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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.

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Fluorescence microscopy and derived techniques are continuously looking for photodetectors able to guarantee increased sensitivity, high spatial and temporal resolution and ease of integration into modern microscopy architectures. Recent advances in single-photon avalanche diodes (SPADs) fabricated with industry-standard microelectronic processes allow the development of new detection systems tailored to address the requirements of advanced imaging techniques (such as image-scanning microscopy). To this aim, we present the complete design and characterization of two bidimensional SPAD arrays composed of 25 fully independent and asynchronously-operated pixels, both having fill-factor of about 50% and specifically designed for being integrated into existing laser scanning microscopes. We used two different microelectronics technologies to fabricate our detectors: the first technology exhibiting very low noise (roughly 200 dark counts per second at room temperature), and the second one showing enhanced detection efficiency (more than 60% at a wavelength of 500 nm). Starting from the silicon-level device structures and moving towards the in-pixel and readout electronics description, we present performance assessments and comparisons between the two detectors. Images of a biological sample acquired after their integration into our custom imagescanning microscope finally demonstrate their exquisite on-field performance in terms of spatial resolution and contrast enhancement. We envisage that this work can trigger the development of a new class of SPAD-based detector arrays able to substitute the typical singleelement sensor used in fluorescence laser scanning microscopy.

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 be made of FRC analysis -in this article we propose novel 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, with no need for prior knowledge of the instrument or sample characteristics. The deconvolution is shown to work exquisitely with both twoand three-dimensional images. We also show how FRC can be used as a powerful metric to observe the progress of iterative deconvolution. While developing the image restoration methods, we also addressed two important limitations in FRC that are of more general interest: how to make FRC work with single images and with three-dimensional images with anisotropic resolution.(1)

The effect of combining the image scanning microscopy (ISM) technique with two-photon fluorescence microscopy is analyzed. The effective spatial frequency cutoff can be doubled, as compared with conventional two-photon fluorescence microscopy, and the magnitude of the optical transfer function near the cutoff of conventional two-photon microscopy is increased by orders of magnitude. For the two-photon case, it is found that the optimum pixel reassignment factor in ISM is not equal to one half, as is often assumed in single-photon fluoresence image scanning microscopy, because the excitation and detection point spread functions are different. The optimum reassignment factor depends on the noise level, and in general the useful cutoff spatial frequency is about 1.8 times that for conventional two-photon microscopy. The effect of altering the reassignment factor in single-photon fluorescence ISM with a Stokes shift is also investigated. Illumination using pupil filters, such as by a Bessel beam, is considered. Using a ring detector array is found to result in good imaging behavior, exhibiting a sharpening of the point spread function by a factor of 1.7 compared with conventional fluorescence. Image formation in ISM can be considered in a four-dimensional spatial frequency space, giving new insight into the imaging properties. This approach is related to phase space representations such as the Wigner distribution function and the ambiguity function. A noniterative algorithm for image restoration is proposed.

We introduce the pSTED-SPLIT microscopy technique, which explores the fluorescence dynamics in the phasor space to achieve sub-diffraction resolution images, with no need of high illumination intensity.

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.