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Single-cell metabolite analysis plays an important role in biological study. While mass spectrometry is a powerful tool for identification and quantitation of metabolites, the low absolute analyte amounts in single cell and difficulty in sampling represent significant challenges in single cell analysis. In this study, we developed an effective method with a simple sampling procedure for analyzing single cells. A single cell was driven to a capillary tip through electro-migration, followed by releasing the cell contents through electroporation, into a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent mass spectrometry analysis was performed directly with nanoelectrospray ionization. This method was applied for analyzing a variety of cells and monitoring the metabolic changes in response to perturbed cell culturing conditions. This method opens a new avenue for easy and rapid analysis of single cells with high sensitivity.

Two-Photon Microscopy has become an invaluable tool for biological and medical research, providing high sensitivity, molecular specificity, inherent three-dimensional sub-cellular resolution and deep tissue penetration. In terms of imaging speeds, however, mechanical scanners still limit the acquisition rates to typically 10-100 frames per second. Here we present a high-speed non-linear microscope achieving kilohertz frame rates by employing pulse-modulated, rapidly wavelength-swept lasers and inertia-free beam steering through angular dispersion. In combination with a high bandwidth, single-photon sensitive detector, this enables recording of fluorescent lifetimes at speeds of 88 million pixels per second. We show high resolution, multi-modal - two-photon fluorescence and fluorescence lifetime (FLIM) - microscopy and imaging flow cytometry with a digitally reconfigurable laser, imaging system and data acquisition system. These high speeds should enable high-speed and high-throughput image-assisted cell sorting.

The freshwater flagellate alga Euglena agilis Carter was exposed to the polycyclic aromatic hydrocarbon (PAH) anthracene for 96 h under optimal photosynthetically active radiation (PAR), and responses of growth, photosynthetic pigment production, and photosynthetic efficiency were assessed. Anthracene reduced the growth rate (μ) and levels of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids. The growth rate was more sensitive than photosynthetic parameters, with a median effective concentration (EC 50 ) of 4.28 mg L -1 . Between 5 and 15 mg L -1 , anthracene inhibited the maximum quantum yield (F v /F m ) of photosystem II (PSII) and the maximum photosynthetic electron transport rate through PSII (rETR max ) with EC 50 values of 14.88 and 11.8 mg L -1 , respectively. At all anthracene concentrations, intracellular reactive oxygen species (ROS) were elevated, indicating increased oxidative stress. Anthracene presumably reduced the PSII efficiency of photochemical energy regulation and altered the photochemistry through intracellular ROS formation. Acute exposure to PAHs may induce severe physiological changes in phytoplankton cells, which may influence vital ecological processes within the aquatic environments. Additionally, growth and Chl a content may serve as sensitive risk assessment parameters of anthracene toxicity in water management since EC 50 values for both overlap with anthracene levels (8.3 mg L -1 ) permitted by the US Environmental Protection Agency (USEPA).

In nature, protozoa play a major role in controlling bacterial populations. This paper proposes a microfluidic device for the study of protozoa behaviors change due to their chemotactic response in the presence of bacterial cells. A three-channel microfluidic device was designed using a nitrocellulose membrane into which channels were cut using a laser cutter. The membrane was sandwiched between two glass slides; a Euglena suspension was then allowed to flow through the central channel. The two side channels were filled with either, 0.1% peptone as a negative control, or a Listeria suspension respectively. The membrane design prevented direct interaction but allowed Euglena cells to detect Listeria cells as secretions diffused through the nitrocellulose membrane. A significant number of Euglena cells migrated toward the chambers near the bacterial cells, indicating a positive chemotactic response of Euglena toward chemical cues released from Listeria cells. Filtrates collected from Listeria suspension with a series of molecular weight cutoffs (3k, 10k and 100k) were examined in Euglena chemotaxis tests. Euglena cells were attracted to all filtrates collected from the membrane filtration with different molecular weight cutoffs, suggesting small molecules from Listeria might be the chemical cues to attract protozoa. Headspace volatile organic compounds (VOC) released from Listeria were collected, spiked to 0.1% peptone and tested as the chemotactic effectors. It was discovered that the Euglena cells responded quickly to Listeria VOCs including decanal, 3,5- dimethylbenzaldehyde, ethyl acetate, indicating bacterial VOCs were used by Euglena to track the location of bacteria., Competing Interests: The authors have declared that no competing interests exist.

