological function. Optical super-resolution techniques provide the spatial resolution necessary to visualize and to investigate individual protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) builds on the detection of single fluorophore labels, which next to the generation of high-resolution images provides access to quantitative molecular information. We developed various tools for quantitative SMLM (qSMLM), an imaging method that both super-resolves individual protein clusters and reports on molecular numbers by analyzing the kinetics of single emitter blinking. This method is compatible with both fluorescent proteins and organic fluorophores. With qSMLM, we quantify protein copy numbers in single clusters, and we study how changes in the stoichiometry of protein complexes translates into function.

Photoluminescence images can be acquired with detection schemes that have both single-photon sensitivity and nanosecond scale temporal resolution, enabling the study of possible structural bases for photoluminescence lifetimes and other features of the photon arrival statistics. Within the context of super-resolution (SR) imaging, this has been demonstrated with detection schemes that collect images with a bundle of optical fibers that are coupled to individual single-photon counting avalanche photodiode detectors. Recently, our group used a bundle of four optical fibers to collect these “time-resolved photon arrival” images. Despite the paucity of information contained in a four-pixel image, we precisely located the emission centroid of quantum dots (QDs) and observed correlations between centroid location, photoluminescence lifetime, and intensity within clusters of QDs that were suggestive of electronic interactions among them. This proceedings paper details the approach that we used to locate the emission centroid based on the counts in the four detectors.

The emerging field of quantum imaging introduces new methods to overcome classical limitations in optical microscopy. A detection apparatus capable of analyzing the quantum signature of light, is a crucial component in the heart of any such method. We present a novel quantum imaging modality, based on a state-of-the-art single photon avalanche diode (SPAD) array in a confocal setup. This modality enables unprecedented simplicity and scalability in imaging temporal photon correlations. We demonstrate the potential of this approach by measuring temporal correlations of classical and quantum sources, as well as demonstrate a quantum super-resolution technique.

We propose Modloc, a new single molecule localization strategy based on a time signature. By shifting a structured excitation pattern, it induces a time modulated emission of the fluorophores where the phase holds its position. An efficient demodulation optical assembly compatible with short emitters On-time has been designed. Performances of ModLoc, will be discussed, in particular its 2 fold lateral precision improvement and its robustness to optical aberrations. By applying the modulation in a tilted direction, we will show that an almost isotropic 3D localization (~11 nm) is obtained over the whole capture range up to 30 µm in depth

Super-resolved microscopy techniques have overcome the diffraction limit to provide image resolutions approaching the scale of fluorescent labels. However, many of these techniques require significant experimental resources and expertise and impose long image data acquisition times, making it difficult to acquire super-resolved data from sufficiently large sample numbers to overcome intrinsic biological variation. We have worked to make stimulated emission depletion (STED) microscopy and single molecule localisation microscopy (SMLM) more straightforward to implement and more practical to image larger numbers of cells. Here we present work in progress developing easySLM STED and easySTORM, including a new modular microscope frame that we believe can make it easier to prototype microscopy techniques and to implement and maintain them in lower resourced settings.

Superresolution microscopy methods have revolutionized far-field optical fluorescence microscopy by manipulating state transitions of the emitters, offering potentially unlimited resolution. In practice, however, the resolution of an image is limited by the finite photon budget of fluorescent probes. The recently introduced localization concept, termed MINFLUX, tackles this limitation by rendering each emitted photon more informative, achieving single digit nanometer resolution. Here, we present a MINFLUX strategy with high photon efficiency in arbitrarily large regions that allows imaging in fixed and living cells. This allows isotropic localization precision and surpasses the typical ∝1⁄√N dependence. A multi-color modality for 3D-MINFLUX imaging will be also presented, together with several biological applications.

Measuring 3D orientation properties of single fluorescent emitters including their angle wobbling, as well as their position, is a challenge that would enrich super-resolution techniques with structural molecular information. We present a polarized microscopy technique that provides all 3D orientation parameters unambiguously, using four-polarization splitting of the image plane and intensity filtering in the back focal plane. Using an inverse problem approach we can retrieve 3D orientation, wobbling and 2D position of the fluorophores with high precision. We validated the technique using fluorescent nano-beads and applied it to the structural study of fluorescently labelled F-actin filaments.

