Optics Express

In single-molecule localization microscopy (SMLM), achieving precise localization hinges on obtaining an authentic point spread function (PSF) influenced by system and sample-induced aberrations. Here, we introduce VISPR (Vectorial in-situ PSF retrieval) retrieving precise 3D PSF models considering both system and sample-induced aberrations under SMLM conditions. By employing the theory of vectorial PSF model and maximum likelihood estimation (MLE) phase retrieval, VISPR is capable of reconstructing an accurate 3D PSF model achieving the theoretically minimum uncertainty and accurately reflecting three-dimensional information of single molecules. This capability empowers accurate 3D super-resolution reconstruction in 3D SMLM. Additionally, VISPR applies to low signal-to-noise ratio circumstances and is adept at retrieving high-frequency details of the experimental PSF across an extensive depth range--a challenging feat for alternative approaches. As an effective tool, VISPR enables the quantitative assessment of aberrations induced by the system and sample environment. From the simulations and experiments, we verified the superiority and effectiveness of VISPR. It is essential to highlight that VISPR applies to various SMLM microscope modalities.

Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing the STED beam intensity, it theoretically improves the spatial resolution to any desired value. However, the higher is the dose of stimulating photons, the stronger are the photo-bleaching and photo-toxicity effects, which potentially compromise live-cell and long-term imaging. For this reason the scientific community is looking for strategies to reduce the STED beam intensity needed to achieve a target resolution. Here, we show how the combination of STED microscopy with image scanning microscopy (ISM) meets this request. In particular, we introduce a new STED-ISM architecture - based on our recent single-photon-avalanche-diode (SPAD) detector array - which allows covering the near-diffraction limit resolution range with reduced STED beam intensity. We demonstrate this ability both with simulated data and in live-cell experiments. Because of (i) the minimal changes in the optical architecture of the typical point-scanning STED microscope; (ii) the parameter-free, robust and real-time pixel-reassignment method to obtain the STED-ISM image; (iii) the compatibility with all the recent progresses in STED microscopy, we envisage a natural and rapid upgrade of any STED microscope to the proposed STED-ISM architecture.

Confocal Brillouin microscopy (CBM) enables high-resolution mechanical imaging but has slow acquisition speeds due to its point-by-point scanning strategy. Line-scanning Brillouin microscopy (LSBM) offers imaging acquisition speed improvements but faces challenges such as beam distortion in biaxial configurations and insufficient extinction ratio due to the single-stage VIPA spectrometer. To overcome these limitations, we developed a coaxial line-scanning Brillouin microscopy (cLSBM) system by using a two-stage parallel VIPA spectrometer. The coaxial design minimizes image distortion, and the two-stage parallel VIPA spectrometer significantly enhances the rejection of non-Brillouin noises. Experiment results showed that the first VIPA, served as a filter for noise rejection, has a rejection ability of 18 dB. The system was characterized by standard materials including ethanol and water, achieving a precision of 7.5 MHz and 12.6 MHz respectively. In the next step, we will optimize the system to further enhance noise rejection and utilize this setup to investigate the evolution of tissue mechanics during embryonic development.

Single-molecule localization microscopy methods extensively leverage the microscope point spread function (PSF) for fitting the molecules. Calibrating an accurate PSF model is especially difficult in the presence of depth-dependent aberrations which alter the PSF shape depending on the imaging depth. The aberrations at depths of a few micrometers become substantial enough to considerably impoverish the conventional calibration methods performance. In our work, we propose a novel spline model which enables the depth-dependent PSF model calibration by interpolating between the beads at arbitrary depths. We show that diffspline reduces the PSF intensity overestimation by 67.8 percentage points and underestimation by 21.8 percentage points. Moreover, it eliminates the depth-dependent bias and improves the localization precision two-fold compared to previous approaches.

