Universal inverse modeling of point spread functions for SMLM localization and microscope characterization.

The point spread function (PSF) of a microscope describes the image of a point emitter. Knowing the accurate PSF model is essential for various imaging tasks, including single-molecule localization, aberration correction and deconvolution. Here we present universal inverse modeling of point spread functions (uiPSF), a toolbox to infer accurate PSF models from microscopy data, using either image stacks of fluorescent beads or directly images of blinking fluorophores, the raw data in single-molecule localization microscopy (SMLM). Our modular framework is applicable to a variety of microscope modalities and the PSF model incorporates system- or sample-specific characteristics, for example, the bead size, field- and depth- dependent aberrations, and transformations among channels. We demonstrate its application in single or multiple channels or large field-of-view SMLM systems, 4Pi-SMLM, and lattice light-sheet microscopes using either bead data or single-molecule blinking data.

Selective-plane-activation structured illumination microscopy.

The background light from out-of-focus planes hinders resolution enhancement in structured illumination microscopy when observing volumetric samples. Here we used selective plane illumination and reversibly photoswitchable fluorescent proteins to realize structured illumination within the focal plane and eliminate the out-of-focus background. Theoretical investigation of the imaging properties and experimental demonstrations show that selective plane activation is beneficial for imaging dense microstructures in cells and cell spheroids.

Experimental validation of numerical point spread function calculation including aberration estimation.

Image reconstruction in fluorescence microscopy is highly sensitive to the accuracy of the impulse response, defined as the point spread function (PSF), of the optical system under which the image to reconstruct was acquired. In our previous work, we developed a MATLAB toolbox for accurately calculating realistic vector Fourier-based PSF accounting for any type of aberrations [arXiv, arXiv:2301.13515 (2023)10.48550/arXiv.2301.13515]. In this work, we present a fundamental experimental validation of these numerical methods. The simulated results are found to fit experimental data under different image acquisition conditions at an accuracy higher than 0.97 in normalized cross-correlation. These methods enable a relative contrast of up to 95%.

Spinal cord synaptic plasticity by GlyRß release from receptor fields and syndapin I-dependent uptake.

Glycine receptor-mediated inhibitory neurotransmission is key for spinal cord function. Recent observations suggested that by largely elusive mechanisms also glycinergic synapses display synaptic plasticity. We imaged receptor fields at ultra-high resolution at freeze-fractured membranes, tracked surface and internalized glycine receptors (GlyR) and studied differential regulations of GlyRß interactions with the scaffold protein gephyrin and the F-BAR domain protein syndapin I and thereby reveal key principles of this process. S403 phosphorylation of GlyRß, known to be triggered by synaptic signaling, caused a decoupling from gephyrin scaffolds but simultaneously promoted association of syndapin I with GlyRß. In line, kainate-treatments used to trigger rearrangements of glycine receptors in murine syndapin I KO spinal cords (mixed sex) showed even more severe receptor field fragmentation than already observed in untreated syndapin I KO spinal cords. Syndapin I KO furthermore resulted in more dispersed receptors and increased receptor mobility also pointing out an important contribution of syndapin I in the organization of GlyRß fields. Strikingly, syndapin I KO also led to a complete disruption of kainate-induced GlyRß internalization. Accompanying quantitative ultra-high resolution studies in dissociated spinal cord neurons strongly suggested that the observed defects in GlyR internalization observed in syndapin I KO spinal cords are directly caused by syndapin I deficiency within murine spinal cord neurons. Together our results unveiled important mechanisms organizing and altering glycine receptor fields during both steady-state and particularly upon kainate-induced synaptic rearrangement - principles organizing and fine-tuning synaptic efficacy and plasticity of glycinergic synapses in the spinal cord.SIGNIFICANCE STATEMENTInitial observations suggested that also glycinergic synapses - key for spinal cord and brain stem functions - may display some form of synaptic plasticity. Imaging receptor fields at ultra-high resolution at freeze-fractured membranes, tracking surface and internalized glycine receptors (GlyR) and studying regulations of GlyRß interactions we here reveal key principles of these kainate-inducible adaptations. A switch from gephyrin-mediated receptor scaffolding to syndapin I-mediated GlyRß scaffolding and internalization allows for modulating synaptic receptor availability. In line, kainate-induced GlyRß internalization was completely disrupted and GlyRß receptor fields were distorted upon syndapin I KO. These results unveiled important mechanisms during both steady-state and kainate-induced alterations of synaptic GlyR fields - principles underlying synaptic efficacy and plasticity of synapses in the spinal cord.

