# Contrast Enhancement for Topographic Imaging in Confocal Laser Scanning Microscopy
## 1 Introduction  
The patent by [PERSON] in 1961 was the first step in the development of confocal laser scanning microscopy (CLSM) [1]. It was further investigated by [PERSON] et al. giving one of the first theoretical descriptions for example, also by [PERSON] et al. who showed one of the first tissue and cell investigations with CLSM and compared them to conventional systems [2; 3]. Its optical sectioning capability enables a true axial resolution and increases contrast and resolving power, giving the CLSM an advantage over wide-field microscopy [4; 5]. Due to this, CLSM has gained a reputation for medical and biological applications, as well as for material science [6; 7; 8]. Multiple approaches were discovered to improve CLSM, for example with the use of synthetic holography, two-photon absorption or laser induced fluorescence effects [9; 10; 11]. One crucial part of the confocal microscope is the pinhole. It enables optical sectioning, enhances the contrast and thus improves the lateral resolution [4; 12]. Thus the pinhole was analyzed in various publications dealing with the shape [13], size [14; 15] and misalignment in lateral [16; 17] and axial direction [18]. The attributes of the pinhole's behavior, its influence on the point spread function as well as the optical transfer function of the CLSM are well-known and described in detail [15; 18; 19]. Effects of laterally displacing the pinhole were investigated in References [16; 17; 20; 21] describing an increase in Rayleigh resolution, edge effects and dark field microscopy effects for certaindisplacements and systems. The lateral light intensity distribution in the pinhole plane is commercially used in airy scanning [22] or computationally enhanced and exploited in pixel reassignment [23]. Axial displacement of the pinhole was analyzed by [PERSON] et al. [18], showing the change of the axial intensity distribution and describing the point spread function mathematically but not the effect on images acquired by the CLSM. Other publications [20; 21] showed edge effects with defocus of the sample and due to their finite tube systems a defocus of the light at the pinhole plane, respectively. In References [20; 21] the axial misalignment for extended focus imaging and autofocus imaging was tested. The results show an overshoot of intensity at the edges giving suggestions for using this as a localization method. In Reference [7] a piezo moves the objective lens for ensuring that the target is in the focal plane while the resulting defocus in the pinhole plane is not addressed. For improving the optical sectioning, techniques such as two-pinhole confocal fluorescence and differential confocal microscopy (DCM) are used [24; 25]. In the first technique, two different pinhole types are implemented, one conventional and a specifically designed pupil filter. The combination results in a synthetic image with increased sectioning capability. In DCM the sample is defocused in such a way that its axial plane corresponds to a position on the linear slope of the axial intensity distribution. This results in a high sensitivity to height changes and, after a calibration, an increase in measurement accuracy for these [25; 26]. Nevertheless, all those methods need additional components, post processing or more acquisition time. Others defocus the sample and thus decrease the resolution or do not address the influence of pinhole displacement on the images. In this paper, we show an easy to integrate method of contrast enhancement in reflection CLSM for topographic structures with low height differences and thus low contrast. For this purpose, we apply an axial shift of the pinhole or the associated lens. In contrast to other techniques, the resulting images do not suffer from decreased resolution by sample defocus, the resolution of the system is still diffraction limited. There is no need to add additional scanning procedures or post processing as in the previously mentioned approaches.