According to Rayleighs criterion, two incoherent emitters with a separation below the diffraction limit are not resolvable with a conventional fluorescence microscope. One method of Super-Resolution Microscopy (SRM) circumvents the diffraction-limited resolution by precisely estimating the position of spatiotemporally independent emitters. However, these methods of SRM techniques are not optimal for estimating the separation of two simultaneously excited emitters. Recently, a number of detection methods based on modal imaging have been developed to achieve the quantum Cramer-Rao lower bound (QCRB) to estimate the separations between two nearby emitters. The QCRB determines the minimum achievable precision for all possible detection methods. Current modal imaging techniques assume a scalar field generated from a point source, such as a distant source from an optical fiber or a pinhole. However, for fluorescently labeled samples, point emitters are single fluorophores that are modeled as dipole emitters and, in practice, are often freely rotating. Dipole radiation must be described by vectorial theory, and the assumption of a scalar field no longer holds. Here, we present a method to numerically calculate the QCRB for measuring the separation of two dipole emitters, incorporating the vectorial theory. Furthermore, we propose a near-quantum optimal detection scheme based on one of the modal imaging techniques, super-localization by image inversion interferometry (SLIVER), for estimating the separation of two freely rotating dipoles. In the proposed method, we introduce a vortex wave plate before the SLIVER detection to separate the radial and azimuthal components of the dipole radiation. With numerical simulations, we demonstrated that our method achieves non-divergent precision at any separation between two dipole emitters. We investigated several practical effects relevant to experimental measurements in super-resolution microscopy, including numerical aperture, detection bandwidth, number of estimation parameters, background, and misalignment on separation estimation. Our proposed measurement provides a near quantum-limited detection scheme for measuring the separation of two freely-rotating dipole emitters, such as fluorescently tagged molecules, which are commonly used in super-resolution microscopy.

Bessel Beams (BBs) and BB lattices are structured-light excitation profiles frequently applied in material processing, nonlinear spectroscopy and in many fluorescence microscopy methods such as Light Sheet Microscopy (LSM). In LSM, BBs and BB-lattices offer wider excitation profiles, higher acquisition rate, enhanced resolution, and improved signal-to-noise ratio, while reducing the overall phototoxicity. However, this performance improvement typically comes at the cost of layout complexity and spatial constraints, originating from the optical arrangement required for obtaining BB features and for multiplexing the BB in a lattice of beamlets. Here, we introduce a novel method for encoding in a single flat element all the optical operations required to generate a BB lattice, including those of the excitation objective. We assessed the effective capabilities of this approach, using Meta-Surface (MS) technology to fabricate the corresponding flat optical element and to characterize its optical figures. Finally, we demonstrated its actual application in LSM, recording neuronal activity at cellular resolution in the zebrafish larval brain using fluorescence based neuronal activity reporters. In perspective, this approach, applied here for LSM, prompts a step forward in the BB versatility and in the BB application scenarios.

We present a novel method to improve the imaging efficiency of tiling light sheet microscopy. In the method, scanning non-coaxial beam arrays synchronized with regional virtual confocal slits are used to illuminate imaging plane. There are two advantages. One is the imaging efficiency increases proportional to the number of excitation beams within the non-coaxial beam array. The other is the width of the regional virtual confocal slits could be very wide without admitting off-focus fluorescence generated by the non-coaxial beam array, which makes the method easy to adopt and very robust in practice. We describe the method in detail, characterize the method via numerical simulations. The results suggest that the imaging efficiency and feasibility of the tiling light sheet microscopy could be improved significantly without affecting the 3D imaging ability by using the method. In additions, we propose several configurations to implement the method in practice.

