# Large-Scale Permanent Slide Imaging and Image Analysis for Diatom Morphometrics
## 4 Discussion  
Imaging and image processing methods have been developed and spread rapidly for marine research in the recent years [19; 20; 21; 22]. In contrast to many of these techniques, our approach is conducted ex situ on oxidized material, as has been the common practice in diatom research for nearly 200 years. This enables us to combine traditional preparation methodology with recent advances in imaging and automation, and the application of the latter to existing sets of samples. During the ADIAC project a prototyped automated slide scanning procedure was developed, the main limitation of which consisted of the mechanical parts. Since then, commercial slide scanning systems have become available off the shelf, and advances in camera technology improved the image quality significantly. **Optical resolution:** In the last decade, a variety of slide scanning microscopes have become available, which are capable of producing virtual slides by stitching together overlapping field-of-view images. In many biomedical applications, however, a much lower optical resolution is sufficient, and 40\(\times\) or lower objective magnification is the highest available in many slide scanning systems on the market. For applying to diatoms, however, it is important to select a slide scanning system that can also robustly operate with high-resolution oil immersion objectives. A different high-throughput imaging and measurement method, using unmounted, but still acid-cleaned diatom frustule material, has been proposed previously by [23]. In special settings, like for monospecific cultures, their FlowCam-based method can probably provide a superior throughput compared to our workflow. However, the range of morphometric descriptors seems limited to cell length in the case of the FlowCam. Also, the accuracy of measurements is expected to be higher in the case of images obtained using high-resolution oil immersion objectives from permanent slides, and probably even higher, but definitely more precisely reproducible, than data obtained by time-consuming manual measurement (whether using an ocular micrometer or software-assisted linear measurement between manually selected points in a digital  
Figure 8: Counts of valves of different apical vs. transapical lengths for all MISs. The dashed rectangle depicts the valve size ranges reported in the literature. image). The variation of valve measurements induced by using multiple segmentation methods simultaneously in SHERPA is usually around 1% [5]. **Focus:** Autofocusing at a specified slide position (\(X\), \(Y\)) is realized by capturing images at different focus depths (\(Z\)), determining the value of a focus function at each \(Z\) position and finding the location of its maximum. The focus function used, the \(Z\)-range captured, as well as the steps size, can be controlled by the user. Finding the best focus position is a low-level component of the imaging workflow that is applied at a number of different steps throughout, and autofocus settings for different steps (focus map creation vs. final imaging) and for different objectives need to be optimized separately. An area scan consists of a number of steps (see Figure 2 for a graphical overview): After selection of the target area, the \(Z\) position providing the maximum of a focus function is determined over a grid covering the target area. Using linear or spline interpolation, a focus map is created estimating the height (\(Z\) coordinate) of the optimal focus position across the whole target area. The way the focus grid is created can be controlled by the user in fine detail. Respective parameters regard the resolution of the grid for focus map creation, the autofocus settings applied in this step, as well as the interpolation method. The target area is divided into overlapping fields-of-view that are captured one by one. For each (\(X\), \(Y\)) position, the \(Z\) position can either be determined from the interpolated focus map, or from a renewed autofocusing around that \(Z\) position (fine focus).