Simultaneous probing of dual intracellular metabolites (ATP and paramylon) in live microalgae using graphene oxide/aptamer nanocomplex.

The development of an intracellular metabolite imaging platform for live microorganisms has been a challenge in the study of microbes. Herein, we performed metabolite imaging in live microalgal cells using a graphene oxide (GO)/aptamer complex. The properties of the GO were characterized using dynamic light scattering (DLS) and atomic force microscopy (AFM), which were determined to have 140 ± 3 nm in mean diameter. An ATP-specific aptamer was mixed with GO to form a GO/aptamer complex, and the feasibility of the complex was tested in vitro. The high correlation between the fluorescence intensity and concentration of ATP was observed in the range 0-10 mM. Next, the feasibility of the complex was confirmed in vivo. Under both phototrophic and heterotrophic culture conditions, Euglena gracilis internalized the complex, and bright fluorescence was observed as the aptamer was bound to the target metabolite (ATP). The fluorescence intensity of cells was correlated to the ATP concentration in the cells. Imaging of dual intracellular metabolites (ATP and paramylon) was achieved by simply using two different aptamers (ATP-specific aptamer and paramylon-specific aptamer) together, showing the great potential of the complex as a dual-sensing/imaging platform. In addition, the GO/aptamer complex exhibited low cytotoxicity; the proliferation and viability of E. gracilis cells were not significantly affected by the complex. Our results suggested that this new imaging platform can be efficiently used for detecting dual intracellular metabolites in live microalgal cells.

Molecular characterization of water extractable Euglena gracilis cellular material composition using asymmetrical flow field-flow fractionation and high-resolution mass spectrometry.

Asymmetrical flow field-flow fractionation (AF4) and high-resolution Orbitrap mass spectrometry (HRMS) were used to separate and characterize cellular fractions of the dark- and light-grown Euglena gracilis cellular material. Biological replicates analyzed by HRMS shared 21-73% of commonly detected m/z values. Greater variability in shared features was found in light-grown cellular fractions (p < 0.05), likely due to small variations in growth stage. Significant differences in molecular composition were observed between AF4 cellular fractions, with dark cell fractions showing a propensity towards carbohydrate-like and tannin-like compounds, and higher double-bond equivalent (DBE) and modified aromatic index (AImod) were associated with light-grown cell fractions. Fractionation and high-resolution mass spectrometry aided characterization demonstrated the power of the AF4 to selectively cater to certain compounds/cellular entities with distinct compositional classes and double-bond equivalents and aromaticity index characteristics. Graphical abstract.

Rapid, Untargeted Chemical Profiling of Single Cells in Their Native Environment.

We report a method that enables untargeted, high throughput, and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture-mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into an LVC-MS sampling probe. Once exposed to an appropriate solvent, the cell is lysed, extracted, and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly, and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.

Single-Cell Analysis of Morphological and Metabolic Heterogeneity in Euglena gracilis by Fluorescence-Imaging Flow Cytometry.

Microalgal biofuels and biomass have ecofriendly advantages as feedstocks. Improved understanding and utilization of microalgae require large-scale analysis of the morphological and metabolic heterogeneity within populations. Here, with Euglena gracilis as a model microalgal species, we evaluate how fluorescence- and brightfield-derived-image-based descriptors vary during environmental stress at the single-cell level. This is achieved with a new multiparameter fluorescence-imaging cytometric technique that allows the assaying of thousands of cells per experiment. We track morphological changes, including the intensity and distribution of intracellular lipid droplets, and pigment autofluorescence. The combined fluorescence-morphological analysis identifies new metrics not accessible with traditional flow cytometry, including the lipid-to-cell-area ratio (LCAR), which shows promise as an indicator of oil productivity per biomass. Single-cell metrics of lipid productivity were highly correlated ( R2 > 0.90, p < 0.005) with bulk oil extraction. Such chemomorphological atlases of algal species can help optimize growth conditions and selection approaches for large-scale biomass production.

Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe.