Standard SMLM facilitates the reconstruction of super-resolution map (both location and localization precision) of the target single molecules. In fact, single molecule data does provide information related to the orientation of single molecules, which can be derived from the knowledge of PSF shape and its direction. This information is vital to probe the sub-domain of macromolecules that undergo orientation and conformational changes and provides essential clues on their catalytic activity. Accessing this information in real-time opens up a powerful new window to look into the link between the orientation of macromolecules and the output function. Here, we decode the orientation of single molecules from the knowledge of PSF shape and its direction. The method is primarily based on field-dipole interaction and the fact that the distribution of emitted photons strongly depends on the orientation of the dipole (fluorophore) with respect to the polarization of light. Accordingly, the photon emission from the specimen and the resultant PSF distribution model is developed. Computational studies show changes in the shape and orientation of the recorded PSF (in the image / detector plane). Specifically, a set of three distinct distributions (Gaussian, bivariate-Gaussian and skewed-Gaussian) are recognized from the study, apart from a superset of all possible (a total of 16) distributions. Experiments were conducted on Dendra2-Actin and Dendra2-HA transfected cells that validate the emission model. We report a localization precision of[~] 20 nm and an orientation precision of {+/-}5{degrees}. In addition, the distinct orientation of single molecules is noted for Actin and HA in a cell (Influenza type-A model). Further analysis suggests a preferred directional distribution of Dendra2-Actin single molecules, while Dendra2-HA molecules seem to be randomly oriented in a cluster. The availability of orientation information in SMLM without the need for additional optics adds a new feature, which can be explored to reveal the state of a single molecule (orientation and conformational changes) in cellular sub-domains / partitions. The study implies that the orientation of single molecules that has more profound implications for the functioning of macromolecules. The orientation information revealed by oSM LM technique gives it a wide-spread appeal and expands the reach of localization microscopy.

In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 x 315 x 80 m3. The axial extent of point-spread-function (PSF) was 17.6 {+/-} 1.6 m and 90.4 {+/-} 2.1 m with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.

Light nanoscopy is attracting widespread interest for the visualization of fluorescent structures at the nanometer scale, especially in cellular biology. To achieve nanoscale resolution, one has to surpass the diffraction limit--a fundamental phenomenon determining the spot size of focused light. Recently, a variety of methods have overcome this limit, yet in practice they are often constrained by the requirement of special fluorophores, nontrivial data processing, or high price and complex implementation. For this reason, confocal fluorescence microscopy that yields relatively low resolution is still the dominant method in biomedical sciences. It was shown that image scanning microscopy (ISM) with an array detector instead of a point detector could improve the resolution of confocal microscopy. Here we review the principles of the confocal microscopy and present a simple method based on ISM with a different image reconstruction approach, which can be easily implemented in any camera-based laser-scanning set-up to experimentally obtain the theoretical resolution limit of the confocal microscopy. Our method, Single Pixel Reconstruction Imaging (SPiRI) enables high-resolution 3D imaging utilizing image formation only from a single pixel of each of the recorded frames. We achieve experimental axial resolution of 330 nm, which was not shown before by basic confocal or ISM-based systems. Contrary to the majority of techniques, SPiRI method exhibits a low lateral-to-axial FWHM aspect ratio, which means a considerable improvement in 3D fluorescence imaging of cellular structures. As a demonstration of SPiRI application in biomedical sciences, we present a 3D structure of bacterial chromosome with excellent precision.

Molecules capable of emitting a large number of photons (also known as fortunate molecules) are crucial for achieving resolution close to a single molecule limit (the actual size of a single molecule). We propose a long-exposure single molecule localization microscopy (leSMLM) technique that enables detection of fortunate molecules, which is based on the fact that detecting a relatively small subset of molecules with large photon emission increases its localization [Formula]. Fortunate molecules have the ability to emit a large burst of photons over a prolonged time (> average triplet-state lifetime). So, a long exposure time allows the time window necessary to detect these elite molecules. The technique involves the detection of fortunate molecules to generate enough statistics for a quality reconstruction of the target protein distribution in a cellular system. Studies show a significant PArticle Resolution Shift (PAR-shift) of about 6 nm and 11 nm towards Single-molecule-limit (away from diffraction-limit) for an exposure time window of 60 ms and 90 ms, respectively. In addition, a significant decrease in the fraction of fortunate molecules (single molecules with small localization precision) is observed. Specifically, 8.33% and 3.43% molecules are found to emit in 30 - 60 ms and 60 - 90 ms, respectively, when compared to SMLM. The long exposure has enabled better visualization of Dendra2HA molecular cluster, with sub-clusters within a large cluster. Thus, the proposed technique (leSMLM) facilitates a better study of cluster formation in fixed samples. Overall, the method enables better spatial resolution at the cost of relatively poor temporal resolution.

