The images of fluorophores observed with a microscope are blurred by the wave-like properties of light. (a) The image of a single fluorophore (red circle) has a width greater than approximately 250 nm when viewed with visible light, despite the fact that the fluorophore itself is only a few nanometers in size. The image of such a point emitter is called the point-spread function (PSF). The position of the fluorophore in this case can be determined by measuring the center position of the image, which is equivalent to the PSF in this case. (b) When multiple fluorophores are located in close proximity, their images overlap and it becomes difficult to distinguish the individual fluorophores from one another. It is the width of the PSF that limits the ability of the microscope to resolve closely spaced fluorophores. The fluorophore positions cannot be determined accurately in this case.