Temporal changes in zooplankton indicators highlight a bottom-up process in the Bay of Marseille (NW Mediterranean Sea).

Sixteen years (2005-2020) of zooplankton monitoring in the Bay of Marseille (N-W Mediterranean Sea) are analyzed in relation to physical, meteorological, climatic and biotic data. Samples were collected every two weeks by a vertical haul (0-55 m) of a 200 µm plankton net. Different indices characterizing the mesozooplankton are compared: biomass dry weight of four size fractions between 200 and 2000 µm; abundances of the whole of the mesozooplankton and of 13 main taxonomic groups defined from plankton imagery; seasonal onset timing of each zooplankton group; and two other types of indices: the first characterized diversity based on abundance data, and the second was derived from zooplankton size spectra shape. The clearest pattern in the environmental compartment was an overall decreasing trend in nutrients, shifts in phytoplankton metrics (i.e. size structure and particulate organic matter), and changes in winter conditions (i.e. increasing temperatures, precipitation and NAO). Interannual patterns in the mesozooplankton community were: (i) a decrease of total abundance (ii) a decrease in biomass for the four size fractions, with an earlier decrease for the 1000-2000 µm size fraction (in 2008); (iii) a reduced dominance of copepods (calanoids and oithonoids) and a concomitant increase in abundance of other taxonomic groups (crustaceans, pteropods, chaetognaths, salps) which induced higher diversity; (iv) a first shift in size spectra towards smaller sizes in 2009, when the 1000-2000 µm size fraction biomass decreased, and a second shift towards larger sizes in 2013 along with increased diversity; and (iv) a later onset in the phenology for some zooplankton variables and earlier onset for salps. Concomitant changes in the phytoplankton compartment, winter environmental conditions, zooplankton community structure (in size and diversity) and zooplankton phenology marked by a shift in 2013 suggest bottom-up control of the pelagic ecosystem.

Trajectories of nutrients concentrations and ratios in the French coastal ecosystems: 20 years of changes in relation with large-scale and local drivers.

Along with their important diversity, coastal ecosystems receive various amounts of nutrients, principally arising from the continent and from the related human activities (mainly industrial and agricultural activities). During the 20th century, nutrients loads have increased following the increase of both the global population and need of services. Alongside, climate change including temperature increase or atmospheric circulation change has occurred. These processes, Ecosystem state changes are hard to monitor and predict. To study the long-term changes of nutrients concentrations in coastal ecosystems, eleven French coastal ecosystems were studied over 20 years as they encompass large climatic and land pressures, representative of temperate ecosystems, over a rather small geographical area. Both univariate (time series decomposition) and multivariate (relationships between ecosystems and drivers) statistical analyses were used to determine ecosystem trajectories as well as typologies of ecosystem trajectories. It appeared that most of the French coastal ecosystems exhibited trajectories towards a decrease in nutrients concentrations. Differences in trajectories mainly depended on continental and human influences, as well as on climatic regimes. One single ecosystem exhibited very different trajectories, the Arcachon Bay with an increase in nutrients concentrations. Ecosystem trajectories based on ordination techniques were proven to be useful tools to monitor ecosystem changes. This study highlighted the importance of local environments and the need to couple uni- and multi-ecosystem studies. Although the studied ecosystems were influenced by both local and large-scale climate, by anthropogenic activities loads, and that their trajectories were mostly similar based on their continental influence, non-negligible variations resulted from their internal functioning.

Enhanced resolution of marine viruses with violet side scatter.

Marine viruses make up an essential compartment of the marine ecosystem. They are the most abundant organisms and represent one of the biggest sources of unknown biodiversity. Viruses also have an important impact on bacterial and algal mortality in the ocean, and as such have a major influence on microbial diversity and biogeochemical cycling. However, little is known about the abundance and distribution patterns of viruses across the oceans and seas. Over the last 20 years, flow cytometry has been the technique of choice to detect and count the viral particles in natural samples. Nevertheless, due to their small size, the detection of marine viruses is still extremely challenging. In this article we describe how a new generation of flow cytometer which uses the side scatter (SSC) of violet photons from a 405 nm laser beam helps to improve the resolution for detecting marine viruses. To the best of our knowledge, this is the first report where virioplankton has been detected in aquatic samples using flow cytometry with a 405 nm violet SSC instead of a 488 nm blue SSC.

Summertime Tintinnid Community in the Surface Waters Across the North Pacific Transition Zone.