PtyLab.m/py/jl: a cross-platform, open-source inverse modeling toolbox for conventional and Fourier ptychography.

Conventional (CP) and Fourier (FP) ptychography have emerged as versatile quantitative phase imaging techniques. While the main application cases for each technique are different, namely lens-less short wavelength imaging for CP and lens-based visible light imaging for FP, both methods share a common algorithmic ground. CP and FP have in part independently evolved to include experimentally robust forward models and inversion techniques. This separation has resulted in a plethora of algorithmic extensions, some of which have not crossed the boundary from one modality to the other. Here, we present an open source, cross-platform software, called PtyLab, enabling both CP and FP data analysis in a unified framework. With this framework, we aim to facilitate and accelerate cross-pollination between the two techniques. Moreover, the availability in Matlab, Python, and Julia will set a low barrier to enter each field.

Hybrid 2D Correlation-Based Loss Function for the Correction of Systematic Errors.

We present the derivation of a new kind of loss function from the symmetry rules of synchronous and asynchronous two-dimensional correlation maps. This loss function, which takes into account correlations that are based on causal relations among the members of a series of spectra, can be employed to solve non-linear inverse problems that are plagued by systematic multiplicative errors. This possibility results from the correlation-based loss function being practically insensitive to such systematic errors, which often arise in spectroscopy because sample spectra are usually ratioed against reference spectra. Using dispersion analysis, a sophisticated method of band fitting, of the spectra of poly(methyl methacrylate) films deposited on gold, we demonstrate the applicability and validity of the new loss function. If gold is used as a substrate, experimental spectra are often unphysical, that is, they display reflectance values larger than unity. In such cases, our correlation-based loss function not only helps to achieve accurate fits but also provides corrections to obtain physically meaningful spectra, which leads to results that are superior to conventional correction methods. The validity of the results is checked and proved with help of the results of dispersion analysis of spectra of films of poly(methyl methacrylate) on calcium fluoride (CaF2) and silicon (Si), which do not suffer from the systematic errors.

Light-sheet microscopy of cleared tissues with isotropic, subcellular resolution.

We present cleared-tissue axially swept light-sheet microscopy (ctASLM), which enables isotropic, subcellular resolution imaging with high optical sectioning capability and a large field of view over a broad range of immersion media. ctASLM can image live, expanded, and both aqueous and non-aqueous chemically cleared tissue preparations. Depending on the optical configuration, ctASLM provides up to 260 nm of axial resolution, a three to tenfold improvement over confocal and other reported cleared-tissue light-sheet microscopes. We imaged millimeter-scale cleared tissues with subcellular three-dimensional resolution, which enabled automated detection of multicellular tissue architectures, individual cells, synaptic spines and rare cell-cell interactions.

Saturated-excitation image scanning microscopy.

Image scanning microscopy (ISM) overcomes the trade-off between spatial resolution and signal volume in confocal microscopy by rearranging the signal distribution on a two-dimensional detector array to achieve a spatial resolution close to the theoretical limit achievable by infinitesimal pinhole detection without sacrificing the detected signal intensity. In this paper, we improved the spatial resolution of ISM in three dimensions by exploiting saturated excitation (SAX) of fluorescence. We theoretically investigated the imaging properties of ISM, when the fluorescence signals are nonlinearly induced by SAX, and show combined SAX-ISM fluorescence imaging to demonstrate the improvement of the spatial resolution in three dimensions. In addition, we confirmed that the SNR of SAX-ISM imaging of fluorescent beads and biological samples, which is one of the challenges in conventional SAX microscopy, was improved.

