Data analysis of sperm morphology is critical for evaluating bull fertility, yet it is commonly performed using light microscopy and staining techniques in a subjective and manual manner. In this study, we introduce a scalable, high-resolution approach combining label-free Imaging Flow Cytometry with deep learning for automated classification of bovine sperm morphology. We analyzed 401,535 single-cell images obtained out 1.8 million events acquired at 40 × magnification from three bull breeds - Kazakh Whitehead, Auliekol, and Simmental, from fresh and cryopreserved sperm - providing a uniquely large and diverse dataset. The dataset was used for training and evaluation of deep learning models. Multiple classification strategies and architectures were evaluated using consistent training and data preparation strategies, including MobileNetV3-Large, EfficientNetV2-S, ResNet-50, and ConvNeXt-Tiny. Among these, the ConvNeXt-Tiny yielded superior results, achieving an accuracy 91.1% and a macro F1-score of 0.91 after training with a Linear Probing and Fine-Tuning (LP-FT) strategy, and was chosen as the primary model to classify spermatozoa into eight distinct morphological categories. Testing across different conditions and breeds resulted in a 10–15% drop in generalization performance, highlighting the limitations in generalization due to dataset variability and underscoring the need for larger, standardized imaging protocols. The proportion of morphologically abnormal spermatozoa differed by seasons and following cryopreservation. This study highlights the advantages of integrating IFC and artificial intelligence algorithms for robust, high-throughput, and objective label-free assessment of spermatozoa morphology in both fresh and cryopreserved sperm, offering a promising tool for improving fertility diagnostics and breeding strategies in veterinary practice.

The autofluorescence of algal pigments enables non-invasive, high-throughput characterization of microalgae at single-cell resolution. We applied full-spectrum cytometry, imaging flow cytometry, and cell sorting to analyze the spectral and morphological diversity among major microalgal groups and 102 Chlorophyta strains. The distinct spectral signatures from chlorophylls, carotenoids, and phycobiliproteins enabled clear separation of major pigment-defined algal groups, particularly those containing phycobiliproteins. Furthermore, principal component analysis of Volvocales (Chlorophyta) revealed three spectral clusters supported by corresponding differences in cell size and shape. Additionally, in Gonium cultures, we observed that spectral signatures in the yellow-green region were altered in the presence of bacteria, suggesting that interactions between the algal host and bacteria affect pigment-related fluorescence. Spectral heterogeneity observed within monocultures was linked to pigment accumulation, cell size, and morphological variability. These findings establish full-spectrum cytometry as a powerful method for profiling pigment composition, physiology, and structural diversity in microalgae, with broad applications in microbial ecology, environmental monitoring, and biotechnology.

Background: Focal Adhesions (FAs) play a key role in cell-substrate adhesion and migration. Modern high-throughput live cell microscopy provides experimental data on FA’s behavior with increasingly high temporal resolution for an ever-expanding variety of cell cultures and experimental conditions. For this kind of data, manual analysis is challenging in terms of reproducibility and sheer volume of it, so fully automated software-based FA tracking algorithms become crucially important[1]. Materials and methods: Cell cultures with fluorescently labeled FA proteins (vinculin-RFP and paxillin-EGFP) were obtained, live cells were imaged by spinning disk confocal microscopy, and FAs were quantitatively analyzed via custom MATLAB-based software. Results: We optimized the algorithm for tracking FAs all the way to microscope resolution limit, accessing dynamics (growth, disassembly and lifespan) for events starting from half a minute in duration, while also tracking through FA splitting, merging, and translocation. After initial validation on a panel of 6 cell cultures, we used this algorithm to quantify differences in regional FA behavior in A549 cells. Newly-formed small FAs underwent frequent merging/fusing events, consistent with liquid-liquid phase condensation. FA formation in the regions of rapid edge protrusion was upregulated by an order of magnitude in respect to other cell edges. On retracting edge long-range FA translocations were observed. By simultaneous tracking of vinculin and paxillin in double transfected cells differences in the dynamics of these proteins inside FAs were highlighted, including the phenomenon of FA layer separation. Conclusion: Our automated algorithm proved to be a powerful tool for high-throughput analysis of FA behavior in cells and highlighted several previously poorly documented features of FA dynamics. Acknowledgement: This research has been funded by the grant number AP23488797 from the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan Key words: cytoskeleton, focal contacts, cell migration, confocal microscopy, automated analysis. References: [1] Mathew E Berginski, Eric A Vitriol, Klaus M Hahn, Shawn M Gomez. High- resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS ONE 6(7): e22025 (2011).