Located from 35° to 45° latitude in both hemispheres, the transition zone is an important region with respect to the planktonic biogeography of the sea. However, to the best of our knowledge, there have been no reports on the existence of a tintinnid community in the transition zone. In this research, tintinnids along two transects across the North Pacific Transition Zone (NPTZ) were investigated in summer 2016 and 2019. Eighty-three oceanic tintinnid species were identified, 41 of which were defined as common oceanic species. The common oceanic species were further divided into five groups: boreal, warm water type I, warm water type II, transition zone, and cosmopolitan species. Undella californiensis and Undella clevei were transition zone species. Other species, such as Amphorides minor, Dadayiella ganymedes, Dictyocysta mitra, Eutintinnus pacificus, Eutintinnus tubulosus, Protorhabdonella simplex, and Steenstrupiella steenstrupii, were the most abundant in the NPTZ but spread over a much larger distribution region. Species richness showed no obvious increase in the NPTZ. Boreal, transition zone, and warm water communities were divided along the two transects. Tintinnid transition zone community mainly distributed in regions with water temperatures between 15 and 20°C. The tintinnid lorica oral diameter size classes were dominated by the 24-28 µm size class in three communities, but the dominance decreased from 66.26% in the boreal community to 48.85% in the transition zone community and then to 22.72% in the warm water community. Our research confirmed the existence of tintinnid transition zone species and community. The abrupt disappearance of warm water type I species below 15°C suggested that this group could be used as an indicator of the northern boundary of the NPTZ.

Surface ocean microbiota determine cloud precursors.

One pathway by which the oceans influence climate is via the emission of sea spray that may subsequently influence cloud properties. Sea spray emissions are known to be dependent on atmospheric and oceanic physicochemical parameters, but the potential role of ocean biology on sea spray fluxes remains poorly characterized. Here we show a consistent significant relationship between seawater nanophytoplankton cell abundances and sea-spray derived Cloud Condensation Nuclei (CCN) number fluxes, generated using water from three different oceanic regions. This sensitivity of CCN number fluxes to ocean biology is currently unaccounted for in climate models yet our measurements indicate that it influences fluxes by more than one order of magnitude over the range of phytoplankton investigated.

Virioplankton distribution in the tropical western Pacific Ocean in the vicinity of a seamount.

The shallow Caroline Seamount is located in the tropical western Pacific Ocean. Its summit is 57 m below the surface and penetrates the euphotic zone. Therefore, it is ideal for the study of the influence of seamount on plankton distribution. Here, virioplankton abundance and distribution were investigated by flow cytometry (FCM) in the Caroline Seamount in August and September 2017. The total abundance of virus-like particles (VLP) was in the range of 0.64 × 106 -18.77 × 106  particles/ml and the average was 5.37 ± 3.75 × 106  particles/ml. Three to four distinct viral subclusters with similar side scatter but different green fluorescence intensities were identified. Above the deep chlorophyll maximum (DCM), two medium fluorescence virus (MFV) subclusters were discriminated. Between the DCM and the deeper layers, only one MFV subcluster was resolved. In general, low fluorescence viruses (LFV) comprised the most abundant subclusters. In the 75-150 m water column, however, the MFV abundance was higher than the LFV abundance. High fluorescence viruses (HFV) constituted the least abundant subcluster throughout the entire water column. Virioplankton abundance was significantly enhanced at the seamount stations. Environmental factors including water temperature and nitrate concentration were the most correlated with the variation in virioplankton abundance at the seamount stations. Interactions between shallow seamounts and local currents can support large virus standing stocks, causing a so-called indirect "seamount effect" on the virioplankton.

Impact of sterilization methods on dissolved trace metals concentrations in complex natural samples: Optimization of UV irradiation.

Sterilization is essential for discriminating biotic responses from abiotic reactions in laboratory experiments investigating biogeochemical processes of complex natural samples. However, the conventional methods used to effectively sterilize materials or culture media do not allow sterilizing complex natural samples while maintaining biogeochemical balances. The aim of this study was to develop a low-cost and easy-to-use method to obtain geochemically unmodified and sterilized samples from complex lacustrine or coastal marine ecosystems. In preliminary assays, the impact of several sterilization methods (autoclaving, chemical poisoning, microwave, UV irradiation) on the trace metals balances was studied using borosilicate glass (BG), fluorinated ethylene-propylene (FEP) or polyethylene terephthalate (PET) bottles. Unlike other methods, UV sterilization had minor effects on the distribution of dissolved trace metals. Additional tests using complex lacustrine and coastal marine samples under 10 g/L sediments were performed using a homemade UV sterilization chamber designed to simultaneously irradiate a large number samples. Results showed: •very reproducible UV tests in BG and FEP bottles•faster sterilization using FEP bottles than using BG bottles•low variations of dissolved trace metals concentrations, except for Al, Cu, Fe and Zn.