Microalgae are one of the most suitable subjects for testing the potentiality of light microscopy and image analysis, because of the size of single cells, their endogenous chromaticity, and their metabolic and physiological characteristics. Microscope observations and image analysis can use microalgal cells from lab cultures or collected from water bodies as model to investigate metabolic processes, behavior/reaction of cells under chemical or photic stimuli, and dynamics of population in the natural environment in response to changing conditions. In this paper we will describe the original microscope we set up together with the image processing techniques we improved to deal with these topics. Our system detects and recognizes in-focus cells, extracts their features, measures cell concentration in multi-algal samples, reconstructs swimming cell tracks, monitors metabolic processes, and measure absorption and fluorescent spectra of subcellular compartments. It can be used as digital microscopy station for algal cell biology and behavioral studies, and field analysis applications., (© 2016 Wiley Periodicals, Inc.)

We demonstrate on-chip gas/liquid sensing by using the chemotaxis of live bacteria (Euglena gracilis) confined in an isolated micro-aquarium, and gas/liquid permeation through porous polydimethylsiloxane (PDMS). The sensing chip consisted of one closed micro-aquarium and two separated bypass microchannels along the perimeter of the micro-aquarium. Test gas/liquid and reference samples were introduced into the two individual microchannels separately, and the gas/liquid permeated through the PDMS walls and dissolved in the micro-aquarium water, resulting in a chemical concentration gradient in the micro-aquarium. By employing the closed micro-aquarium isolated from sample flows, we succeeded in measuring the chemotaxis of Euglena for a gas substance quantitatively, which cannot be achieved with the conventional flow-type or hydro-gel-type microfluidic devices. We found positive (negative) chemotaxis for CO2 concentrations below (above) 15%, with 64 ppm as the minimum concentration affecting the cells. We also observed chemotaxis for ethanol and H2O2. By supplying culture medium via the microchannels, the Euglena culture remained alive for more than 2 months. The sensing chip is thus useful for culturing cells and using them for environmental toxicity/nutrition studies by monitoring their motion.

Evolution has selected a system of two intertwined cell cycles: the cell division cycle (CDC) and the daily (circadian) biological clock. The circadian clock keeps track of solar time and programs biological processes to occur at environmentally appropriate times. One of these processes is the CDC, which is often gated by the circadian clock. The intermeshing of these two cell cycles is probably responsible for the observation that disruption of the circadian system enhances susceptibility to some kinds of cancer. The core mechanism underlying the circadian clockwork has been thought to be a transcription & translation feedback loop (TTFL), but recent evidence from studies with cyanobacteria, synthetic oscillators and immortalized cell lines suggests that the core circadian pacemaking mechanism that gates cell division in mammalian cells could be a post-translational oscillator (PTO).

We demonstrate "depth of field multiplexing" by a high resolution spatial light modulator (SLM) in a Fourier plane in the imaging path of a standard microscope. This approach provides simultaneous imaging of different focal planes in a sample with only a single camera exposure. The phase mask on the SLM corresponds to a set of superposed multi-focal off-axis Fresnel lenses, which sharply image different focal planes of the object to non-overlapping adjacent sections of the camera chip. Depth of field multiplexing allows to record motion in a three dimensional sample volume in real-time, which is exemplarily demonstrated for cytoplasmic streaming in plant cells and rapidly swimming protozoa.

Little is known about the photoreceptors involved in the photoperiodism of unicellular organisms, which we elucidated by deriving their action spectra. The flagellated alga Euglena gracilis exhibits photoperiodism, with a long-day response in cell reproduction. The underlying clock is a circadian rhythm with photoinductive capability, peaking at subjective dusk and occurring at the 26th hour in continuous darkness (DD) when transferred from continuous light (LL); it regulates photoinduction, a high-irradiance response (HIR), of a dark-capability of progressing through cell division. We derived the action spectra by irradiating E. gracilis with monochromatic light for 3h at around the 26th hour; the action maxima occurred at 380, 450-460, 480, 610, 640, 660, 680, and 740nm. Except for the maximum at 450-460nm, which was always a major maximum, the maxima greatly depended on the red (R)/far-red (FR) ratio of the prior LL. The high R/FR ratio resulted in a dominant major peak at 640nm and minor peaks at 480 and 680nm, whereas the low ratio resulted in dominant major peaks at 610 and 740nm and minor peaks at 380 and 660nm; the critical fluence was minimally about 60mmolm(-2). These HIRs resulted from the accumulation of corresponding low-fluence responses (LFRs) because we found that repetition of a 3-min light/dark cycle, with critical fluences of 1mmolm(-2), lasting for 3h resulted in the same photoinduction as the continuous 3-h irradiation. Moreover, these LFRs expressed photoreversibility. Thus, photoperiodic photoinduction involves Euglena-phytochrome (640 and 740nm) and blue photoreceptor (460nm). Although 380, 480, 610, 660, and 680nm may also represent Euglena-phytochrome, a definite conclusion awaits further study.