Focal adhesions (FAs) are multi-protein complexes that mediate cell attachment to the extracellular matrix. Their formation and maturation depend on intracellular tension generated by actin filaments interacting with phosphorylated myosin II. Using live-cell and confocal microscopy, we investigated how FA dynamics are regulated by actin polymerization and myosin II-driven contractility. We found that knockdown of myosin II resulted in complete and irreversible disassembly of FAs. However, partial inhibition of myosin II, through either ROCK or myosin light chain kinase (MLCK) inhibitors, leads to gradual FA shrinkage. In contrast, complete inhibition of myosin II phosphorylation causes disassembly of existing FAs, followed by the formation of new, small FAs at the cell periphery. In both cases, FAs formed after inhibition of myosin II phosphorylation exhibited significantly longer lifespans than FAs in control cells. Similarly, partial inhibition of actin polymerization using nanomolar concentrations of latrunculin B or cytochalasin D also promoted the formation of small FAs. Complete and irreversible FA disassembly occurred only when actin filaments were fully disrupted, leading to cell lamella retraction. These findings suggest that actin polymerization at the cell edge is the minimal and sufficient requirement for the assembly of small FAs. Notably, our data demonstrate for the first time that perturbation of the actin-myosin system results in stabilization and prolonged lifespan of small FAs, whereas larger FAs, formed in the presence of myosin II activity, are more dynamic. Together, these results emphasize the essential role of cortical actin organization and myosin II phosphorylation in the maintenance and turnover of FAs.

Aging is interrelated with changes in red blood cell parameters and functionality. In this article, we focus on red blood cells (RBCs) and provide a review of the known changes associated with the characterization of RBC deformability in aging and related pathologies. The biophysical parameters complement the commonly used biochemical parameters and may contribute to a better understanding of the aging process. The power of the deformability measurement approach is well established in clinical settings. Measuring RBCs' deformability has the advantage of relative simplicity, and it reflects the complex effects developing in erythrocytes during aging. However, aging and related pathological conditions also promote heterogeneity of RBC features and have a certain impact on the variance in erythrocyte cell properties. The possible applications of deformability as an early biophysical biomarker of pathological states are discussed, and modulating PIEZO1 as a therapeutic target is suggested. The changes in RBCs' shape can serve as a proxy for deformability evaluation, leveraging single-cell analysis with imaging flow cytometry and artificial intelligence algorithms. The characterization of biophysical parameters of RBCs is in progress in humans and will provide a better understanding of the complex dynamics of aging.

Cryopreservation of sperm is an essential technique in assisted reproduction in cattle. The objective of the study was to systematically review and synthesize the literature on bull semen quality evaluation based on the comparison of morphological and metabolic parameters of cryopreserved bovine spermatozoa such as DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphology (% of abnormal cells). The electronic databases PubMed, Web of Sciences, Scopus, and Google Scholar were searched up to December 2023. Studies and references were included if they reported the following parameters: DNA integrity, mitochondrial status, plasma membrane alterations, total motility, and morphological aberrations (% of abnormal cells) for conventional cryopreserved bovine spermatozoa. After an electronic search, out of 1,526 original studies, only 40 were included in the meta-analysis. Standardized mean differences (SMD) with 95% confidence intervals were estimated for the chosen studies, and a meta-analysis was performed using a random effects model. The tau-squared (tau2) and inconsistency index (I2) quantified heterogeneity among different studies. The regression analysis for the evaluated parameters showed a positive correlation between mitochondrial membrane potential (MMP), total motility, and abnormal morphology and a negative correlation between DNA fragmentation index (DFI) and total motility and MMP. Moreover, subgroup analysis demonstrated similar associations for dairy and non-dairy bull breeds, albeit with lower I2 values. The presence of publication bias was confirmed by Egger’s test, except for the MMP parameter. A multi-parametric analysis of morphological and metabolic parameters can address the existing limitations of cryopreserved bovine spermatozoa quality assessment. Combining imaging flow cytometry (IFC) with standardization of sperm pre-processing and optimization of the experimental protocols may help to differentiate sperm from cellular debris and cytoplasmic droplets of similar size and alleviate limitations demonstrated by conventional sperm analysis.

