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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 microscopy with a small pinhole, while the resolution is improved compared with conventional (wide-field) microscopy. In previous studies, it has usually been assumed that pixels should be reassigned by a constant factor, to a point midway between the illumination and detection spots. Here it is shown that the peak intensity of the effective point spread function (PSF) can be further increased by 4% by a new choice of the pixel reassignment factor. For an array of two Airy units, the peak of the effective PSF is 1.90 times that of a conventional microscope, and the transverse resolution is 1.53 times better. It is confirmed that image scanning microscopy gives optical sectioning strength identical to that of a confocal microscope with a pinhole equal to the size of the detector array. However, it is shown that image scanning microscopy exhibits axial resolution superior to a confocal microscope with a pinhole the same size as the detector array. For a two-Airy-unit array, the axial resolution is 1.34 times better than in a conventional microscope for the standard reassignment factor, and 1.28 times better for the new reassignment factor. The axial resolution of a confocal microscope with a two-Airy-unit pinhole is only 1.04 times better than conventional microscopy. We also examine the signal-to-noise ratio of a point object in a uniform background (called the detectability), and show that it is 1.6 times higher than in a confocal microscope.

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 confocal microscopy with a small pinhole, while the resolution is improved compared with conventional fluorescence microscopy. Here we consider two cases in which the illumination and detection point spread functions are dissimilar: illumination with a Bessel beam and multiphoton microscopy. It has been shown previously that for Bessel beam illumination in image scanning microscopy with a large array, the imaging performance is degraded. On the other hand, it is also known that the resolution of confocal microscopy is improved by Bessel beam illumination. Here we analyze image scanning microscopy with Bessel beam illumination together with a small array and show that an improvement in transverse resolution (width of the point spread function) by a factor of 1.78 compared with a conventional fluorescence microscope can be obtained. We also examine the behavior of image scanning microscopy in two- or three-photon fluorescence and for two-photon excitation also with Bessel beam illumination. The combination of the optical sectioning effect of image scanning microscopy with multiphoton microscopy reduces background from the sample surface, which can increase penetration depth. For a detector array size of two Airy units, the resolution of two-photon image scanning microscopy is a factor 1.85 better and the peak of the point spread function 2.84 times higher than in nonconfocal two-photon fluorescence. The resolution of three-photon image scanning microscopy is a factor 2.10 better, and the peak of the point spread function is 3.77 times higher than in nonconfocal three-photon fluorescence. The resolution of two-photon image scanning microscopy with Bessel beam illumination is a factor 2.13 better than in standard two-photon fluorescence. Axial resolution and optical sectioning in two-photon or three-photon fluorescence are also improved by using the image scanning modality.

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.

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-resolution FLIM and improves multicolor, live-cell and in-depth imaging, thereby paving the way for a massive transition from confocal microscopy to ISM. A single-photon detector array enables robust and versatile image scanning microscopy (ISM) on any confocal microscope. This implementation makes super-resolution FLIM possible and eases a transition from confocal microscopy to ISM.

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 detector array, which allows straightforward upgrade of an existing confocal microscope, without compromising any of its functionalities. In contrast to all-optical ISM implementations, our approach provides access to the raw scanned images, opening the way to adaptive reconstruction methods, capable of considering different imaging conditions and distortions. We demonstrate its utility in the context of fluorescence lifetime, deep, multicolor and live-cell imaging. This implementation will pave the way for a transparent and massive transition from conventional CLSM to ISM.confocal microscopy | time-resolved spectroscopy | image scanning microscopy | single-photon detector array

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 reported. It is demonstrated how the FNDs can be used as dual-contrast labels-and together with automatic image registration tool SuperTomo, for precise image correlation-in high-resolution stimulated emission depletion (STED)/confocal and transmission electron microscopy (TEM) correlative microscopy experiments. It is shown how FNDs can be employed in experiments with both live and fixed cells as well as simple test samples. The fluorescence imaging can be performed either before TEM imaging or after, as the robust FNDs survive the TEM sample preparation and can be imaged with STED and other fluorescence microscopes directly on the TEM grids.

