Atomic force microscopy (AFM) is a method that provides the nanometer-resolution three-dimensional imaging of living cells in their native state in their natural physiological environment. In addition, AFM’s sensitivity to measure interaction forces in the piconewton range enables researchers to probe surface properties, such as elasticity, viscoelasticity, hydrophobicity and adhesion. Despite the growing number of applications of AFM as a method to study biological systems, AFM is not yet an established technique for studying microalgae. Following a brief introduction to the basic principles and operation modes of AFM, this review highlights the major contributions of AFM in the field of microalgae research. A pioneering AFM study on microalgae was performed on diatoms, revealing the fine structural details of diatom frustule, without the need for sample modification. While, to date, diatoms are the most studied class of microalgae using AFM, it has also been used to study microalgae belonging to other classes. Besides using AFM for the morphological characterization of microalgae at the single cell level, AFM has also been used to study the surface properties of microalgal cells, with cell elasticity being most frequently studied one. Here, we also present our preliminary results on the viscoelastic properties of microalgae cell (Dunaliella tertiolecta), as the first microrheological study of microalgae. Overall, the studies presented show that AFM, with its multiparametric characterization, alone or in combination with other complementary techniques, can address many outstanding questions in the field of microalgae.

The impact of the heavy metal cadmium on the lightly silicified marine diatom Cylindrotheca closterium was studied with regard to growth dynamics, organic matter production and morphological characteristics. C. closterium cultures were able to sustain positive growth in the tested range of 0–1000 μg Cd l^–1 with a significant decrease in specific growth rate in the exponential growth phase at the highest tested Cd concentration. The concentrations of both dissolved (DOC_cell) and particulate organic carbon per cell (POC_cell) in C. closterium cultures exposed to 500 and 1000 µg l^–1 Cd more than doubled compared with the control culture. Enhanced organic matter production as a feedback response to Cd exposure may thus contribute to cell detoxification, supported by the electrochemical data showing that Cd was not significantly accumulated by or associated with diatom cells except for the culture exposed to 1000 μg Cd l^–1. In order to gain more insight into the effects of cadmium on diatom morphological features, the cell surface of C. closterium was characterized at the nanoscale on a single cell level using atomic force microscopy (AFM). Changes induced by Cd were observed as irregular patterns of silica spheres on more silicified parts of the cell, girdle band and around the raphe. These changes clearly show that beside the physiological response, C. closterium also showed a morphological response which is probably due to the interference of Cd with the diatom biosilification process. This study overall contributes to better understanding of the effects of cadmium on diatoms and shows that morphological characteristics assessed by AFM may be a valuable indicator for metal contamination.

The aim of this study was to examine the effect of positively charged (amine-modified) and negatively charged (carboxyl-modified) polystyrene nanoplastics (PS NPs) on the nanostructural, nanomechanical, and antioxidant responses of the marine diatom Cylindrotheca closterium. The results showed that both types of PS NPs, regardless of surface charge, significantly inhibited the growth of C. closterium during short-term exposure (3 and 4 days). However, longer exposure (14 days) to both PS NPs types did not significantly inhibit growth, which might be related to the detoxifying effect of the microalgal extracellular polymers (EPS) and the higher cell abundance per PS NPs concentration. The exposure of C. closterium to both types of PS NPs at concentrations above the corresponding concentrations that resulted in a 50% reduction of growth (EC₅₀) demonstrated phytotoxic effects, mainly due to the excessive production of reactive oxygen species, resulting in increased oxidative damage to lipids and changes to antioxidant enzyme activities. Diatoms exposed to nanoplastics also showed a significant decrease in cell wall rigidity, which could make the cells more vulnerable. Atomic force microscopy images showed that positively charged PS NPs were mainly adsorbed on the cell surface, while both types of PS NPs were incorporated into the EPS that serves to protect the cells. Since microalgal EPS are an important food source for phytoplankton grazers and higher trophic levels, the incorporation of NPs into the EPS and interactions with the cell walls themselves may pose a major threat to marine microalgae and higher trophic levels and, consequently, to the health and stability of the marine ecosystem.

Microbial eukaryotes have essential roles in aquatic ecosystems, yet their diversity and ecological functions in extreme environments remain understudied compared to prokaryotes. This study aims to thoroughly characterize the composition and diversity of microbial eukaryotic communities in 14 geothermal waters across Croatia. Physicochemical analysis revealed significant variations in temperature (36–55 °C), pH (6.5–8.3), and nutrient concentrations, with all sites displaying anoxic conditions except for one. Sequencing of the V9 18S rRNA gene identified 134 taxa, predominantly from the Alveolata, Stramenopiles, and Opisthokonta supergroups. The highest diversity and richness were observed in aquifer groups with moderate temperatures and nutrient levels, while extreme sites exhibited reduced diversity. Among the key environmental factors shaping these communities, temperature, pH, and nitrate concentrations were most significant. Photoautotrophic and mixotrophic taxa, such as Ochrophyta, Dinoflagellata, and Chlorophyta, were prominent, reflecting their roles in primary production and nutrient cycling. Decomposers, including Basidiomycota and Ascomycota, were linked to organic matter degradation. Microeukaryotes showed adaptations to extreme conditions, such as thermotolerance and evolutionary shifts from phototrophy to heterotrophy, highlighting their ecological versatility. These findings underscore the potential of microbial eukaryotes in biotechnological applications, such as bioremediation and biofuel production. Genera like Tribonema and Navicula demonstrated promising capabilities in nutrient removal and CO₂ fixation. However, further research is necessary to investigate and confirm their suitability for these purposes. To summarize, our research provides new insights into understudied microbial eukaryotes in Croatian hot springs that represent a valuable model for exploring microbial diversity, ecological interactions, and industrial applications in extreme environments.

Karst freshwater systems represent unique ecological niches where physicochemical and biological interactions promote intensive calcium carbonate precipitation and the formation of tufa barriers. Here, we studied the composition, diversity, and functional potential of prokaryotic and microeukaryotic communities involved in early‐stage tufa formation at two sites within the Plitvice Lakes National Park, Croatia. Over a 5‐day period in two different seasons, tufa and water samples were collected at Prošćansko Lake and Novakovića Brod to examine temporal and spatial microbial dynamics. High‐throughput sequencing revealed early stabilization of prokaryotic communities within tufa biofilms, dominated by genera, such as Bacillus , Delftia , Hyphomicrobium , and Methylobacterium–Methylorubrum , which are linked to carbonate precipitation processes. With biofilm maturation, shifts toward Acinetobacter and Rhodoferax indicated increasing heterotrophic activity and organic matter degradation. In contrast, microeukaryotic communities were more variable, with diatoms and Zygnemophyceae contributing to extracellular polymeric substance production, crucial for carbonate entrapment. Site‐specific patterns reflected environmental influences, such as hydrodynamics and terrestrial organic input. The results underscore the importance of microbial succession and community specialization in the formation and stability of tufa barriers, offering new insights into microbial contributions to biogeochemical processes in karst freshwater systems.