###### Abstract
The approach utilized a rather straight forward experimental setup consisting of an Argon laser focused into a cuvette with a combination of macromolecular DNA with ethidium bromide (EtBr) solution and the emission registered by a photomultiplier tube. The key to the technology was the electronic apparatus enabling correlation analysis of the recorded fluorescence called hardware correlation. Since its inception the experimental set-ups enabling FCS have evolved regarding both optical systems and hardware electronics. The technological advancement in the field of optical microscopy accelerated the momentum of FCS research. The combination of FCS with confocal laser scanning microscopy demonstrated by [PERSON] in 1993 was a breakthrough [14]. Because the detection volume in confocal microscopy is restricted to a diffraction limited point in the sample, the recorded number of diffusing molecules could be substantially reduced, which is an advantage for the analysis. FCS combined with confocal laser scanning microscopy has demonstrated being particularly promising for physicochemical studies concerning, e.g., photophysical characterization [15], diffusion [15; 16; 17] and chemical kinetics [12; 18; 19]. The application of FCS for biological studies has been introduced utilizing both laser scanning confocal optics [9; 17], as well as combined with MPM [8; 9; 16; 20]. The combination with MPM is particularly advantageous in the context of biological samples since it operates in NIR. In fact, MPM combined with FCS has been introduced as an \"ideal tool\" [21] for exploring the dynamics of fluorescent molecules inside cells and other biological samples [7; 16; 22; 23; 24]. However, despite previous reports promoting the applicability of the method, its implementation is far from straightforward. A majority of the previous reports tend to focus on the applications of MPM-FCS rather than systematically describing the experimental conditions required to perform the experiments. This lack of transfer of know-how restricts the feasibility to a few research groups and hampers translation and broader exploration of the technology. For example, knowledge of experimental preconditions and validation experiments are required. The aim of this paper is thus to bridge this gap. One of the major challenges in the biomedical context is combining imaging and FCS in biological complex matter. In general, selection of objective lens with a suitable NA is important because NA defines the ability to collect the photons and defines the resolution of the microscope [25; 26]. When MPM is adopted for imaging, objective lenses with larger field-of-view and long working distances are normally preferred [27; 28; 2]. Generally long working distance objective lenses 20-40\(\times\) are utilized having NA in the range 0.7-1.0. However, in order to optimize data acquisition and signal-to-noise ratio in FCS most reports employ high resolution objective lenses (>40\(\times\)) with NA typically above 1.0 which significantly restricts both field of view as well as working distance [7; 14; 16; 21]. In order to facilitate the combination of imaging and FCS it would be desirable to use the same objective lens for both measurements. To date, only a limited number of studies have been investigating the relationship between focal volume optics and FCS data [16]. However, to the best of our knowledge there are no studies systematically comparing how FCS performance will be affected by the choice of objective lens under the same conditions using the same experimental system, which is a central part of the present investigation. Another important factor is the concentration range in which the FCS method can operate. It is known that FCS is restricted to low concentrations in the range of nanomolar [7; 16].