Synthetic biology has advanced in creating artificial microbial and algal communities, but technical and evolutionary complexities still pose significant challenges. Traditional methods, like microscopy and pigment analysis, are limited in throughput and resolution. In contrast, advancements in full-spectrum cytometry enabled high-throughput, multidimensional analysis of single cells based on size, complexity, and spectral fingerprints, offering more precision and flexibility than conventional flow cytometry. This study uses full-spectrum cytometry to analyze synthetic algal-microbial communities, enabling rapid species identification and enumeration. The workflow involves recording individual spectral signatures from monocultures, using autofluorescence to capture populations of interest, and creating a spectral library for further analysis. This spectral library was used for the analysis of the synthetic phytoplankton communities, revealing differences in spectral signatures. Moreover, the synthetic consortium experiment monitored algal growth, comparing results from different instruments, highlighting the advantages of the spectral virtual filter system for precise population separation and abundance tracking. By capturing the entire emission spectrum of each cell, this method enhances understanding of algal-microbial community dynamics and responses to environmental stressors. The development of standardized spectral libraries would improve the characterization of algal communities, further advancing synthetic biology and phytoplankton ecology research.

The Aral Sea was once the fourth-largest inland water body in the world. However, the lake rapidly shrank over the past six decades, mainly due to the loss of inflow from one of its tributaries, the Amu Darya River. Lakes and reservoirs are traditionally characterized by static chemical and morphological parameters, leaving untouched a dynamic impact of phytoplankton changes. We used an integrated approach combining traditional microscopy and FlowCam-based imaging flow cytometry to study phytoplankton communities during the 2018 and 2019 expeditions in the Aral Sea remnant lakes system. The residual Aral Sea water bodies experienced different environmental conditions, forming hypersaline South Aral, North Aral Sea that is constantly getting freshwater, and brackish Chernyshev Bay and Tushchybas Lake with 2-8 times amplitude of salinity changes attributed to the variability in the precipitation and periodical influx of freshwater. The salinity fluctuations had an impact on the phytoplankton communities in Chernyshev Bay, making it similar to the phytoplankton of North Aral in 2018 while resembling the hypersaline South Aral phytoplankton assemblages in 2019. Multivariate analysis revealed that salinity, water temperature, ammonium, and nitrates were major contributors to explaining the variance in the sampling data. We conclude that drastic phytoplankton fluctuations occur in the two brackish water bodies in the middle of the former Aral Sea, reflecting changes in salinity. ### Competing Interest Statement The authors have declared no competing interest. * ABD : area-based diameter ANOVA : analysis of variance CCA : canonical correspondence analysis DIC : differential interference contrast DO : dissolved oxygen FGs : functional groups IFC : imaging flow cytometry PCy : picocyanobacteria SD : standard deviation TP : total phosphorus