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 sample at two optical angles. The system is realized by using an atomic force microscope (AFM) chip as a microreflector to deflect the STED beams near the region-of-interest (ROI), thus allowing observations at an angle ∠. Consequently, the superior lateral resolution can be utilized to resolve details in the axial direction of the main optical axis of the microscope. Here, fluorescent nanoparticles 90 nm apart and biological structures 80 nm apart along axial direction were distinguished by utilizing an off-the-shelf, commercial STED microscope, coupled with an AFM and an AFM chip micro-reflector.

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 problems due to near infrared excitation and upconversion photoluminescence. We introduce a nanosensor based on upconversion resonance energy transfer (UC-RET) between an upconverting nanoparticle (UCNP) and a fluorogenic pH-dependent dye pHrodo™ Red that was covalently bound to the aminosilane surface of the nanoparticles. The sensitized fluorescence of the pHrodo™ Red dye increases strongly with decreasing pH. By referencing the pH-dependent emission of pHrodo™ Red with the pH-insensitive upconversion photoluminescence of the UCNP, we developed a pH-sensor which exhibits a dynamic range from pH 7.2 to 2.5. The applicability of the introduced pH nanosensor for pH imaging was demonstrated by imaging the two emission wavelengths of the nanoprobe in living HeLa cells with a confocal fluorescence microscope upon 980 nm excitation. This demonstrates that the presented pH-nanoprobe can be used as an intracellular pH-sensor due to the unique features of UCNPs: excitation with deeply penetrating near-infrared light, high photostability, lack of autofluorescence and biocompatibility due to an aminosilane coating.

Photo-Acoustic Microscopy (PAM) has raised high interest in in-vivo imaging due to its ability to preserve the near-diffraction limited spatial resolution of optical microscopes, whilst extending the penetration depth to the mm-range. Another advantage of PAM is that it is a label-free technique – any substance that absorbs PAM excitation laser light can be viewed. However, not all sample structures desired to be observed absorb sufficiently to provide contrast for imaging. This work describes a novel imaging method that makes it possible to visualize optically transparent samples that lack intrinsic photo-acoustic contrast, without the addition of contrast agents. A thin, strongly light absorbing layer next to sample is used to generate a strong ultrasonic signal. This signal, when recorded from opposite side, contains ultrasonic transmission information of the sample and thus the method can be used to obtain an ultrasound transmission image on any PAM.

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 conventionally from below and another from the side. The second observation was acquired via a metal-coated silicon mirror, positioned above the region of interest by a custom-built micro-positioner. The acquired two sets of 3D stacks were computationally registered and fused, with our own in-house-developed software, to produce a 3D tomogram with three-dimensional super-resolution. With the presented tomographic super-resolution method we optically investigated actin cytoskeleton through thin and smooth bone layer, particularly at ruffled boarders (RB), which are directly associated with active bone resorption in osteoclasts. Tomographic STED microscopy at RB of osteoclast, cultured on thin bone layer, demonstrated axial resolution of approx. 210 nm, revealing fine axial structures of actin cytoskeleton at RB. Further investigation of the cytoskeleton at RB in relation with associated proteins would provide understanding in the protein roles during the bone resorption.

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 (SiO2) shell. This novel multifunctional probe serves as an alternative nanomaterial to address the existing problems with delivery and subsequent tracing of the particles. Whereas the unique optical properties of ND allows for long-term live cell imaging and tracking of cellular processes, mesoporous silica nanoparticles (MSNs) have proven to be efficient drug carriers. The advantages of both ND and MSNs were hereby integrated in the new composite material, ND@MSN. The optical properties provided by the ND core rendered the nanocomposite suitable for microscopy imaging in fluorescence and reflectance mode, as well as super-resolution microscopy as a STED label; whereas the porous silica coating provided efficient intracellular delivery capacity, especially in surface-functionalized form. This study serves as a demonstration how this novel nanomaterial can be exploited for both bioimaging and drug delivery for future theranostic applications.

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 the CLSM detection volume. However, the detector arrays that have previously been proposed for this purpose require tedious data corrections and preclude the combination of FFS with single-photon techniques, such as fluorescence lifetime imaging. Here, we solve these limitations by integrating a novel single-photon-avalanche-diode (SPAD) array detector in a CLSM system. We validate this new implementation on a series of FFS analyses: spot-variation fluorescence correlation spectroscopy, pair-correlation function analysis, and image-derived mean squared displacement analysis. We predict that the unique combination of spatial and temporal information provided by our detector will make the proposed architecture the method of choice for CLSM-based FFS.