Microalgae offer great potential for the production of biofuel, but high photosynthetic activity is demanded for the practical realisation of microalgal biofuels. To this end, it is essential to evaluate the photosynthetic activity of single microalgal cells in a heterogeneous population. In this study, we present a method to monitor the photosynthetic activity of microalgae (in particular Euglena gracilis, a microalgal species of unicellular, photosynthetic, flagellate protists as our model organism) at single-cell resolution by Raman spectroscopy with deuterium from deuterium oxide (D2 O) as a tracking probe. Specifically, we replaced H2 O in culture media with D2 O up to a concentration of 20 % without disturbing the growth rate of E. gracilis cells and evaluated C-D bond formation as a consequence of photosynthetic reactions by Raman spectroscopy. We used the probe to monitor the kinetics of the C-D bond formation in E. gracilis cells by incubating them in D2 O media under light irradiation. Furthermore, we demonstrated Raman microscopy imaging of each single E. gracilis cell to discriminate deuterated cells from normal cells. Our results hold great promise for Raman-based screening of E. gracilis and potentially other microalgae with high photosynthetic activity by using D2 O as a tracking probe.

Study of the behavior of Euglena viridis, Euglena gracilis and Lepadella patella cultured in all-glass microaquarium.

In the paper, the microaquarium fabricated in a form of entirely glass lab-on-a-chip for culturing and microscale study of microorganisms has been presented. A new approach towards cellular studies that brings a significant improvement over commonly utilized - polymer-based solutions has been shown. For the first time, all-borosilicate glass chip was applied for the culturing of the selected microorganisms and enabled notable population growth and behaviorism investigation. The chip fabrication method in comparison to typical glass chip technology was notably simplified, including quick patterning and low temperature bonding in 80 °C. In the studies, both a single-cell (Euglena gracilis and Euglena viridis) and multi-cell microorganisms (Lepadella patella) were cultured in the microaquarium. Behaviorism of the selected microorganisms was investigated by supplying various proportions of carbon dioxide, nitrogen and air into the chip. Tests included studies of microorganisms chemotaxis, viability (mostly based on photosynthesis process) and coexistence in the lab-on-a-chip environment. The experiments confirmed that the developed chip is a tool that fits the requirements for the culturing and behavioral studies of microorganisms and constitute ground-works to propel its further application in broadly defined cellular study field.

Scaling body size fluctuations.

The size of an organism matters for its metabolic, growth, mortality, and other vital rates. Scale-free community size spectra (i.e., size distributions regardless of species) are routinely observed in natural ecosystems and are the product of intra- and interspecies regulation of the relative abundance of organisms of different sizes. Intra- and interspecies distributions of body sizes are thus major determinants of ecosystems' structure and function. We show experimentally that single-species mass distributions of unicellular eukaryotes covering different phyla exhibit both characteristic sizes and universal features over more than four orders of magnitude in mass. Remarkably, we find that the mean size of a species is sufficient to characterize its size distribution fully and that the latter has a universal form across all species. We show that an analytical physiological model accounts for the observed universality, which can be synthesized in a log-normal form for the intraspecies size distributions. We also propose how ecological and physiological processes should interact to produce scale-invariant community size spectra and discuss the implications of our results on allometric scaling laws involving body mass.

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis.

Euglena gracilis has the ability to accumulate a storage polysaccharide, a ß-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The Km and Vmax values of EgFBPaseIII were 16.5 ± 1.6 µM and 30.4 ± 7.2 µmol min(-1) mg protein(-1), respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.

Optimization of complex medium composition for heterotrophic cultivation of Euglena gracilis and paramylon production.

Heterotrophic cultivation of Euglena gracilis was carried out on synthetic (Hutner medium) and complex cultivation media in order to optimize production of ß-1,3-glucan (paramylon). For preparation of complex media, various industrial by-products (e.g., molasses, corn steep solid, yeast extract, and beef extract) were used with or without addition of pure compounds [glucose, galactose, fructose, lactose, maltose, sucrose, and (NH4)2HPO4]. Heterotrophic cultivation of E. gracilis was performed in Erlenmeyer flasks and additionally confirmed during research in the stirred tank bioreactor. The results clearly show that E. gracilis can easily metabolize glucose and fructose as carbon sources and corn steep solid as complex nitrogen and growth factors source for biomass growth and paramylon synthesis. Furthermore, it was also proved that addition of (NH4)2HPO4, beef extract, or gibberellic acid did not have positive effect on the biomass growth and paramylon synthesis. After optimization of complex medium composition and verification in the stirred tank bioreactor, it was concluded that medium composed of glucose (20 g/L) and corn steep solid (30 g/L) is the most suitable complex medium for industrial cultivation of E. gracilis and paramylon production.