Irradiance spectra change spatiotemporally, and angiosperms adapt accordingly, mainly through phytochromes. This study challenges the long-held belief that the flagellated alga Euglena gracilis lacks phytochromes and is therefore unaffected by spectral changes. We photoautotrophically cultured the alga under continuous light (LL), then transferred it to darkness. After about 26h in darkness, different irradiations for 3h enabled cell division in dark-arrested G2 cells evoking a high-irradiance response (HIR). The spectral characteristics of the irradiation during the LL period (pre-irradiation) defined the spectral sensitivity in the subsequent dark period. LL with light rich in the red spectrum led to a HIR to the red spectrum (R-HIR), whereas light rich in the far-red spectrum (FR) led to a FR-HIR. Finishing the period of pre-irradiation consisting of continuous cool-white fluorescent light (rich in R) by a FR pulse enhanced the characteristics of the FR-HIR 26h later. By contrast, a R pulse given at the end of the pre-irradiation rich in FR potentiated the R-HIR. The effects were completely photoreversible between R and FR with critical fluences of about 2mmolm(-2), satisfying the classic diagnostic feature of phytochromes. The action spectrum of the FR effect at the end of pre-irradiation consisting of continuous cool-white fluorescent light (rich in R) had a main peak at 740nm and a minor peak at 380nm, whereas antagonization of the FR effect had a main peak at 640nm and a minor peak at 480nm. Wavelengths of 610 and 670nm appeared in both spectra. We also demonstrated the photoreversibility of 380/640, 480/740, and (610 and 670)/(640 and 740) nm. We conclude that Euglena displays phytochrome-like responses similar to the 'shade avoidance' and 'end-of-day FR' effects reported in angiosperms.

To provide new information on the series of structural changes that Euglena photoreceptive proteins undergo inside the photoreceptor in response to light, we measured in vivo emission fluorescence spectra in the stable intermediates of its photocycle. Our emission spectra give a certain indication that fluorescent proteins are present in the Euglena photoreceptor and that they undergo a photocycle. On the basis of our data, we suggested that at least two stable intermediates, one of which is fluorescent, can be discriminated at room temperature and with our time resolution.

We found a Gs protein coupled to adenylyl cyclase in a free-living protist, Euglena gracilis. This Gs protein of approximately 42 kDa is substrate for cholera toxin and is recognized by an antibody against the C-terminal decapeptide of Gs. Furthermore, this protein is coupled to adenylyl cyclase, as shown by: (a) the activation of the enzyme by GTP-analogues and (b) the effect of cholera toxin on cAMP accumulation in intact cells and the continuous activation of adenylyl cyclase activity in membranes. These data indicate that the Gs-adenylyl cyclase-coupled system is already apparent in the protist kingdom.

To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density. We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity. The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression. Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.

The localization of DNA in the condensed interphase chromosomes of Euglena was determined by immunoelectron microscopy. Deposits of gold particles that coincided with the localization of DNA followed threads that corresponded to the chromatin fibers. The threads were 55-80nm in diameter and were assumed to be supersolenoids. The localization of gold deposits on chromosomes that had been sectioned in various directions suggested that the chromatin fibers coiled around the surface of chromosomes, with a wide central axial region of the chromosomes remaining free of DNA. These findings are discussed in relation to current models of chromosomal structure.

We have localized LHCP II apoprotein in the Golgi and thylakoids of Euglena gracilis Klebs var. bacillaris Cori and strain Z Pringsheim by electron microscopy using a specific antibody and protein A-gold. Using synchronized cells (light, 14 h:dark, 10 h) we show that thylakoids are always immunoreactive. There is no reaction in the Golgi at 0 h (the beginning of the light period) but immunoreaction appears in the Golgi soon thereafter, rises to a peak at 8 h and declines to zero by 16 h (2 h into the dark period). The peak in immunoreaction in the Golgi immediately precedes the peak in cellular 14C-labeling of thylakoid LHCP II apoprotein seen by Brandt and von Kessel (Plant Physiol. (1983) 72, 616), supporting our suggestion that processing in the Golgi precedes deposition of LHCP apoprotein in the thylakoids. Substitution of preimmune serum for antiserum eliminates the immunoreaction in the Golgi, and thylakoids of synchronized cells of mutant Gr1BSL which lacks LHCP II apoprotein show no immunoreaction in the Golgi or thylakoids at any stage. Random observations indicate that the compartmentalized osmiophilic structure (COS) shows an immunoreaction with anti-LHCP II apoprotein antibody at 1 h into the light period (when the Golgi is not immunoreactive) and at 10 h into the light period (when the Golgi is fully reactive), suggesting that the COS remains immunoreactive throughout the cell cycle.

