Prof. Colin Sheppard; School of Optometry and Vision Science, UNSW Sydney, NSW2052, Australia

Leuchs-Russell-Auditorium, A.1.500, Staudtstr. 2

Location details

Abstract:
Image scanning microscopy (ISM) is becoming the preferred embodiment for performing confocal microscopy. There are now numerous commercial systems using this approach. In this implementation, the confocal pinhole is replaced by a detector array, in which each pixel behaves as a confocal microscope with a small pinhole, and then these signals are combined, in the simplest implementation, by the process of pixel reassignment and summation [1–3]. The result is a greatly increased signal strength, combined with an improved transverse spatial resolution compared with a confocal microscope with a finite size of pinhole. In fact, spatial resolution is usually better even than in a confocal microscope with a vanishingly small pinhole. Optical sectioning for a given size of detector array is equal to that for a confocal microscope with a pinhole the same size as the detector array [4]. Usually ISM is performed in a fluoresecence mode, f-ISM, when the improved signal strength is a very important property. The theory of image formation in f-ISM has been described in several papers [1-4]. It should be noted that the improvement in resolution compared even with a confocal microscope with a vanishingly small pinhole is not predicted by a simple Gaussian beam theory, but is a result of pupils with hard-edged apertures [1]. 
A recent paper demonstrated CARS-ISM [5]. This paper used an interferometric technique to coherently sum, after pixel reassignment, images from elements of a detector array. The same group had earlier reported coherent summation of second harmonic generation (SHG) images (SHG-ISM) [6]. SHG-ISM using summation of intensity images has also been presented [7-9]. Some papers have described ISM implemented in a reflectance mode, r-ISM [10,11]. By reflectance we mean non-fluorescence, and including both reflection and scattering interactions. In fact r-ISM was proposed and analyzed in the first paper on ISM [1].
It was explained in Ref.[1] that there are two different ways to perform pixel reassignment in a non-fluorescence microscope of reflection or transmission geometry. Each individual detector pixel acts as a confocal microscope with a small pinhole (called a true confocal microscope), so that it results in purely coherent imaging. Then after pixel reassignment, the signals can be summed either as (a) complex amplitudes (coh-r-ISM), recorded interferometrically [12], or (b) as intensities (pc-r-ISM). Many of the recent implementations for reflectance or SHG have summed intensities. Some have summed complex amplitudes, but it has been suggested that summing intensities is nonlinear and therefore not valid. Our view is that summing intensities results in an overall partially coherent image (hence the preface pc-), as occurs in a conventional bright field microscope, which is a valid approach, although admittedly usually the results are not as good as when summing complex amplitudes. Partially coherent image formation in pc-r-ISM is analyzed, and compared with coh-r-ISM.

References
1. Sheppard, C.J.R. Super-resolution in confocal imaging. Optik 80, 53–54 (1988).
2. Müller, C.B.; Enderlein, J. Image scanning microscopy. Phys. Rev. Lett. 104, 198101 (2010).
3. Sheppard, C.J.R.; Mehta, S.B.; Heintzmann, R. Superresolution by image scanning microscopy using pixel reassignment. Optics Letters 38, 2889–2892 (2013).
4. Sheppard, C.J.R.; Castello, M.; Tortarolo, G.; Zunino, A.; Slenders, E.; Bianchini, P.; Vicidomini, G.; Diaspro, A. Signal strength and integrated intensity in confocal and image scanning microscopy. J. Opt. Soc. Am. A, 40, 138–148 (2023).
5. Zhitnitsky, A.; Bitton, O.; Benjamin, E.; Oron, D. Super-resolved coherent anti-Stokes Raman scattering microscopy by coherent image scanning, Nature Communications 15, 10073 (2024).
6. Raanan, D.; Song, M.S.; Tisdale, W.A.; Oron, D. Super-resolved second harmonic generation imaging by coherent image scanning microscopy. Applied Physics Letters 120, 071111 (2022).
7. Wang, W.; Wu, B.; Zhang, B.; Zhang, Z.; Li, X.; Zhen, S.; Fan, Z.; Tan, J. Second harmonic generation microscopy using pixel reassignment. Journal of Microscopy 281, 97–105 (2020).
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9. Zhang, C.; Lin, F.; Zhang, Y.; Yang, H.; Lin, D.; He, J.; Liao, C.; Weng, X.; Liu, L.; Wang, Y.; et al. Super-resolution secondharmonic generation imaging with multifocal structured illumination microscopy. Nano Letters 23, 7975–7982 (2023).
10. DuBose, T.B.; LaRocca, F.; Farsiu, S.; Izatt, J.A. Super-resolution retinal imaging using optically reassigned scanning laser ophthalmoscopy. Nature Photonics 13, 257–263 (2019).
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12. Hamilton, D.; Sheppard, C. A confocal interference m2ic7r0oscope. Optica Acta 29, 1573–1577 (1982).