This study comprises the comprehensive toxicological assessment of thiolated organosilica nanoparticles (NPs) synthesised from 3-mercaptopropyltrimethoxysilane (MPTS). We investigated the influence of three different types of nanoparticles synthesised from 3-mercaptopropyltrimethoxysilane: the starting thiolated silica (Si-NPSH) and their derivatives prepared by surface PEGylation with PEG 750 (Si-NP-PEG750) and 5000 Da (Si-NPPEG5000) on biological subjects from in vitro to in vivo experiments to explore the possible applications of those nanoparticles in biomedical research. As a result of this study, we generated a comprehensive understanding of the toxicological properties of these nanoparticles, including their cytotoxicity in different cell lines, hemolytic properties, in vitro localisation, mucosal irritation properties and biodistribution in BALB/c mice. Our findings indicate that all three types of nanoparticles can be considered safe and have promising prospects for use in biomedical applications. Nanoparticles did not affect the viability of HPF, MCF7, HEK293 and A549 cell lines at low concentrations (up to 100 mu g/mL); moreover, they did not cause organ damage to BALB/c mice at concentrations of 10 mg/kg. The outcomes of this study enhance our understanding of the impact of organosilica nanoparticles on health and the environment, which is vital for developing silica nanoparticle-based drug delivery systems and provides opportunities to expand the applications of organosilica nanoparticles.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

Focal adhesions (FAs) are multi-protein structures mediating interaction between cells and extracellular matrix. The formation and maturation of FAs depend on intracellular tension generated through the of actin filaments interacting with phosphorylated myosin II. Using live cell and confocal microscopy we evaluated the regulation of FA dynamics by actin polymerization and myosin II-mediated tension in motile cells. Our findings confirm that while partial inhibition of myosin II, through ROCK kinase or myosin II light chain kinase (MLCK) inhibitors, leads to the gradual shrinkage but increased stability of FAs, complete inhibition of myosin II phosphorylation results in disassembly of existing FAs followed by the formation of new small FAs at the cell edges. These FAs are rather stable, supported by cortical actin structures. Partial inhibition of actin polymerization with nanomolar doses of latrunculin B or cytochalasin D results in the similar effect, while complete inhibition leads to the rapid and irreversible disassembly of FAs and shrinkage of cell lamella. Overall, our results show that small FAs, which are normally short-living structures at the advancing cell edge, could be stabilized and persist, driven only by partial actin polymerization. These insights highlight the role of the interplay between actin and myosin in maintaining FA formation and dynamics.

Epithelial-to-mesenchymal transition (EMT) is a crucial process that occurs during normal development, such as embryogenesis and organ formation. If it is inappropriately activated, it can lead to pathological processes like metastasis. Although EMT is well studied at the morphological and transcriptome level, cytoskeleton changes during this process are less understood. In our work we aimed to describe the morphological changes that occurred in MCF-7, A-549 and HaCaT cells after EMT, analyze microtubule dynamics, spatial distribution and its contribution to cell motility, identify changes in actin filament organization and study focal adhesion turnover in the cells after EMT. We hypothesized that the dynamics of microtubules in cells undergoing EMT might change. Cells undergoing EMT were expected to have more dynamic microtubules. Also, cells undergoing EMT are expected to adhere more efficiently to diverse substrates, and therefore spread and move more easily. Focal contacts in cells undergoing EMT were expected to be more pronounced and dynamic than in cells that not undergoing EMT. The research methods used in the study included inducing EMT in modified MCF-7 cells through an inserted inducible Tet-on system and stimulation of EMT in A-549 and HaCaT cells using TGF-β. The cells were observed using bright field microscopy, and immunofluorescence analysis was conducted to visualize microtubules and actin filaments. Transfection with EB-3–RFP protein was done to describe and measure microtubule dynamics, while transduction with Talin-RFP and transient transfection with Ptag-RFP-vinculin was done to visualize focal adhesions. Time-lapse fluorescent microscopy was used to record films, and the Fiji Image J program was used to analyze the data. All statistical analysis was performed using GraphPad Prism (Dotmatics, USA), and a nonparametric Mann-Whitney U test or parametric t-test with Welch correction. The actin filament measurements were completed using Matlab scripts. The major research findings: In all three-cell models after EMT, cell morphology changed. Cells increased in size. MCF-7 and HaCaT became spread out, while A-549 became elongated. Some of the cells lost cell-cell contacts. All three cell models after EMT had alterations in microtubule organization and dynamics. MCF-7 and HaCaT cells showed more frequently individual MTs at cell edges, while A-549 had less covered nucleus by MTs after EMT. Microtubule dynamics (growth velocity) increased, and the length of microtubule growth tracks became longer. The average angle of MT growth trajectories to cell radius decreased. Actin fibers rearranged into more pronounced stress fibers after EMT. Alterations in the focal adhesion behaviors were different in three models used. These results indicate that cytoskeletal changes during EMT mainly include increased microtubule dynamics.