Exploring the microbiome of the "star" freshwater diatom Asterionella formosa in a laboratory context.

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies.

Resolution of Viable and Membrane-Compromised Free Bacteria in Aquatic Environments by Flow Cytometry.

In aquatic environments, free heterotrophic bacteria play an extremely important role due to their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains-membrane-permeant SYBR Green and membrane-impermeant propidium iodide (PI). The efficiency of the double staining on fresh samples is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI identifies cells with a compromised membrane, partial quenching indicates cells with a slightly damaged membrane, and lack of quenching characterizes cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere. © 2018 by John Wiley & Sons, Inc.

Nanoplanktonic diatoms are globally overlooked but play a role in spring blooms and carbon export.

Diatoms are one of the major primary producers in the ocean, responsible annually for ~20% of photosynthetically fixed CO2 on Earth. In oceanic models, they are typically represented as large (>20 µm) microphytoplankton. However, many diatoms belong to the nanophytoplankton (2-20 µm) and a few species even overlap with the picoplanktonic size-class (<2 µm). Due to their minute size and difficulty of detection they are poorly characterized. Here we describe a massive spring bloom of the smallest known diatom (Minidiscus) in the northwestern Mediterranean Sea. Analysis of Tara Oceans data, together with literature review, reveal a general oversight of the significance of these small diatoms at the global scale. We further evidence that they can reach the seafloor at high sinking rates, implying the need to revise our classical binary vision of pico- and nanoplanktonic cells fueling the microbial loop, while only microphytoplankton sustain secondary trophic levels and carbon export.

Evolution of the scattering properties of phytoplankton cells from flow cytometry measurements.

After the exponential growth phase, variability in the scattering efficiency of phytoplankton cells over their complete life cycle is not well characterised. Bulk measurements are impacted by senescent cells and detritrus. Thus the analysis of the evolution of the optical properties thanks to their morphological and/or intra-cellular variations remains poorly studied. Using the Cytosense flow cytometer (CytoBuoy b.v., NL), the temporal course of the forward and sideward efficiencies of two phytoplankton species (Thalassiosira pseudonana and Chlamydomonas concordia) were analyzed during a complete life-cycle. These two species differ considerably from a morphological point of view. Over the whole experiment, the forward and sideward efficiencies of Thalassiosira pseudonana were, on average, respectively 2.2 and 1.6 times higher than the efficiencies of Chlamydomonas concordia. Large intra-species variability of the efficiencies were observed over the life cycle of the considered species. It highlights the importance of considering the optical properties of phytoplankton cells as a function of the population growth stage of the considered species. Furthermore, flow cytometry measurements were combined with radiative transfer simulations and biogeochemical and optical measurements. Results showed that the real refractive index of the chloroplast is a key parameter driving the sideward signal and that a simplistic two-layered model (cytoplasm-chloroplast) seems particularly appropriate to represent the phytoplankton cells.

Cryptic oxygen cycling in anoxic marine zones.

Oxygen availability drives changes in microbial diversity and biogeochemical cycling between the aerobic surface layer and the anaerobic core in nitrite-rich anoxic marine zones (AMZs), which constitute huge oxygen-depleted regions in the tropical oceans. The current paradigm is that primary production and nitrification within the oxic surface layer fuel anaerobic processes in the anoxic core of AMZs, where 30-50% of global marine nitrogen loss takes place. Here we demonstrate that oxygenic photosynthesis in the secondary chlorophyll maximum (SCM) releases significant amounts of O2 to the otherwise anoxic environment. The SCM, commonly found within AMZs, was dominated by the picocyanobacteria Prochlorococcus spp. Free O2 levels in this layer were, however, undetectable by conventional techniques, reflecting a tight coupling between O2 production and consumption by aerobic processes under apparent anoxic conditions. Transcriptomic analysis of the microbial community in the seemingly anoxic SCM revealed the enhanced expression of genes for aerobic processes, such as nitrite oxidation. The rates of gross O2 production and carbon fixation in the SCM were found to be similar to those reported for nitrite oxidation, as well as for anaerobic dissimilatory nitrate reduction and sulfate reduction, suggesting a significant effect of local oxygenic photosynthesis on Pacific AMZ biogeochemical cycling.

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa.

We report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replication.

Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium.

Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

Consequence of a sudden wind event on the dynamics of a coastal phytoplankton community: an insight into specific population growth rates using a single cell high frequency approach.

