# A Low-Cost Automated Digital Microscopy Platform for Automatic Identification of Diatoms
## 1 Introduction  
### What Is a Diatom and How Can We See It? Diatoms are microscopic unicellular algae present on Earth since at least 180 millions years ago. They survive in any aquatic ecosystem with enough light--sea, lakes, rivers, even mud--where they live in the body of water as plankton or attached to plants, rocks, or small sand particles. Diatoms are the most diverse protists on the planet (i.e., unicellular organisms with a nucleus) with around 20,000 known species with 90% of them--according to estimates--remaining still unveiled. It is estimated that diatoms are responsible for 20% of total carbon fixation on Earth, being more productive than all the rainforests on the planet. Thus, they are essential to the whole ecosystem equilibrium of our planet [1; 2; 3]. Due to the microscopic size of diatoms--ranging from 2 \(\upmu\)m to 2 mm in length for most species--they are invisible to the naked eye; hence, scientists use light microscopes (LM) or scanning electron microscopes (SEM) to reveal diatoms' secrets. Diatoms are unique living beings whose cell walls are based on inorganic opaline silica, quite resistant to decay, heat, and acids. It is as if the cell were inside a Petri dish, called the frustule. The shell consists of two interlocking halves, the epitheca and the--slightly smaller--hypotheca (see Figure 1 for details). As shown in Figure 1, each thece is formed by a structure called the valve--the main element--and several connected bands attached to it, also known as the girdle bands. Morphologically, diatoms can be separated in two types: centric--with radial symmetry--and pennate--with a bilateral symmetry. Because of their microscopic size, diatoms are not collected individually, but in samples with an enormous number of specimens, even though they are not visible until the samples are processed. The preparation of samples by diatomists involves a careful cleaning process carried out with several oxidizing substances to eliminate all organic detritus in the sample [4; 5]. All that is left after cleaning the sample are the siliceous skeletons of the diatoms, usually the disassociated elements of their frustules (i.e., valves and girdle elements; see Figure 1). Currently, the identification and classification of diatoms is carried out using the optical microscope and digital cameras attached to it for image acquisition. With optical microscopy, it is possible to reach practical resolution values of about 0.25 \(\upmu\)m, capable of distinguishing striae density, to 4/\(\upmu\)m. It is worth noting that valves are transparent structures, so the image contrast from an optical microscope is a consequence of differences in the refractive index (RI) between the subject and its surroundings. The diatoms siliceous frustules have an RI of 1.4-1.43, and usually, they are mounted in a medium such as Naphrax--a synthetic resin for slide preparation--with an RI of about 1.6-1.7. Thus, what is seen of a diatom frustule depends on reflection, refraction, and diffraction at the frustule surface, and this is greatly affected by the focus over its three-dimensional morphology [6; 7; 8]. Magnification--usually \(\times\)100, \(\times\)60, \(\times\)40, and \(\times\)20 are used--is very important to capture the microscopic features of a valve, though illumination and focus are crucial to get images with good contrast. Brightfield and oblique illumination have proven their suitability for capturing high quality diatoms images. Both illumination and focus, rather than absolute choices, are relative choices with respect to the specimen under observation and its three-dimensional character. In recent studies,  
Figure 1: (**a**) Scanning electron micrographs (SEM) of four diatoms with diverse morphologies (images courtesy of [PERSON], San Diego Univ. ); (**b**) parts of a circular frustule; and (**c**) parts of a pennate frustule. multifocus and multiexposure fusion techniques have been applied to enhance digital images of diatoms [9; 10; 11; 12; 13].