Epithelial cells undergoing EMT experience significant alterations at transcriptional and morphological levels. However, changes in the cytoskeleton, especially cytoskeleton dynamics are poorly described. Addressing the question we induced EMT in three cell lines (MCF-7, HaCaT and A-549) and analyzed morphological and cytoskeletal changes there using immunostaining and life cell imaging of cells transfected with microtubule and focal adhesion markers. In all studied cell lines, cell area after EMT increased, MCF-7 and A-549 cells became elongated, while HaCaT cells kept the aspect ratio the same. We next analyzed three components of the cytoskeleton: microtubules, stress fibers and focal adhesions. The following changes were observed after EMT in cultured cells: (i) Organization of microtubules becomes more radial; and the growth rate of microtubule plus ends was accelerated; (ii) Actin stress fibers become co-aligned forming the longitudinal cell axis; and (iii) Focal adhesions had decreased area in all cancer cell lines studied and became more numerous in HaCaT cells. We conclude that among dynamic components of the cytoskeleton, the most significant changes during EMT happen in the regulation of microtubules.

Multi-nuclearity is a common feature for cells in different cancers. Also, analysis of multi-nuclearity in cultured cells is widely used for evaluating the toxicity of different drugs. Multi-nuclear cells in cancer and under drug treatments form from aberrations in cell division and/or cytokinesis. These cells are a hallmark of cancer progression, and the abundance of multi-nucleated cells often correlates with poor prognosis.The use of standard bright field or fluorescent microscopy to analyze multi-nuclearity at the quantitative level is laborious and can suffer from user bias. Automated slide-scanning microscopy can eliminate scorer bias and improve data collection. However, this method has limitations, such as insufficient visibility of multiple nuclei in the cells attached to the substrate at low magnification.Since quantification of multi-nuclear cells using microscopic methods might be difficult, imaging flow cytometry (IFC) is a method of choice for this. We describe the experimental protocol for the preparation of the samples of multi-nucleated cells from the attached cultures and the algorithm for the analysis of these cells by IFC. Images of multi-nucleated cells obtained after mitotic arrest induced by taxol, as well as cells obtained after cytokinesis blockade by cytochalasin D treatment, can be acquired at a maximal resolution of IFC. We suggest two algorithms for the discrimination of single-nucleus and multi-nucleated cells. The advantages and disadvantages of IFC analysis of multi-nuclear cells in comparison with microscopy are discussed.

Spectral flow cytometry is a new technology that enables measurements of fluorescent spectra and light scattering properties in diverse cellular populations with high precision. Modern instruments allow simultaneous determination of up to 40+ fluorescent dyes with heavily overlapping emission spectra, discrimination of autofluorescent signals in the stained specimens, and detailed analysis of diverse autofluorescence of different cells-from mammalian to chlorophyll-containing cells like cyanobacteria. In this paper, we review the history, compare modern conventional and spectral flow cytometers, and discuss several applications of spectral flow cytometry.

Fluorescence methods are widely used for the study of marine and freshwater phytoplankton communities. However, the identification of different microalgae populations by the analysis of autofluorescence signals remains a challenge. Addressing the issue, we developed a novel approach using the flexibility of spectral flow cytometry analysis (SFC) and generating a matrix of virtual filters (VF) which allowed thorough examination of autofluorescence spectra. Using this matrix, different spectral emission regions of algae species were analyzed, and five major algal taxa were discriminated. These results were further applied for tracing particular microalgae taxa in the complex mixtures of laboratory and environmental algal populations. An integrated analysis of single algal events combined with unique spectral emission fingerprints and light scattering parameters of microalgae can be used to differentiate major microalgal taxa. We propose a protocol for the quantitative assessment of heterogenous phytoplankton communities at the single-cell level and monitoring of phytoplankton bloom detection using a virtual filtering approach on a spectral flow cytometer (SFC-VF).