Phytoplankton is a key component in marine ecosystems. It is responsible for most of the marine primary production, particularly in eutrophic lagoons, where it frequently blooms. Because they are very sensitive to their environment, the dynamics of these microbial communities has to be observed over different time scales, however, assessment of short term variability is often out of reach of traditional monitoring methods. To overcome these limitations, we set up a Cytosense automated flow cytometer (Cytobuoy b.v.), designed for high frequency monitoring of phytoplankton composition, abundance, cell size, and pigment content, in one of the largest Mediterranean lagoons, the Berre lagoon (South-Eastern France). During October 2011, it recorded the cell optical properties of 12 groups of pico-, nano-, and microphytoplankton. Daily variations in the cluster optical properties were consistent with individual changes observed using microscopic imaging, during the cell cycle. We therefore used an adaptation of the size-structured matrix population model, developed by Sosik et al. (2003) to process the single cell analysis of the clusters and estimate the division rates of 2 dinoflagellate populations before, during, and after a strong wind event. The increase in the estimated in situ daily cluster growth rates suggest that physiological changes in the cells can prevail over the response of abundance.

Onset of the spring bloom in the northwestern Mediterranean Sea: influence of environmental pulse events on the in situ hourly-scale dynamics of the phytoplankton community structure.

Most of phytoplankton influence is barely understood at the sub meso scale and daily scale because of the lack of means to simultaneously assess phytoplankton functionality, dynamics and community structure. For a few years now, it has been possible to address this objective with an automated in situ high frequency sampling strategy. In order to study the influence of environmental short-term events (nutrients, wind speed, precipitation, solar radiation, temperature, and salinity) on the onset of the phytoplankton bloom in the oligotrophic Bay of Villefranche-sur-Mer (NW Mediterranean Sea), a fully remotely controlled automated flow cytometer (CytoSense) was deployed on a solar-powered platform (EOL buoy, CNRS-Mobilis). The CytoSense carried out single-cell analyses on particles (1-800 µm in width, up to several mm in length), recording optical pulse shapes when analyzing several cm(3). Samples were taken every 2 h in the surface waters during 2 months. Up to 6 phytoplankton clusters were resolved based on their optical properties (PicoFLO, Picoeukaryotes, Nanophytoplankton, Microphytoplankton, HighSWS, HighFLO). Three main abundance pulses involving the 6 phytoplankton groups monitored indicated that the spring bloom not only depends on light and water column stability, but also on short-term events such as wind events and precipitation followed by nutrient pulses. Wind and precipitation were also determinant in the collapse of the clusters' abundances. These events occurred within a couple of days, and phytoplankton abundance reacted within days. The third abundance pulse could be considered as the spring bloom commonly observed in the area. The high frequency data-set made it possible to study the phytoplankton cell cycle based on daily cycles of forward scatter and abundance. The combination of daily cell cycle, abundance trends and environmental pulses will open the way to the study of phytoplankton short-term reactivity to environmental conditions.

Antifouling coatings influence both abundance and community structure of colonizing biofilms: a case study in the Northwestern Mediterranean Sea.

When immersed in seawater, substrates are rapidly colonized by both micro- and macroorganisms. This process is responsible for important economic and ecological prejudices, particularly when related to ship hulls or aquaculture nets. Commercial antifouling coatings are supposed to reduce biofouling, i.e., micro- and macrofoulers. In this study, biofilms that primarily settled on seven different coatings (polyvinyl chloride [PVC], a fouling release coating [FRC], and five self-polishing copolymer coatings [SPC], including four commercial ones) were quantitatively studied, after 1 month of immersion in summer in the Toulon Bay (Northwestern Mediterranean Sea, France), by using flow cytometry (FCM), microscopy, and denaturing gradient gel electrophoresis. FCM was used after a pretreatment to separate cells from the biofilm matrix, in order to determine densities of heterotrophic bacteria, picocyanobacteria, and pico- and nanoeukaryotes on these coatings. Among diatoms, the only microphytobenthic class identified by microscopy, Licmophora, Navicula, and Nitzschia were determined to be the dominant taxa. Overall, biocide-free coatings showed higher densities than all other coatings, except for one biocidal coating, whatever the group of microorganisms. Heterotrophic bacteria always showed the highest densities, and diatoms showed the lowest, but the relative abundances of these groups varied depending on the coating. In particular, the copper-free SPC failed to prevent diatom settlement, whereas the pyrithione-free SPC exhibited high picocyanobacterial density. These results highlight the interest in FCM for antifouling coating assessment as well as specific selection among microbial communities by antifouling coatings.

Hyperspectral cytometry.

