Pixel reassignment in image scanning microscopy: a re-evaluation

Abstract

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.

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

“…Notably, this simulation is closer to the APR-ISM than to the PR-ISM, since it automatically considers the wavelength Stokes shifts, and it does not impose a common pixel reassignment factor for the different detector elements. As expected, since no distortions or aberrations are included in the simulations, our approach generates shift-vectors similar to the (theoretical) static values [41] for elements close (≤ 1 AU) to the central reference [14].…”

“…Since the number of elements of our SPAD array is limited, an optimal magnification M of the system needs to be found, as it is desirable to maximize the photon collection efficiency, but in such a way that out-of-focus background rejection and ISM reassignment performance are not compromised [14]. With this in mind, we calculated the PSF for both 2PE-ISM and 2PE as a function of the effective SPAD array size (in Airy unit of the focused fluorescence light).…”

“…In computational ISM, the single-point detector of a regular point-scanning CLSM is substituted with a detector array, which collects a small image at each sampling position. Because the detector array has a field-of-view of 1-1.5 AU, no separate pinhole is required to reject out-of-focus light [14], and thus the important optical-sectioning ability of CLSM is maintained. The ISM result is obtained computationally post-acquisition, by pixel reassignment or deconvolution.…”

Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application

“…Yeh et al combined SHGM with structured illumination based on point-scanning, which improves the resolution by factors of~1.4 in the lateral and 1.56 in the axial directions, respectively [74]. Sheppard proposed pixel reassignment, in which the point detector of the traditional confocal microscopy is replaced by a multi-element array detector [79]. Combining with deblurring operation, this method showed a spatial resolution enhancement of about 1.87 compared with conventional SHGM [80].…”

Second harmonic generation microscopy using pixel reassignment

“…At the same time, Sheppard proposed that the spatial resolution of scanning confocal microscopy could be enhanced effectively by replacing the point detector of a conventional confocal microscope by an array detector consisting of many elements (Sheppard, 1988). And the raw data postprocessing is based on pixel reassignment (Roth et al ., 2013; Sheppard et al ., 2013; Sheppard et al ., 2020). This elegant method was realised experimentally by image scanning microscopy (ISM) (Müller & Enderlein, 2010).…”