Abstract Fourier ring correlation (FRC) has recently gained popularity among fluorescence microscopists as a straightforward and objective method to measure the effective image resolution. While knowledge of numeric resolution value is helpful in e.g., interpreting imaging results, much more practical use can be made FRC analysis—in this article we propose blind restoration methods enabled by it. We apply perform de-noising frequency domain filtering. novel linear non-linear deconvolution...

Ratiometric referenced luminescent pH nanosensors based on an upconversion resonance energy transfer (UC-RET) process enable imaging applications with NIR excitation.

Recent advances within materials science and its interdisciplinary applications in biomedicine have emphasized the potential of using a single multifunctional composite material for concurrent drug delivery biomedical imaging. Here we present novel consisting photoluminescent nanodiamond (ND) core with porous silica (SiO2) shell. This probe serves as an alternative nanomaterial to address existing problems subsequent tracing particles. Whereas unique optical properties ND allows long-term...

Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation imaging results.STED theoretically provides molecular resolution, but practically different factors limit its resolution.Because these are related to both sample and system, estimation not straightforward.Here we show method based on Fourier ring correlation (FRC), which estimates an absolute value directly from any STED and, more...

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition several samples-essentially images-of CLSM detection volume. However, arrays that have previously been proposed for this purpose require tedious data corrections preclude with single-photon...

Abstract To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage specimens. Moreover, out-of-focus background may degrade signal stemming from focal plane. Here, we propose a new method mitigate these limitations without drawbacks. First, enhance STED microscope with detector array, enabling...

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution poor, below confocal microscopy. Here, we propose a straightforward implementation 2PE image (2PE-ISM) that, by leveraging our recently introduced single-photon avalanche diode (SPAD) array detector and novel blind reconstruction method, shown enhance effective resolution, as well overall quality With adaptive pixel...

Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up events in time. Manually finding right to be analyzed, or eliminated from data are common day-to-day problems microscopy research today, and constantly growing size image datasets does not help matter. We propose a simple method software tool sorting within dataset, according their relative quality. demonstrate applicability our good quality STED sample preparation optimization...

Image scanning microscopy is a technique based on confocal microscopy, in which the pinhole replaced by detector array, and resulting image reconstructed, usually process of pixel reassignment. The array collects most fluorescent light, so signal-to-noise ratio much improved compared with small pinhole, while resolution conventional (wide-field) microscopy. In previous studies, it has been assumed that pixels should be reassigned constant factor, to point midway between illumination...

Abstract Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access an entirely set of information. Together with the blooming SP LSM techniques and development tailored there growing need for (i) DAQ systems capable handling high-throughput high-resolution photon information generated by these (ii) incorporating protocols in existing fluorescence LSMs. We developed open-source, low-cost, multi-channel...

Stress fibers are major contractile actin structures in non-muscle cells, where they have an important role adhesion, morphogenesis and mechanotransduction. Palladin is a multidomain protein, which associates with stress variety of cell-types. However, the exact palladin fiber assembly maintenance has remained obscure, whether it functions as filament cross-linker or scaffolding protein was unknown. We demonstrate that specifically required for non-contractile dorsal fibers, consequently...

Abstract Development of fluorescent and electron dense markers is essential for the implementation correlative light microscopy, as dual‐contrast landmarks are required to match details in multimodal images. Here, a novel method microscopy that utilizes nanodiamonds (FNDs) probes reported. It demonstrated how FNDs can be used labels—and together with automatic image registration tool SuperTomo, precise correlation—in high‐resolution stimulated emission depletion (STED)/confocal transmission...

Image scanning microscopy is a technique of confocal in which the pinhole replaced by detector array, and image reconstructed most straightforwardly pixel reassignment. In fluorescence mode, array collects fluorescent light, so signal-to-noise ratio much improved compared with small pinhole, while resolution conventional microscopy. Here we consider two cases illumination detection point spread functions are dissimilar: Bessel beam multiphoton It has been shown previously that for large...

Summary A method for generating three‐dimensional tomograms from multiple axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed top standard microscopic sample, used to record projection at an oblique angle relation main optical axis. Combining with regular STED image into single view by tomographic reconstruction, shown result tomogram three‐to‐four‐fold improved apparent resolution....

Abstract Fourier ring correlation (FRC) has recently gained some popularity among (super-resolution) fluorescence microscopists as a straightforward and objective method to measure the effective resolution of microscopy image. While knowledge numeric value is helpful in e.g. interpreting imaging results, much more practical use can be made FRC analysis – this article we propose novel blind image restoration methods enabled by it. We apply perform de-noising frequency domain filtering. linear...

Abstract Stimulated emission depletion (STED) microscopy is one of the most influential nanoscopy techniques; by increasing STED beam intensity, it theoretically improves spatial resolution to any desired value. However, higher dose stimulating photons, stronger are photo-bleaching and photo-toxicity effects, which potentially compromise live-cell long-term imaging. For this reason scientific community looking for strategies reduce intensity needed achieve a target resolution. Here, we show...

Abstract Actin and myosins (IIA, IIB, X) generate mechanical forces in osteoclasts that drive functions such as migration membrane trafficking. In neurofibromatosis, these processes are perturbed due to a mutation neurofibromatosis type 1 (NF1) gene. This leads generation of hyperactive bone-resorbing increases incidence skeletal dysplasia e.g. early-onset osteoporosis patients suffering from neurofibromatosis. To study the density function actin clusters mutated cells we introduce new...

In image scanning microscopy, the pinhole of a confocal microscope is replaced by detector array. The point spread function for each element can be interpreted as probability density signal, peak giving most likely origin. This thus allows form maximum likelihood restoration, and compensation aberrations, with similarities to adaptive optics. As an example aberration, we investigate theoretically experimentally illumination vortex doughnut beam. After reassignment summation over array,...

Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability interface other - this makes correlation multi-modal data extremely challenging. We introduce a new software tool simultaneous STimulated Emission Depletion (STED) an Atomic Force...

ABSTRACT Two-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo . However, resolution in two-photon poor, below confocal microscopy, and lack an optical pinhole becomes apparent complex samples as reduced quality sectioning. Here, we propose a straightforward implementation 2PE image scanning (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on new single-photon avalanche...

Image scanning microscopy (ISM) improves the spatial resolution of conventional confocal laser-scanning (CLSM), but current implementations reduce versatility and restrict its combination with fluorescence spectroscopy techniques, such as lifetime. Here, we describe a natural design ISM based on fast single-photon detector array, which allows straightforward upgrade an existing microscope, without compromising any functionalities. In contrast to all-optical implementations, our approach...

We present a tomographic Stimulated Emission Depletion (STED) microscopy method with three-dimensional superresolution, and its application to osteoclast bone resorption study. In order improve axial resolution in standard STED system by tomography, two projections were obtained imaging sample at different angles; one conventionally from below another the side. The second observation was acquired via metal-coated silicon mirror, positioned above region of interest custom-built...