Quality control of image sensors using gaseous tritium light sources.

We propose a practical method for radiometrically calibrating cameras using widely available gaseous tritium light sources (betalights). Along with the gain (conversion factor) and read noise level, the predictable photon flux of the source allows us to gauge the quantum efficiency. The design is easily reproducible with a 3D printer (three-dimensional printer) and three inexpensive parts. Suitable for common image sensors, we believe that the method has the potential to be a useful tool in microscopy facilities and optical laboratories alike. This article is part of the theme issue 'Super-resolution structured illumination microscopy (part 2)'.

Super-resolution microscopy: a brief history and new avenues.

Super-resolution microscopy (SRM) is a fast-developing field that encompasses fluorescence imaging techniques with the capability to resolve objects below the classical diffraction limit of optical resolution. Acknowledged with the Nobel prize in 2014, numerous SRM methods have meanwhile evolved and are being widely applied in biomedical research, all with specific strengths and shortcomings. While some techniques are capable of nanometre-scale molecular resolution, others are geared towards volumetric three-dimensional multi-colour or fast live-cell imaging. In this editorial review, we pick on the latest trends in the field. We start with a brief historical overview of both conceptual and commercial developments. Next, we highlight important parameters for imaging successfully with a particular super-resolution modality. Finally, we discuss the importance of reproducibility and quality control and the significance of open-source tools in microscopy. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Polarized illumination coded structured illumination microscopy (picoSIM): experimental results.

The need for acquiring at least three images to reconstruct an optical section of a sample limits the acquisition rate in structured illumination microscopy (SIM) for optical sectioning. In polarized illumination coded structured illumination microscopy (picoSIM) the three individual light patterns are encoded in a single polarized illumination light distribution, enabling the acquisition of the complete SIM data in a single exposure. Here, we describe our experimental set-up and show experimental results acquired with sequential and single-shot picoSIM. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Structured illumination ophthalmoscope: super-resolution microscopy on the living human eye.

In this paper, we present the prototype of an ophthalmoscope that uses structured illumination microscopy (SIM) to enable super-resolved imaging of the human retina, and give first insights into clinical application possibilities. The SIM technique was applied to build a prototype that uses the lens of the human eye as an objective to 'super-resolve' the retina of a living human. In our multidisciplinary collaboration, we have adapted this well-established technique in ophthalmology and successfully imaged a human retina using significantly lower light intensity than a state-of-the-art ophthalmoscope. Here, we focus on the technical implementation and highlight future perspectives of this method. A more application-oriented note for physicians on the diagnostic and disease-preventive value of this method, as well as the medical results of the clinical study carried out, will be published in a report addressed to an appropriate specialist audience. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

UCsim2: two-dimensionally structured illumination microscopy using UC2.

State-of-the-art microscopy techniques enable the imaging of sub-diffraction barrier biological structures at the price of high costs or a lack of transparency. We try to reduce some of these barriers by presenting a super-resolution upgrade to our recently presented open-source optical toolbox UC2. Our new injection moulded parts allow larger builds with higher precision. The 4× lower manufacturing tolerance compared to three-dimensional printing makes assemblies more reproducible. By adding consumer-grade available open-source hardware such as digital mirror devices and laser projectors, we demonstrate a compact three-dimensional multimodal setup that combines image scanning microscopy and structured illumination microscopy. We demonstrate a gain in resolution and optical sectioning using the two different modes compared to the widefield limit by imaging Alexa Fluor ® 647- and Silicon Rhodamine-stained HeLa cells. We compare different objective lenses and by sharing the designs and manuals of our setup, we make super-resolution imaging available to everyone. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 2)'.