Identification and functional analysis of peroxiredoxin isoforms in Euglena gracilis.

Euglena gracilis lacks catalase and contains ascorbate peroxidase (APX) which is localized exclusively in the cytosol. Other enzymes that scavenge reactive oxygen species (ROS) in Euglena have not yet been identified; therefore, ROS metabolism, especially in organelles, remains unclear in Euglena. The full-length cDNAs of four Euglena peroxiredoxins (EgPrxs) were isolated in this study. EgPrx1 and -4 were predicted to be localized in the cytosol, and EgPrx2 and -3 in plastids and mitochondria, respectively. The catalytic efficiencies of recombinant EgPrxs were similar to those of plant thiol-peroxidases, but were markedly lower than those of APX from Euglena. However, transcript levels of EgPrx1, -2, and -3 were markedly higher than those of APX. The growth rate of Euglena cells, in which the expression of EgPrx1 and -4 was suppressed by gene silencing, was markedly reduced under normal conditions, indicating physiological significance of Prx proteins.

Sub-pixel resolving optofluidic microscope for on-chip cell imaging.

We report the implementation of a fully on-chip, lensless, sub-pixel resolving optofluidic microscope (SROFM). The device utilizes microfluidic flow to deliver specimens directly across a complementary metal oxide semiconductor (CMOS) sensor to generate a sequence of low-resolution (LR) projection images, where resolution is limited by the sensor's pixel size. This image sequence is then processed with a pixel super-resolution algorithm to reconstruct a single high resolution (HR) image, where features beyond the Nyquist rate of the LR images are resolved. We demonstrate the device's capabilities by imaging microspheres, protist Euglena gracilis, and Entamoeba invadens cysts with sub-cellular resolution and establish that our prototype has a resolution limit of 0.75 microns. Furthermore, we also apply the same pixel super-resolution algorithm to reconstruct HR videos in which the dynamic interaction between the fluid and the sample, including the in-plane and out-of-plane rotation of the sample within the flow, can be monitored in high resolution. We believe that the powerful combination of both the pixel super-resolution and optofluidic microscopy techniques within our SROFM is a significant step forwards toward a simple, cost-effective, high throughput and highly compact imaging solution for biomedical and bioscience needs.

Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis.

The unicellular flagellate Euglena gracilis shows a negative gravitactic behavior. This is based on physiological mechanisms which in the past have been indirectly assessed. Meanwhile, it was possible to isolate genes involved in the signal transduction chain of gravitaxis. The DNA sequences of five calmodulins were found in Euglena, one of which was only known in its protein structure (CaM.1); the other four are new. The biosynthesis of the corresponding proteins of CaM.1-CaM.5 was inhibited by means of RNA interference to determine their involvement in the gravitactic signal transduction chain. RNAi of CaM.1 inhibits free swimming of the cells and pronounced cell-form aberrations. The division of cells was also hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Blockage of CaM.3 to CaM. 5 did not impair gravitaxis. In contrast, the blockage of CaM.2 has only a transient and not pronounced influence on motility and cell form, but leads to a total loss of gravitactic orientation for more than 30 days. This indicates that CaM.2 is an element in the signal transduction chain of gravitaxis in E. gracilis. The results are discussed with regard to the current working model of gravitaxis in E. gracilis.

Parallel on-axis holographic phase microscopy of biological cells and unicellular microorganism dynamics.

We apply a wide-field quantitative phase microscopy technique based on parallel two-step phase-shifting on-axis interferometry to visualize live biological cells and microorganism dynamics. The parallel on-axis holographic approach is more efficient with camera spatial bandwidth consumption compared to previous off-axis approaches and thus can capture finer sample spatial details, given a limited spatial bandwidth of a specific digital camera. Additionally, due to the parallel acquisition mechanism, the approach is suitable for visualizing rapid dynamic processes, permitting an interferometric acquisition rate equal to the camera frame rate. The method is demonstrated experimentally through phase microscopy of neurons and unicellular microorganisms.

