A robust and versatile platform for image scanning microscopy enabling super-resolution FLIM.

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.

Pixel reassignment in image scanning microscopy with a doughnut beam: example of maximum likelihood restoration.

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 for aberrations, with similarities to adaptive optics. As an example of an aberration, we investigate theoretically and experimentally illumination with a vortex doughnut beam. After reassignment and summation over the detector array, the point spread function is compact, and the resolution and signal level higher than in a conventional microscope.

Image scanning microscopy with multiphoton excitation or Bessel beam illumination.

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.

Pixel reassignment in image scanning microscopy: a re-evaluation.

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.

STED-TEM Correlative Microscopy Leveraging Nanodiamonds as Intracellular Dual-Contrast Markers.

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.

Palladin promotes assembly of non-contractile dorsal stress fibers through VASP recruitment.

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 obscure, and whether it functions as an actin filament crosslinker or scaffolding protein was unknown. We demonstrate that palladin is specifically required for the assembly of non-contractile dorsal stress fibers, and is, consequently, essential for the generation of stress fiber networks and the regulation of cell morphogenesis in osteosarcoma cells migrating in a three-dimensional collagen matrix. Importantly, we reveal that palladin is necessary for the recruitment of vasodilator stimulated phosphoprotein (VASP) to dorsal stress fibers and that it promotes stress fiber assembly through VASP. Both palladin and VASP display similar rapid dynamics at dorsal stress fibers, suggesting that they associate with stress fibers as a complex. Thus, palladin functions as a dynamic scaffolding protein that promotes the assembly of dorsal stress fibers by recruiting VASP to these structures.

Focus image scanning microscopy for sharp and gentle super-resolved microscopy.

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

The BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy.

Fluorescence laser-scanning microscopy (LSM) is experiencing a revolution thanks to new single-photon (SP) array detectors, which give access to an entirely new set of single-photon information. Together with the blooming of new SP LSM techniques and the development of tailored SP array detectors, there is a growing need for (i) DAQ systems capable of handling the high-throughput and high-resolution photon information generated by these detectors, and (ii) incorporating these DAQ protocols in existing fluorescence LSMs. We developed an open-source, low-cost, multi-channel time-tagging module (TTM) based on a field-programmable gate array that can tag in parallel multiple single-photon events, with 30 ps precision, and multiple synchronisation events, with 4 ns precision. We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. We expect that our BrightEyes-TTM will support the microscopy community in spreading SP-LSM in many life science laboratories.

Confocal-based fluorescence fluctuation spectroscopy with a SPAD array detector.

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.

Two-photon image-scanning microscopy with SPAD array and blind image reconstruction.

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 recently introduced single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure ∼1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of ∼2 - and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique.

Fourier ring correlation simplifies image restoration in fluorescence microscopy.

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 we propose blind image restoration methods enabled by it. We apply FRC to perform image de-noising by frequency domain filtering. We propose novel blind linear and non-linear image deconvolution methods that use FRC to estimate the effective point-spread-function, directly from the images. We show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. We also address two important limitations in FRC that may be of more general interest: how to make FRC work with single images (within certain practical limits) and with three-dimensional images with highly anisotropic resolution.

Image Quality Ranking Method for Microscopy.

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 propose a simple method and a software tool for sorting images within a dataset, according to their relative quality. We demonstrate the applicability of our method in finding good quality images in a STED microscope sample preparation optimization image dataset. The results are validated by comparisons to subjective opinion scores, as well as five state-of-the-art blind image quality assessment methods. We also show how our method can be applied to eliminate useless out-of-focus images in a High-Content-Screening experiment. We further evaluate the ability of our image quality ranking method to detect out-of-focus images, by extensive simulations, and by comparing its performance against previously published, well-established microscopy autofocus metrics.

Photon upconversion sensitized nanoprobes for sensing and imaging of pH.

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.

Core-shell designs of photoluminescent nanodiamonds with porous silica coatings for bioimaging and drug delivery II: application.

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.