With a limited effective voxel rate, to date, each laser-scanning mesoscopic multiphoton microscope (MPM), despite securing an ultra-large field of view (FOV) and an ultra-high optical resolution simultaneously, experiences a fundamental issue with digitization; i.e., inability to satisfy the Nyquist-Shannon sampling criterion to resolve the optics-limited sub-micron resolution over the whole FOV. Such a system either neglects the criterion degrading the digital resolution to twice the pixel size, or significantly reduces the imaging area and/or the imaging speed to respect the digitization. Here we introduce a Nyquist figure of merit parameter to assess this issue, further to comprehend a maximum aliasing-free FOV and a cross-over excitation wavelength for a laser scanning MPM system. Based on our findings we demonstrate an ultra-high voxel rate acquisition in a custom-built mesoscopic MPM system to exceed the Nyquist-rate for a >3800 FOV-resolution ratio while not compromising the imaging speed as well as the photon-budget.

We present a robust, "real-time" optical autofocus system for microscopy that provides high accuracy (<230 nm) and long range ([~]130 {micro}m) with a 1.4 numerical aperture oil immersion objective lens. This autofocus can operate in a closed loop, single-shot functionality over a range of {+/-}37.5 {micro}m and can also operate as a 2-step process up to {+/-}68 {micro}m. A real-time autofocus capability is useful for experiments with long image data acquisition times, including single molecule localization microscopy, that may be impacted by defocusing resulting from drift of components, e.g., due to changes in temperature or mechanical drift. It is also vital for automated slide scanning or multiwell plate imaging where the sample may not be in the same horizontal plane for every field of view during the image data acquisition. To realise high precision and long range, we implement orthogonal optical readouts using cylindrical lenses. We demonstrate the performance of this new optical autofocus system with automated multiwell plate imaging and single molecule localisation microscopy and illustrate the benefit of using a superluminescent diode as the autofocus light source.

Photon emission by single molecules is a random event with a well-defined distribution. This calls for event-based detection in single-molecule localization microscopy. The detector has the advantage of providing a temporal change in photons and emission characteristics within a single blinking period (typically, [~] 30 ms) of a single molecule. This information can be used to better localize single molecules within a user-defined collection time (shorter than average blinking time) of the event detector. The events collected over every short interval of time / collection time ([~] 3 ms) give rise to several independent temporal photon distributions (tPSFs) of a single molecule. The experiment showed that single molecules intermittently emit photons. So, capturing events over a shorter period / collection time than the entire blinking period gives rise to several realizations of the temporal PSFs (tPSFs) of a single molecule. Specifically, this translates to a sparse collection of active pixels per frame on the detector chip (image plane). Ideally, multiple realizations of single-molecule tPSF give several position estimates of the single-molecules, leading to multiple tPSF centroids. Fitting these centroid points by a circle provides an approximate position (circle center) and geometric localization precision (determined by the FWHM of the Gaussian) of a single molecule. Since the single-molecule estimate (position and localization precision) is directly driven by the data (photon detection events on the detector pixels) and the recorded tPSF, the estimated value is purely experimental rather than theoretical (Thomsons formula). Moreover, the temporal nature of the event camera and tPSF substantially reduces noise and background in a low-noise environment. The method is tested on three different test samples (1) Scattered Cy3 dye molecules on a coverslip, (2) Mitochondrial network in a cell, and (3) Dendra2HA transfected live NIH3T3 cells (Influenza-A model). A super-resolution map is constructed and analyzed based on the detection of events (temporal change in the number of photons). Experimental results on transfected NIH3T3 cells show a localization precision of [~] 10 nm, which is [~] 6 fold better than standard SMLM. Moreover, imaging HA clustering in a cellular environment reveals a spatio-temporal PArticle Resolution (PAR) (2.3lp x{tau} ) of 14.11 par where 1 par = 10-11 meter.second. However, brighter probes (such as Cy3) are capable of [~] 3.16 par. Cluster analysis of HA molecules shows > 81% colocalization with standard SMLM, indicating the consistency of the proposed eventSMLM technique. The single-molecule imaging on live cells reveals temporal dynamics (migration, association, and dissociation) of HA clusters for the first time over 60 minutes. With the availability of event-based detection and high temporal resolution, we envision the emergence of a new kind of microscopy that is capable of high spatio-temporal particle resolution in the sub-10 par regime.