Effect of aluminum on cellular division and photosynthetic electron transport in Euglena gracilis and Chlamydomonas acidophila.

The present study investigated aluminum's effect on cellular division and the photosynthetic processes in Euglena gracilis and Chlamydomonas acidophila at pH 3.0, at which Al is present mostly as Al(3+), AlSO(4) (+), and Al(SO(4))(2) (-). These algal species were exposed to 100, 188, and 740 microM Al, and after 24 h cell-bound Al was significantly different from control only for the highest concentration tested. However, very different effects of Al on algal cellular division, biomass per cell, and photosynthetic activity were found. Aluminum stimulated cell division but decreased at some level biomass per cell in C. acidophila. Primary photochemistry of photosynthesis, as Photosystem II quantum yield, and energy dissipation via nonphotochemical activity were slightly affected. However, for E. gracilis, under the same conditions, Al did not show a stimulating effect on cellular division or photosynthetic activity. Primary photochemical activity was diminished, and energy dissipation via nonphotochemical pathways was strongly increased. Therefore, when Al is highly available in aquatic ecosystems, these effects may indicate very different response mechanisms that are dependent on algal species.

Chromium- and copper-induced inhibition of photosynthesis in Euglena gracilis analysed on the single-cell level by fluorescence kinetic microscopy.

Here, we investigated effects of copper (Cu) and chromium (Cr) toxicity on two contrasting strains of Euglena gracilis, with and without chloroplasts, grown in culture media promoting either phototrophic or heterotrophic growth. This led to insights into Cr/Cu toxicity mechanisms and into the regulation of phototrophic vs heterotrophic metabolism. Our data strongly suggest that in Cu(2+) and Cr(6+) stressed Euglena photosynthesis is the primary target of damage. In the applied light conditions, this was mainly damage to the photosystem II reaction centre, as shown by single-cell measurements of photochemical fluorescence quenching. Respiration and photosynthetic dark reactions were less sensitive. The malfunctioning photosynthesis enhanced production of reactive oxygen species (mainly superoxide), leading to elevated amounts of carotenoid degradation products. At higher metal concentrations in chloroplast-containing cells, but not white cells, this oxidative stress resulted in increased respiratory oxygen uptake, likely by damage to mitochondria. During growth in nutrient solution promoting heterotrophic metabolism, the cells were able to repair the metal-induced damage to photosynthesis, moderating the inhibition of photochemistry. Growth in medium forcing the cells into photosynthesis increased the investment in photosynthetic pigments. Comparison of the two Euglena strains surprisingly showed that the previously metal-resistant strain lost this resistance during culture.

Freeze etching of cells without cryoprotectants.

The technique of spray-freeze etching was applied to unicellular organisms. The superior freezing rates obtainable with this method gave excellent cryofixation on Chlorella, Euglena, and spermatozoa without the use of antifreeze agents, and cell damage due to ice crystal formation was never observed. In many instances the resultant morphology differed significantly from that obtained from glycerol-treated, freeze-etched cells. Furthermore, viability studies of spray-frozen Chlorella compared favorably with cells frozen by other methods.

Phototaxis and sensory transduction in Euglena.