Hyperspectral cytometry is an emerging technology for single-cell analysis that combines ultrafast optical spectroscopy and flow cytometry. Spectral cytometry systems utilize diffraction gratings or prism-based monochromators to disperse fluorescence signals from multiple labels (organic dyes, nanoparticles, or fluorescent proteins) present in each analyzed bioparticle onto linear detector arrays such as multianode photomultipliers or charge-coupled device sensors. The resultant data, consisting of a series of characterizing every analyzed cell, are not compensated by employing the traditional cytometry approach, but rather are spectrally unmixed utilizing algorithms such as constrained Poisson regression or non-negative matrix factorization. Although implementations of spectral cytometry were envisioned as early as the 1980s, only recently has the development of highly sensitive photomultiplier tube arrays led to design and construction of functional prototypes and subsequently to introduction of commercially available systems. This chapter summarizes the historical efforts and work in the field of spectral cytometry performed at Purdue University Cytometry Laboratories and describes the technology developed by Sony Corporation that resulted in release of the first commercial spectral cytometry system-the Sony SP6800. A brief introduction to spectral data analysis is also provided, with emphasis on the differences between traditional polychromatic and spectral cytometry approaches.

Generalized unmixing model for multispectral flow cytometry utilizing nonsquare compensation matrices.

Multispectral and hyperspectral flow cytometry (FC) instruments allow measurement of fluorescence or Raman spectra from single cells in flow. As with conventional FC, spectral overlap results in the measured signal in any given detector being a mixture of signals from multiple labels present in the analyzed cells. In contrast to traditional polychromatic FC, these devices utilize a number of detectors (or channels in multispectral detector arrays) that is larger than the number of labels, and no particular detector is a priori dedicated to the measurement of any particular label. This data-acquisition modality requires a rigorous study and understanding of signal formation as well as unmixing procedures that are employed to estimate labels abundance. The simplest extension of the traditional compensation procedure to multispectral data sets is equivalent to an ordinary least-square (LS) solution for estimating abundance of labels in individual cells. This process is identical to the technique employed for unmixing spectral data in various imaging fields. The present study shows that multispectral FC data violate key assumptions of the LS process, and use of the LS method may lead to unmixing artifacts, such as population distortion (spreading) and the presence of negative values in biomarker abundances. Various alternative unmixing techniques were investigated, including relative-error minimization and variance-stabilization transformations. The most promising results were obtained by performing unmixing using Poisson regression with an identity-link function within a generalized linear model framework. This formulation accounts for the presence of Poisson noise in the model of signal formation and subsequently leads to superior unmixing results, particularly for dim fluorescent populations. The proposed Poisson unmixing technique is demonstrated using simulated 8-channel, 2-fluorochrome data and real 32-channel, 6-fluorochrome data. The quality of unmixing is assessed by computing absolute and relative errors, as well as by calculating the symmetrized Kullback-Leibler divergence between known and approximated populations. These results are applicable to any flow-based system with more detectors than labels where Poisson noise is the dominant contributor to the overall system noise and highlight the fact that explicit incorporation of appropriate noise models is the key to accurately estimating the true label abundance on the cells. © 2013 International Society for Advancement of Cytometry.

Hyperspectral cytometry at the single-cell level using a 32-channel photodetector.

Despite recent progress in cell-analysis technology, rapid classification of cells remains a very difficult task. Among the techniques available, flow cytometry (FCM) is considered especially powerful, because it is able to perform multiparametric analyses of single biological particles at a high flow rate-up to several thousand particles per second. Moreover, FCM is nondestructive, and flow cytometric analysis can be performed on live cells. The current limit for simultaneously detectable fluorescence signals in FCM is around 8-15 depending upon the instrument. Obtaining multiparametric measurements is a very complex task, and the necessity for fluorescence spectral overlap compensation creates a number of additional difficulties to solve. Further, to obtain well-separated single spectral bands a very complex set of optical filters is required. This study describes the key components and principles involved in building a next-generation flow cytometer based on a 32-channel PMT array detector, a phase-volume holographic grating, and a fast electronic board. The system is capable of full-spectral data collection and spectral analysis at the single-cell level. As demonstrated using fluorescent microspheres and lymphocytes labeled with a cocktail of antibodies (CD45/FITC, CD4/PE, CD8/ECD, and CD3/Cy5), the presented technology is able to simultaneously collect 32 narrow bands of fluorescence from single particles flowing across the laser beam in <5 µs. These 32 discrete values provide a proxy of the full fluorescence emission spectrum for each single particle (cell). Advanced statistical analysis has then been performed to separate the various clusters of lymphocytes. The average spectrum computed for each cluster has been used to characterize the corresponding combination of antibodies, and thus identify the various lymphocytes subsets. The powerful data-collection capabilities of this flow cytometer open up significant opportunities for advanced analytical approaches, including spectral unmixing and unsupervised or supervised classification.