# Large-Scale Permanent Slide Imaging and Image Analysis for Diatom Morphometrics
## 2 Materials and Methods  
We implemented a complete workflow for semi-automated light-microscopic imaging, measurement and analysis of diatom valves (Figure 1, the numbering refers to the workflow steps as they are described below). Content to be analysed consists of cleaned frustules mounted on permanent slides (step 1); the according raw material can originate from planktonic or benthic samples, sediment traps, sediment cores or cultures. Data collection and processing consists of two cycles of slide scanning and image analysis via SHERPA: The first imaging cycle (steps 2-4) serves the purpose of locating valves of interest at low magnification. During the second cycle (steps 5-7), only these valves of interest are imaged at high magnification and measured at high precision. This method provides a good trade-off between manual effort, time consumption and data economy. Final analysis of the morphometric features measured by SHERPA is realized via R scripts customized to the particular problem (step 8). **For image acquisition**, we use a Metafer slide scanning system (MetaSystems, Altlussheim, Germany) operating with a ZEISS AxioImager.Z2 using ZEISS objectives Plan-APOCHROMAT 10x/0.45, Plan-NEOFLUAR 20x/0.5 and Plan-APOCHROMAT 63x/1.4 with oil immersion. Images are obtained with a CoolCube 1m monochrome camera (MetaSystems). In combination with the system's three-colour (red, green, blue) LED illumination, colour images can be recorded by taking three images of each position and combining them in silico. Since permanent slides prepared of oxidized diatom frustules contain no relevant colour information, we solely use blue LED illumination in all imaging procedures to achieve the highest possible optical resolution. Regarding diatom analysis, the Metafer slide scanning system is capable of two main modes of operation. In area scan mode, a user defined rectangular or circular region on a slide is imaged in overlapping fields-of-view; these images can be combined (stitched) into large panoramic overview images (virtual slides) using the VSlide software (version 1.1.101, MetaSystems). In position list mode, a list of user-defined positions, spread arbitrarily over the slide, can be imaged. The vertical position of objects can be determined by automatic creation of a focus map, i.e., based on interpolation between reference points, or by autofocusing at each position captured, or using a combination of both Each object/field-of-view can optionally be photographed at multiple focus depths, and the resulting images can, again optionally, be combined (stacked) to extended focus depth images. All parameters regarding the operation of the slide scanner are recorded in so-called classifiers. These include settings for illumination (intensity, colour) and imaging (exposition, number and distances of focal planes to image) as well as parameters related to stacking focal plane images. Further  
Figure 1: Schematic overview of our imaging/image analysis workflow, composed of the following steps: (**1**) Sample preparation, (**2**) low-resolution scan for locating valves of interest, (**3**) stitching of the virtual slide, (**4**) selecting objects of interest for high-resolution scan, (**5**) setting up the position list for high-resolution scan, (**6**) high-resolution scan of selected objects, (**7**) high-precision measurement of morphometric features, and (**8**) data export and post-processing. settings are related to image post-processing and object detection, the latter of which is unfortunately too simplistic to be useful in diatom analyses; these are not applied in the workflows presented in this contribution. The actual classifier settings for each run are saved alongside the images produced, enabling the reproduction of an imaging run or the transparent application of a standardized set of settings to multiple slides. The only important component of an image acquisition workflow that is not explicitly documented in the classifier archive files is the autofocus settings.