All optical microscopes, including conventional widefield, confocal, and two-photon instruments, are limited in the resolution that can be achieved by a series of fundamental physical factors. In a perfect optical system, the resolution is restricted by the numerical aperture of optical components and by the wavelength of light, both incident (excitation) and detected (emission). The concept of resolution is inseparable from contrast, and it is defined as the minimum separation between two points that results in a certain level of contrast between them. In a typical fluorescence microscope, contrast is determined by the number of photons collected from the specimen, the dynamic range of the signal, optical aberrations of the imaging system, and the number of picture elements (pixels) per unit area in the final image (Minsky in Scanning 10:128–138, 1988; Semwogerere and Eric Weeks, in Confocal microscopy, Emory University, Atlanta, Georgia, 2020). A confocal microscope creates sharp images of a specimen that would otherwise appear blurred when viewed with a conventional microscope. This is achieved by excluding most of the light from the specimen that is not from the microscope’s focal plane. The image has less haze and better contrast than that of a conventional microscope and represents a thin cross-section of the specimen (Wilson and Carlini in J Microsc 141:51–66, 1988; Kenneth et al., Davidson copy right 2004–2009 OLYMUS corporation, Florida State University, USA, 2024; Cox and Sheppard in Microsc Res Tech 63:18–22, 2004; Sheppard and Shotton in Image formation in the confocal laser scanning microscope, Springer, New York, 1997).