Spatial light modulation using cost efficient digital mirror arrays (DMA) is finding broad applications in fluorescence microscopy due to the reduction of phototoxicity and bleaching and the ability to manipulate proteins in optogenetic experiments. However, the precise calibration of DMAs and their application to single-molecule localization microscopy (SMLM) remained a challenge because of non-linear distortions between the DMA and camera coordinate system caused by optical components. Here we develop a fast and easy to implement calibration procedure that determines these distortions by means of an optical feedback and matches the DMA and camera coordinate system with ~50 nm precision. As a result, a region from a fluorescence image can be selected with a higher precision for illumination compared to manual alignment of the DMA. We first demonstrate the application of our precisely calibrated light modulation by performing a proof-of concept fluorescence recovery after photobleaching experiment with the endoplasmic reticulum-localized protein IRE1 fused to GFP. Next, we develop a spatial feedback photoactivation approach for SMLM in which only regions of the cell are selected for photoactivation that contain photoactivatable fluorescent proteins. The reduced exposure of the cells to 405 nm light increases the possible imaging time by 44% until phototoxic effects cause a dominant fluorescence background and a change in the cells morphology. As a result, the mean number of reliable single molecule localizations is also significantly increased by 28%. Since the localization precision and the ability for single molecule tracking is not altered compared to traditional photoactivation of the entire field of view, spatial feedback photoactivation significantly improves the quality of SMLM images and the precision of single molecule tracking. Our calibration method therefore lays the foundation for improved SMLM with active feedback photoactivation far beyond the applications in this work.\n\nStatement of significanceActively patterned illumination in fluorescence microscopy can reduce bleaching and phototoxicity as well as actively manipulate proteins in optogenetic applications. Matching the coordinate system of the camera and the light patterning device such as digital mirror arrays (DMA) remains a challenge. We developed a fast and easy calibration procedure that determines and corrects for the transformation between the camera and DMA coordinate system with ~50 nm precision. Using this approach, we develop spatial feedback photoactivation for Single Molecule Localization Microscopy (SMLM) to photoswitch only intracellular regions containing photoswitchable fluorophores. Our results show a 44% improvement in the possible data acquisition time before phototoxic effects become detectable and a 28% increase in detected localizations. Spatial feedback photoactivation thus significantly improves SMLM experiments.

Single-molecule orientation and localization microscopy (SMOLM) enables the determination of molecular orientation, wobbling, and position. However, most SMOLM implementations rely on complex point spread function (PSF) fitting, which limits analysis throughput and introduces high computational cost. A way to overcome these limitations is to simplify the analysis using a ratiometric intensity estimation, often relying on polarization projections. While effective in 2D, extending these methods to 3D remains challenging. Here, we introduce a new ratiometric strategy for SMOLM in 3D. Building on the principles of Single Molecule Light Field Microscopy, which captures the 3D position information from a single snapshot by segmenting the back focal plane, we extend this strategy to orientation retrieval. Our approach uses the generalized 3D Stokes formalism to linearly decompose the intensity measurements across the light-field channels, allowing computationally-efficient estimations, while avoiding both complex PSF fitting and polarization projections. This framework, called SMOLM-LFM, enables 6D estimation of single molecules (3D position + 3D orientation) with a simplified optical setup and a large depth-of-field. We present the theoretical foundations, experimental implementation, and validation through measurements on calibration beads, single fluorophores, and cells, thereby demonstrating the methods potential and practical limitations.

