## 1 Introduction  
In microscopic imaging, the imaging of a point-like light source does not appear as a point due to a diffraction pattern termed the point spread function (PSF). The PSF describes the analytical ability of the microscopic imaging of a point source and is a significant parameter for evaluating the resolution and quality of microscopic imaging systems. Therefore, an accurate measurement of the PSF is critical for the evaluation of microscopic systems [1; 2]. The accurate measurement of the PSF is especially important when using super-resolution microscopic technologies [3; 4]. For example, stochastic optical reconstruction microscopy and photo-activated localization microscopy both need accurate PSFs to fit each fluorescent point so that the center of each point can be exactly located [5; 6; 7]. Furthermore, structured illumination microscopy needs accurate PSFs to build the correct optical transfer function for image restoration and to obtain super-resolution images [8; 9; 10; 11].  
There are usually two ways to obtain a PSF: numerical calculations and experimental measurements. Numerical calculations require the PSF mode of lens theory because the difficult estimation of the pupil function often results in the theoretical model not reflecting the real imaging system [12; 13]. Therefore, experimental measurements are the preferred method to obtain PSFs for microscopic systems. The most common methods for experimentally obtaining PSFs are the star point detection, knife-edge, and fluorescent microsphere detection methods [13; 14]. The PSF for microscopic imaging is commonly obtained with the fluorescent microsphere detection method.  
The PSF should be measured on a pre-determined object plane that is perpendicular to the optical axis of the microscopic system (Figure 1). The PSF has a correspondence relationship with an object plane that is perpendicular to the optical axis, and a 3D PSF measurement is used to obtain each PSF on its corresponding perpendicular object plane at the focus depth. Realizing this goal becomes more difficult with super-resolution microscopic measurements. A measured object plane that is not perpendicular to the optical axis results in an obtained PSF that is not homogeneous due to a defocusing effect along with the optical axis direction. Moreover, the accuracy of the PSF is significantly reduced when the PSF does not have a corresponding object plane that is perpendicular to the optical axis. This is especially important when the PSF is employed in 3D super-resolution microscopic image restoration. In this case, all the corresponding object planes within focal depth must be confirmed. Two other microscopic elements are essential when obtaining a PSF: the magnification and the paraxial region. These elements may change with object plane variation along the optical axis. In summary, if the measured object plane that is perpendicular to the optical axis is not determined, the meanings of the PSF, magnification, and paraxial region will be lost for super-resolution microscopic measurements. The common methods for obtaining PSFs stated above are not able to determine the object planes that are perpendicular to the optical axis or the longitudinal height positions of each object plane along the optical axis, as measuring these factors requires more than 3D PSF measurements.  
In this paper, a microscope PSF evaluation method for calibrating the object plane that is perpendicular to the optical axis is proposed. The paraxial region, object plane perpendicularity determination, and 3D PSF evaluation methods are presented. The presented methods, together with the integration of dual-spot position detection technology to realize a 3D PSF on the perpendicular object plane, facilitate the magnification and paraxial region evaluation, as well as the confirmation of any microscopic system. The availability of the proposed method is then experimentally verified.