The accumulation of Euglena gracilis in an illuminated region is brought about by two main mechanisms: orientation and subsequent directed movement (positive phototaxis) toward light scattered from particles in the illuminated zone; and by the trapping of cells in this region because of shock reactions experienced upon the cells encountering a sudden decrease of light intensity at the light-dark boundary (inverse photophobic responses). Phototactic orientation is mediated by inverse photophobic reactions which occur when the shadow of the stigma periodically falls upon the photoreceptor proper. Euglena also exhibits shock reactions when an already high light intensity is increased further (direct photophobic responses). The expression of both types of phobic responses depends upon stimulus intensity and adaptation of the sensory system in a seemingly complex way. A definition of the minimum components of the stimulus transduction system and a systems analytical approach to the study of input-output relationships enables one to construct an electronic analog of the cell's signal processing system that converts the photoreceptor input to commands which activate or inhibit flagellar reorientation. Computer simulation studies show that this model has considerable predictive value. It is hoped that with the approach presented in this article, a generalized model has become available for dealing with the questions of sensory transduction in aneural systems. Certainly, at this point more questions have been raised than have been answered. Where is the processing device located? Are its kinetic properties determined by electrical processes or by the rates of chemical reactions? Is the processor, and thereby the behavior of the orgamism, modulated by natural environmental parameters, and can it be modified permanently through more drastic chemical treatment of the cell? Is the system capable of permanent or transitory modification through repeated response, that is, does it exhibit phenomena analogous to learning and memory in higher organisms? These are only a few of the problems that require study in the future.

Expression of nucleus-encoded genes for chloroplast proteins in the flagellate Euglena gracilis.

Reverse transcription PCRs (RT-PCRs), real-time RT-PCRs and microarrays containing 50-mer oligonucleotides representing nucleus-encoded genes for chloroplast proteins from Euglena gracilis were used to compare light- and dark-grown wild-type mRNA levels to those of light- and dark-grown E. gracilis stable white mutant strains W(gm)ZOflL, W3BUL and W10BSmL. The analyses revealed no light-dependent regulation of mRNA levels. Moreover, the mRNA levels of most genes were unchanged in all white mutants in comparison with wild-type. These results suggest that mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis do not depend on either light or plastid function.

Simple Isolation of Single Cell: Thin Glass Microfluidic Device for Observation of Isolated Single Euglena gracilis Cells.

Single cell analysis has gained attention as a means to investigate the heterogeneity of cells and amplify a cell with desired characteristics. However, obtaining a single cell from a large number of cells remains difficult because preparation of single-cell samples relies on conventional techniques such as pipetting that are labor intensive. In this study, we developed a system combining a 0.6-mm thin glass microfluidic device and machine vision approach to isolate single Euglena gracilis cells, as a model of microorganism with mobility, in a small/thin glass chamber. A single E. gracilis cell in a chamber was cultured for 4 days to monitor its multiplication. With this system, we successfully simplified preparation of single cells of interest and determined that it is possible to combine it with other analytical techniques to observe single cells continuously.

Euglena gracilis growth and cell composition under different temperature, light and trophic conditions.

BACKGROUND: Euglena gracilis, a photosynthetic protist, produces protein, unsaturated fatty acids, wax esters, and a unique ß-1,3-glucan called paramylon, along with other valuable compounds. The cell composition of E. gracilis was investigated in this study to understand how light and organic carbon (photo-, mixo- and heterotrophic conditions) affected growth and cell composition (especially lipids). Comparisons were primarily carried out in cultures grown at 23 °C, but the effect of growth at higher temperatures (27 or 30 °C) was also considered. CELL GROWTH: Specific growth rates were slightly lower when E. gracilis was grown on glucose in either heterotrophic or mixotrophic conditions than when grown photoautotrophically, although the duration of exponential growth was longer. Temperature determined the rate of exponential growth in all cultures, but not the linear growth rate during light-limited growth in phototrophic conditions. Temperature had less effect on cell composition. CELL COMPOSITION: Although E. gracilis was not expected to store large amounts of paramylon when grown phototrophically, we observed that phototrophic cells could contain up to 50% paramylon. These cells contained up to 33% protein and less than 20% lipophilic compounds, as expected. The biomass contained about 8% fatty acids (measured as fatty acid methyl esters), most of which were unsaturated. The fatty acid content of cells grown in mixotrophic conditions was similar to that observed in phototrophic cells, but was lower in cells grown heterotrophically. Heterotrophic cells contained less unsaturated fatty acids than phototrophic or mixotrophic cells. α-Linolenic acid was present at 5 to 18 mg g-1 dry biomass in cells grown in the presence of light, but at < 0.5 mg g-1 biomass in cells grown in the dark. Eicosapentaenoic and docosahexaenoic acids were detected at 1 to 5 mg g-1 biomass. Light was also important for the production of vitamin E and phytol.