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for biomedical studies of active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. Manager is a widely used open-source software platform for controlling many optoelectronic instruments. Due to limited development since its introduction, Manager lacks compatibility with some of the latest microscopy equipment. To address this unmet need, we have developed a novel software-based automation program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent plugin for Manager. Without extra hardware, AMFIP enables the functional synchronization of Manager, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for many hardware and software. Users can customize AMFIP according to their own specific experimental requirements and hardware environments. To verify AMFIPs performance, we applied it to elucidate the relationship between cell spreading and spatial-temporal cellular expression of Yes-associated protein (YAP), a mechanosensitive protein that shuttles between cytoplasm and nucleus upon mechanical stimulation, in an epithelial cell line. We found that the ratio of YAP expression in nucleus and cytoplasm decreases as the spreading area of cells increases, suggesting that the accumulation of YAP in the nucleus decreases throughout the cell spreading processes. In summary, AMFIP provides a new open-source and charge-free solution to integrate multiple hardware and software to satisfy the need of automatic imaging operations in the scientific community.

We have demonstrated 30-Hz three-photon imaging using a single 24-MHz mode-locked Cr:forsterite oscillator with a center wavelength at 1260 nm. By managing the dispersion distribution in the resonator using double-chirped mirrors, we have produced 32-fs pulses with 22-nJ pulse energy. Using the oscillator as a driving source, we have realized multi-color three-photon images using a GFP-labeled Drosophila brain and an AF647-labeled mouse brain. To demonstrate the capability of deep-tissue imaging, we have obtained a 10-times higher SBR from the three-photon images than the two-photon results at different depths in a GFP-labeled Drosophila brain dissection. Furthermore, we have shown the impact of excitation pulse width on three-photon deep-tissue imaging. Our results indicate the superiority of using shorter pulses for deeper-tissue imaging, especially in the Drosophila brain. In addition, we have recorded the three-photon calcium imaging in vivo from the Drosophila mushroom body in response to external electric shocks. We believe our demonstration provides a robust approach for high-speed three-photon microscopy applications, especially for intravital investigations in the Drosophila brain.

Molecular fluorescence microscopy is a leading approach to super-resolution and nanoscale imaging in life and material sciences. However, super-resolution fluorescence microscopy is often bottlenecked by system-specific calibrations and long acquisitions of sparsely blinking molecules. We present a deep-learning approach that reconstructs super-resolved images directly from a single diffraction-limited camera frame. The model is trained exclusively on synthetic data encompassing a wide range of optical and sample parameters, enabling robust generalization across microscopes and experimental conditions. Applied to dense terrylene samples with 150 ms acquisition time, our method significantly reduces reconstruction error compared to Richardson-Lucy deconvolution, ThunderSTORM multi-emitter fitting, and DECODE based on deep learning. The results confirm the ability to resolve emitters separated by 35 nm at 580 nm wavelength, corresponding to seven-fold resolution improvement beyond the Rayleigh criterion. Furthermore, we demonstrate strong generalization ability of the developed model and its resilience across a broad range of noise levels, numerical apertures, and optical aberrations. By delivering unprecedented details from a single short camera exposure without any prior information and calibration, our approach enables plug-and-play super-resolution imaging of fast, dense, or light-sensitive samples on common wide-field microscopy setups.

AO_SCPLOWBSTRACTC_SCPLOWWe report on an Adaptive Optics (AO) Light-Sheet Fluorescence Microscope compatible with neuroimaging, based on direct wavefront sensing without the requirement of a guide star. We demonstrate fast AO correction, typically within 500ms, of in-depth aberrations of the live adult Drosophila brain, enabling to double the contrast when imaging with structural or calcium sensors. We quantify the gain in terms of image quality on multiply neuronal structures part of the sleep network in the Drosophila brain, at various depths, and discuss the optimization of key parameters driving AO such as the number of corrected modes and the photon budget. We present a first design of a compact AO add-on that is compatible with integration into most of reported Light-Sheet setups and neuroimaging.