Anti-Brownian traps enable the measurement of single particles in free solution for long times by actively applying feedback forces based on an observed particle position to counteract Brownian motion. However, current implementations of anti-Brownian traps generally rely on fluorescence emission to detect a particle’s position. This reliance on fluorescence causes particles to be lost from the trap when they enter a fluorescence dark state by blinking or bleaching. Thus, there is a need for non-fluorescent methods of tracking for such traps. Scattered light provides a stable signal free of blinking and bleaching, but is very weak for small particles. However, interferometric scattering, a method of collecting the weak scattered field from a particle and interfering it with a strong reference field reflected from a nearby interface, allows particles to be tracked with sufficient speed and sensitivity. We combine interferometric scattering with our existing anti- Brownian electrokinetic (ABEL) trap to create the interferometric scattering anti-Brownian electrokinetic (ISABEL) trap. This technique enables the trapping of single nanoparticles in free solution for extended durations regardless of fluorescence blinking or bleaching. We verify the scaling of the interferometric scattering signal with the diameter of the particle for gold nanoparticles as small as 20 nm. We also demonstrate the measurement of the fluorescence brightness signal of fluorescent beads as they photobleach, while continuing to trap them with the scattering signal. The ISABEL trap extends the ability of anti-Brownian traps to new samples and new measurements across multiple scientific communities.

Surface enhanced Raman spectroscopy (SERS) has evolved to be a powerful analytical tool for investigating molecular properties of various types of samples. Literature has shown SERS capabilities in both qualitative and quantitative analysis of biomolecules like proteins and DNA as well as single molecules like antiretroviral medication. Central to its application is the synthesis and use of sensing platforms that enhance signal intensity, sensitivity and detection limits. The most popular approach to make such platforms is through fabricating thin film substrates using a combination of polymers and nanomaterials. In this work, we use the self-assembly method to synthesize graphene oxide based scaffolds in a layer-by-layer fashion and characterize them using SERS. The results show a clear difference in Raman spectral fingerprint for the different layers during the self-assembly steps. Lastly, the intensity ratio between the D and G bands of the graphene layer were calculated to measure the layer thickness which was found to be 0.65, this was comparable to thin layer scaffolds reported in literature. Future work will involve the use of atomic force microscopy to confirm surface morphology and layer thickness, followed by screening of antiretroviral medication.

Fluorescence imaging of sub-cellular structures with sizes below the diffraction limit is vital in understanding cel- lular processes. Relying on exciting the sample with different illumination patterns and image processing for the elimination of background fluorescence, Structured Illumination Microscopy (SIM) provides imaging capability beyond diffraction limit using relatively simple optical setups. Here, we present a laser-free, DLP projector-based, and GPU-implemented SIM super resolution microscope. Sub-diffractive biological structures were imaged with a lateral resolution of ∼150 nm. The microscopy system is LED-based and entirely home-built which enables customizable operation at a low cost.

We present a computational method, termed Wasserstein-induced flux (WIF), to robustly quantify the accuracy of individual localizations within a single-molecule localization microscopy (SMLM) dataset without ground- truth knowledge of the sample. WIF relies on the observation that accurate localizations are stable with respect to an arbitrary computational perturbation. Inspired by optimal transport theory, we measure the stability of individual localizations and develop an efficient optimization algorithm to compute WIF. We demonstrate the advantage of WIF in accurately quantifying imaging artifacts in high-density reconstruction of a tubulin network. WIF represents an advance in quantifying systematic errors with unknown and complex distributions, which could improve a variety of downstream quantitative analyses that rely upon accurate and precise imaging. Furthermore, thanks to its formulation as layers of simple analytical operations, WIF can be used as a loss function for optimizing various computational imaging models and algorithms even without training data.

