Sami Koho

Scandit

Super-resolution microscopy is routinely used for fixed and thin samples, while its feasibility for imaging live and thick samples is still limited. In the case of stimulated emission depletion (STED) microscopy, the high-intensity illumination required to achieve effective sub-diffraction resolution can introduce photo-damage, thus reducing the co...

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to the introduction of new asynchronous read-out single-photon (SP) array detectors. These detectors give access to an entirely new set of single-photon information typically lost in conventional fluorescence LSM, thus triggering a new imaging/spectroscopy paradigm – t...

In image scanning microscopy, the pinhole of a confocal microscope is replaced by a detector array. The point spread function for each detector element can be interpreted as the probability density function of the signal, the peak giving the most likely origin. This thus allows a form of maximum likelihood restoration, and compensation of aberratio...

In confocal microscopy, the pinhole throws away valuable signal, limiting how small the pinhole can be, and therefore the attainable spatial resolution. In image scanning microscopy (ISM), most of the available light is detected by a detector array, and reassigned in the image to its most likely origin in the sample. The point spread function (PSF)...

The combination of confocal laser-scanning microscopy (CLSM) and fluorescence fluctuation spectroscopy (FFS) is a powerful tool in studying fast, sub-resolution biomolecular processes in living cells. A detector array can further enhance CLSM-based FFS techniques, as it allows the simultaneous acquisition of several samples–essentially images—of th...

Image scanning microscopy is a technique of confocal microscopy in which the confocal pinhole is replaced by a detector array, and the image is reconstructed most straightforwardly by pixel reassignment. In the fluorescence mode, the detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with c...

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 is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recent...

Image scanning microscopy is a technique based on confocal microscopy, in which the confocal pinhole is replaced by a detector array, and the resulting image is reconstructed, usually by the process of pixel reassignment. The detector array collects most of the fluorescent light, so the signal-to-noise ratio is much improved compared with confocal...

Actin and myosins (IIA, IIB, and X) generate mechanical forces in osteoclasts that drive functions such as migration and membrane trafficking. In neurofibromatosis, these processes are perturbed due to a mutation in neurofibromatosis type 1 (NF1) gene. This mutation leads to generation of hyperactive bone-resorbing osteoclasts that increases incide...

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...

Fourier ring correlation (FRC) has recently gained popularity among fluorescence microscopists as a straightforward and objective method to measure the effective image resolution. While the knowledge of the numeric resolution value is helpful in e.g., interpreting imaging results, much more practical use can be made of FRC analysis-in this article...

Two-photon excitation (2PE) microscopy is the imaging modality of choice, when one desires to work with thick biological samples, possibly in-vivo. However, the resolution in two-photon microscopy is poor, below confocal microscopy, and the lack of an optical pinhole becomes apparent in complex samples as reduced quality of optical sectioning. Here...

Image scanning microscopy (ISM) can improve the effective spatial resolution of confocal microscopy to its theoretical limit. However, current implementations are not robust or versatile, and are incompatible with fluorescence lifetime imaging (FLIM). We describe an implementation of ISM based on a single-photon detector array that enables super-re...

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 a microscopy image. While the knowledge of the numeric resolution value is helpful in e.g. interpreting imaging results, much more practical use can b...

Image scanning microscopy (ISM) improves the spatial resolution of conventional confocal laser-scanning microscopy (CLSM), but current implementations reduce versatility and restrict its combination with fluorescence spectroscopy techniques, such as fluorescence lifetime. Here, we describe a natural design of ISM based on a fast single-photon detec...

Precise knowledge of the effective spatial resolution in a stimulated emission depletion (STED) microscopy experiment is essential for reliable interpretation of the imaging results. STED microscopy theoretically provides molecular resolution, but practically different factors limit its resolution. Because these factors are related to both the samp...

Fluorescent nanodiamonds are presented here as dual-contrast imaging probes for intracellular correlation using super-resolution stimulated emission depletion and transmission electron microscopy. This correlative light and electron microscopy probe is demonstrated to be suitable for live cell imaging, fixed cell imaging, and further, super-resolut...

Development of fluorescent and electron dense markers is essential for the implementation of correlative light and electron microscopy, as dual-contrast landmarks are required to match the details in the multimodal images. Here, a novel method for correlative microscopy that utilizes fluorescent nanodiamonds (FNDs) as dual-contrast probes is report...

Diffraction of light limits the spatial resolution of far-field conventional fluorescence to roughly 200 nm. Confocal laser scanning microscopy allows for a resolution enhancement of √2, while super resolution techniques such as stimulated emission depletion (STED) microscopy reach theoretically unlimited resolution. However, the effective resoluti...

Scientific Reports 6 : Article number: 28962 10.1038/srep28962 ; published online: 01 July 2016 ; updated: 19 August 2016 . In this Article, the PyImageQualityRanking software source code has been omitted from the Methods section under subheading ‘Image Quality Ranking Software’.

Automated analysis of microscope images is necessitated by the increased need for high-resolution follow up of events in time. Manually finding the right images to be analyzed, or eliminated from data analysis are common day-to-day problems in microscopy research today, and the constantly growing size of image datasets does not help the matter. We...

Optical microscopy is living its renaissance. The diffraction limit, although still physically true, plays a minor role in the achievable resolution in far-field fluorescence microscopy. Super-resolution techniques enable fluorescence microscopy at nearly molecular resolution. Modern (super-resolution) microscopy methods rely strongly on software....

A method for generating three-dimensional tomograms from multiple three-dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three-dimensional projection at an oblique ang...

Multi-modal correlative microscopy allows combining the strengths of several imaging techniques to provide unique contrast. However it is not always straightforward to setup instruments for such customized experiments, as most microscope manufacturers use their own proprietary software, with limited or no capability to interface with other instrume...

We present a tomographic Stimulated Emission Depletion (STED) microscopy method with three-dimensional superresolution, and its application to osteoclast bone resorption study. In order to improve axial resolution in standard STED system by tomography, two axial projections were obtained by imaging a sample at two different angles; one conventional...

Acidic pH inside cells indicates cellular dysfunctions such as cancer. Therefore, the development of optical pH sensors for measuring and imaging intracellular pH is a demanding challenge. The available pH-sensitive probes are vulnerable to e.g. photobleaching or autofluorescence background in biological materials. Our approach circumvents these pr...

In stimulated emission depletion (STED) microscopy, the lateral resolution is in the range of tens of nanometers depending on the sample and the instrument. The axial resolution, however, is in standard systems limited by diffraction to about 500 nm. We present an approach to three-dimensional diffraction-unlimited resolution by observing the sampl...

Stress fibers are major contractile actin structures in non-muscle cells, where they have an important role in adhesion, morphogenesis and mechanotransduction. Palladin is a multidomain protein, which associates with stress fibers in a variety of cell-types. However, the exact role of palladin in stress fiber assembly and maintenance has remained o...

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 and biomedical imaging. Here we present a novel composite material consisting of a photoluminescent nanodiamond (ND) core with a porous silica...