Computational image enhancement of multimode fibre-based holographic endo-microscopy: harnessing the muddy modes.

In imaging geometries, which employ wavefront-shaping to control the light transport through a multi-mode optical fibre (MMF), this terminal hair-thin optical component acts as a minimally invasive objective lens, enabling high resolution laser-scanning fluorescence microscopy inside living tissues at depths hardly accessible by any other light-based technique. Even in the most advanced systems, the diffraction-limited foci scanning the object across the focal plane are contaminated by a stray optical signal carrying typically few tens of % of the total optical power. The stray illumination takes the shape of a randomised but reproducible speckle, and is unique for each position of the focus. We experimentally demonstrate that the performance of imaging a fluorescent object can be significantly improved, when resulting images are computationally post-processed, utilising records of intensities of all speckle-contaminated foci used in the imaging procedure. We present two algorithms based on a regularised iterative inversion and regularised direct pseudo-inversion respectively which lead to enhancement of the image contrast and resolution.

Silicon-rhodamine isothiocyanate for fluorescent labelling.

An efficient synthesis for silicon-rhodamines was developed, enabling the preparation and evaluation of silicon-rhodamine isothiocyanate (SITC) as a novel tool for facile fluorescent labeling. Ease of use in conjugation to amino groups, high stability and excellent photophysical properties are demonstrated. SITC-actin was found to be neutral to F-actin polymerization induction and well suited for high resolution fluorescence microscopy.

Answers to fundamental questions in superresolution microscopy.

This article presents answers to the questions on superresolution and structured illumination microscopy (SIM) as raised in the editorial of this collection of articles (https://doi.org/10.1098/rsta.2020.0143). These answers are based on my personal views on superresolution in light microscopy, supported by reasoning. Discussed are the definition of superresolution, Abbe's resolution limit and the classification of superresolution methods into nonlinear-, prior knowledge- and near-field-based superresolution. A further focus is put on the capabilities and technical aspects of present and future SIM methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Super-resolution structured illumination microscopy: past, present and future.

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods. This article is part of the Theo Murphy meeting issue 'Super-resolution structured illumination microscopy (part 1)'.

Impact of deuteration on the ultrafast nonlinear optical response of toluene and nitrobenzene.

Nonlinear pulse propagation inside highly nonlinear media requires accurate knowledge on the temporal response function of the materials used particular in the case of liquids. Here we study the impact of deuteration on the ultrafast dynamics of toluene and nitrobenzene via all optical Kerr gating, showing substantially different electronic and molecular contributions, which was quantified by fitting a multichannel decay model to the data points. Specifically we found that deuteration imposes the time-integrated nonlinearities to reduce particular for toluene which could be caused by both reduced electronic hyperpolarizabilities as well as weaker intermolecular interactions. The results achieved reveal that deuterated organic solvents represent promising materials for infrared photonics since they offer extended infrared transmission compared to their non-deuterated counterparts while maintained strong nonlinear responses.

Super-Resolution Structured Illumination Microscopy.

Super-resolved structured illumination microscopy (SR-SIM) is among the most rapidly growing fluorescence microscopy techniques that can surpass the optical diffraction limit. The strength of SR-SIM is that it can be readily applied to samples prepared for conventional fluorescence microscopy, requiring no sophisticated sample preparation protocols. As an extension of wide-field fluorescence microscopy, it is inherently capable of multicolor imaging and optical sectioning and, with sufficiently fast implementations, permits live cell imaging. Image reconstruction, however, currently relies on sophisticated computational procedures, susceptible to reconstruction artifacts, requiring trained users to recognize and avoid them. Here, we review the latest developments in SR-SIM research. Starting from a historical overview of the development of SR-SIM, we review how this method can be implemented in various experimental schemes, we provide an overview of the important parameters involved in successful image reconstruction, we summarize recent biological applications, and we provide a brief outlook of the directions in which we believe SR-SIM is headed in the future.