The advent of super-resolution microscopy techniques has begun a revolution in the field of optical microscopy. Localization methods have become one of the most dynamically developed areas in this field. The one order of magnitude improvement in the lateral resolution has revealed a lot of new details mainly in the field of biology, which was hidden with conventional microscopes by the diffraction. However, the visualization of these measurements often go hand in hand with information loss and the quantification of such images become limited. Here we present a quantitative evaluation method which uses the raw localization coordinates and the associated precision for quantitative analysis. With our cluster analysis-based method we were able to determine the different properties of the selected clusters, such as their area (or in case of 3D images the volume), spatial distribution and the number of labelled target molecules in them, which is not follows directly from the reconstructed image due the stochastic nature of blinking and the often unknown labelling stoichiometry. The great advantage of our method is that we can gain the latter property from the sample directly. We also applied our method to investigate the repair mechanism of DNA double-strand breaks (DSBs).

Infrared photothermal heterodyne imaging (IR-PHI) represents a convenient, table top approach for conducting super-resolution imaging and spectroscopy throughout the all-important mid infrared (MIR) spectral region. Although IR-PHI provides label-free, super-resolution MIR absorption information, it is not quantitative. In this study, we establish quantitative relationships between observed IR-PHI signals and relevant photothermal parameters of investigated specimens. Specifically, we conduct a size series analysis of different radii polystyrene (PS) beads to quantitatively link IR-PHI signal contrast to specimen heat capacity, thermo-optic coefficient, MIR peak absorption cross-section, and scattering cross-section at IR-PHI’s probe wavelength.

We present a microscopy technique, orbital particle tracking, in which the scanner scans orbits around species, unlike a raster imaging technique in which the scanner scans an area one line at a time. By analyzing the fluorescence emission intensity variation along an orbit, the location of a species in the orbit can be determined with precision of a tenth of a nanometer in a millisecond time scale, and the orbit can be moved to the new location of the species through a feedback loop if any movement is detected. This technique can be extended to two scanning orbits, one above and one below the sample plane to track the sample in 3D space. It can be used in vitro or in vivo to track a motion of a sample or to understand the dynamics of the sample. Additional detectors can help reveal the correlation between events with different emission spectrums. We have performed two different experiments with the system to show the capability of the technique. In the first example, we track a transcription site to understand the relationship between transcription factor - DNA binding and RNA transcription [1, 2]. By labeling a transcription factor with Halo-JF646 and nascent RNA with PP7-GFP, we were able to cross correlate fluorescence intensity to discover temporal coordination between transcription factor DNA binding and resulting gene activation. In the second experiment, we tracked lysosomes in live cells to understand the nature of the transport whether it is an active transport or a free diffusion [3]. Trajectories of a total of 24 lysosomes are recorded during the experiment. The mean squared displacement (MSD) curves of the trajectories showed some clear differences between the behaviors of the lysosomes which were attributed to the active transport along microtubules as opposed to freely diffusing lysosomes.

In this work we report a single molecule imaging method to spread the photon budget of a fluorophore over extended time periods by inserting a non-illuminating interval between consecutive frames. While photobleaching results in loss of fluorescent signals similar to the detachment of a fluorescently labeled biomolecules, we introduced this method to characterize the dwell times of single molecules.

Modulating the polarization of excitation light, resolving the polarization of emitted fluorescence, and point spread function (PSF) engineering have been widely leveraged for measuring the orientation of single molecules. Typically, the performance of these techniques is optimized and quantified using the Cramér-Rao bound (CRB), which describes the best possible measurement variance of an unbiased estimator. However, CRB is a local measure and requires exhaustive sampling across the measurement space to fully characterize measurement precision. We develop a global variance upper bound (VUB) for fast quantification and comparison of orientation measurement techniques. Our VUB tightly bounds the diagonal elements of the CRB matrix from above; VUB overestimates the mean CRB by ~34%. However, compared to directly calculating the mean CRB over orientation space, we are able to calculate VUB ~1000 times faster.