Aim: Flow cytometric diagnosis of lymphoma and leukemia is of high clinical and research importance. However, performing flow cytometry analysis on the day of biopsy might be of challenge due to several reasons, including late sample delivery, problems of preparing the reliable panel for immunophenotyping based on other diagnostic studies, etc. This problem could be partially solved if cell suspension could be fixed and stained on another day or after several days after standard FFPE (formalin-fixed and paraffin-embedded) procedure. Material and methods: Addressing this issue, we compared staining of live lymphocytes in suspension obtained from lymph node biopsies and same specimens fixed using 2-4%-paraformaldehyde, 1-3%-glyoxal, and 0.1-1% glutaraldehyde with subsequent immunostaining on the next day or later. Results: Staining after fixation could be partially representative only after paraformaldehyde fixation for 20 min and subsequent storage of cell suspension in phosphate-buffer saline within not more than 3 days. Probes stained after fixation always shows lower stain index compared to staining of live cells. Conclusion: Staining after fixation cannot be used for determining of the percentage of CD45-positive cells and for testing B-cell lymphomas since antigens against light chains of IgG cannot be properly detected in fixed specimens.

Microcystis is a globally known cyanobacterium causing potentially toxic blooms worldwide. Different morphospecies with specific morphological and physiological characters usually co-occur during blooming, and their quantification employing light microscopy can be time-consuming and problematic. A benchtop imaging flow cytometer (IFC) FlowCam (Yokogawa Fluid Imaging Technologies, USA) was used to identify and quantitate different Microcystis morphospecies from environmental samples. We describe here the FlowCam methodology for sample processing and analysis of five European morphospecies of Microcystis common to the temperate zone. The FlowCam technique allows detection of different Microcystis morphospecies providing objective qualitative and quantitative data for statistical analysis.

Saline lakes are rapidly drying out across the globe, particularly in Central Asia, due to climate change and anthropogenic activities. We present the results of a long-read next generation sequencing analysis of the 16S rRNA-based taxonomic structure of bacteriomes of the Tengiz-Korgalzhyn lakes system. We found that the shallow endorheic, mostly saline lakes of the system show unusually low bacterioplankton dispersal rates at species-level taxonomic resolution. The major environmental factor structuring the lake’s microbial communities was salinity. The dominant bacterial phyla of the lakes with high salinity included a significant proportion of marine and halophilic species. In sum, these results, which can be applied to other lake systems of the semi-arid regions, improve our understanding of the factors influencing lake microbiomes undergoing salinization in response to climate change and other anthropogenic factors. Our results show that finer taxonomic classification can provide new insights and improve our understanding of the environmental factors influencing the microbiomes of lakes undergoing salinization in response to climate change and other anthropogenic factors.### Competing Interest StatementThe authors have declared no competing interest.

Regulated cell death (RCD) is central to the development, integrity, and functionality of multicellular organisms. In the last decade, evidence has accumulated that RCD is a universal phenomenon in all life domains. Cyanobacteria are of specific interest due to their importance in aquatic and terrestrial habitats and their role as primary producers in global nutrient cycling. Current knowledge on cyanobacterial RCD is based mainly on biochemical and morphological observations, often by methods directly transferred from vertebrate research and with limited understanding of the molecular genetic basis. However, the metabolism of different cyanobacteria groups relies on photosynthesis and nitrogen fixation, whereas mitochondria are the central executioner of cell death in vertebrates. Moreover, cyanobacteria chosen as biological models in RCD studies are mainly colonial or filamentous multicellular organisms. On the other hand, unicellular cyanobacteria have regulated programs of cellular survival (RCS) such as chlorosis and post-chlorosis resuscitation. The co-existence of different genetically regulated programs in cyanobacterial populations may have been a top engine in life diversification. Development of cyanobacteria-specific methods for identification and characterization of RCD and wider use of single-cell analysis combined with intelligent image-based cell sorting and metagenomics would shed more light on the underlying molecular mechanisms and help us to address the complex colonial interactions during these events. In this review, we focus on the functional implications of RCD in cyanobacterial communities.