The nucleoplasm, the interiors of cytoplasmic membrane-bound organelles, and the aqueous cytoplasm make up the aqueous compartments of animal cells. The extent to which these compartments are concentrated solutions of macromolecules, metabolites, ions, and other solutes is a matter of some importance to current thinking about cell structure and function. This paper will focus on the aqueous cytoplasm. It will show that the composition and metabolic activities of the cytosol, obtained by methods of cell disruption and fractionation, bear almost no resemblance to those of the aqueous cytoplasm in intact cells. The consequences of this to contemporary views on cell structure and function are considered. A closely related topic concerns the physical properties of the dominant component of these compartments, water: Are these properties the same as those of water in aqueous solutions, or are they altered as a result of interaction with cell architecture? Available evidence strongly suggests that at least a large fraction of the total cell water exhibits properties that markedly differ from those of pure water. Selected examples of these studies will be reviewed, and the roles of cell water will be discussed, notably as they relate to metabolism and cell ultrastructure. Although dimly perceived at present, it appears that living cells exhibit an organization far greater than the current teachings of cell biology reveal.

In photobiotic Euglena gracilis, chlorophyll biosynthesis takes place at a rate which is a direct function of light intensity. There is stoichiometry between chlorophyll accumulation and that of galactosyl diglycerides. This stoichiometry remains relatively invariant, regardless of the wide changes in chlorophyll content brought about in response to controlled variation of light intensity. These findings suggest that the formation of chlorophyll-containing photoreceptor membranes may be paced and stabilized by concurrent synthesis of galactosyl diglycerides.

The euglenoid flagellates are able to change their shape rapidly in response to a variety of stimuli, or sometimes spontaneously. Two extremes of shape can be identified: the "relaxed" form is cylindrical; the contracted form is a somewhat distorted disc. These 2 forms can be interconverted by treatments that alter the Ca2+ concentration of the entire cell. The level of Ca2+ is believed to be normally controlled by a system of calcium-accumulating membranes, identified in Astasia longa by the technique of calcium oxalate precipitation. The system forms a set of parallel tubes of endoplasmic reticulum, one of which lies immediately below each of the ridges of the pellicle. The individual ridges, each with its associated reticulum, microtubules and other elements are suggested to be independent motor units. Local activation of a small number of these units by Ca2+ is made possible by the arrangement of Ca2+ -sequestering reticulum, producing the characteristic squirming euglenoid movement. Uniform activation or suppression of all units produces the 2 extremes of shape. The pellicle of A. longa with its associated microtubules has been purified and shown to contain a Ca2+ -binding site and ATPase activity.

The free-running circadian rhythm of cell division in the algal flagellate, Euglena gracilis (Z) was perturbed by 3-h light signals of varying intensities imposed at different circadian times (CT). Light pulses within the range of 700 to 7,500 lux were found to yield the same 'strong' (Type 0) phase response curve (PRC) comprising both advance and delay phase shifts as great as 15 h. Dark signals generated a PRC of reduced amplitude with very little, if any, phase advance being observed. Light perturbations of lower intensity, however, elicited quite different responses if applied at a quite specific circadian time: A 40- to 400-lux pulse given at approximately CT 0 (late subjective night) induced total arrhythmicity, and the culture reverted to asynchronous, exponential growth. Different degrees of arrhythmicity were induced by the same low-intensity perturbations (I*) given slightly before or after this sensitive phase point (T*), but if imposed at other circadian times, they generated normal type 0 phase resetting. The demonstration of the existence of this critical pulse (T*, I*) provides further evidence that the cell division cycle of Euglena (and presumably other microorganisms) is regulated by a circadian oscillator and, in particular, by one having limit cycle dynamics.

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.

A persisting, free-running, circadian rhythm of cell division in a heterotrophically grown mutant of Euglena gracilis var. bacillaris having impaired photosynthesis is obtained upon placing a culture that has been previously synchronized by a 10,14 light-dark cycle into continuous darkness at 19 degrees C (but not at 25 degrees C). A similar persisting rhythm is initiated in exponentially increasing cultures (growing in darkness at 19 degrees C) by a single "switch-up" in irradiance to continuous bright illumination. The results implicate an endogenous biological clock which "gates" the specific event of cell division in the cell developmental cycle.

Simple washing of live Euglena gracilis cleans the pellicle of external coating material. When these cleaned cells are viewed with a scanning electron microscope, organelle outlines can be seen through the thinned pellicle. To view organelles directly, fixed Euglena cells are gently broken first or first frozen and then broken as part of their preparation for scanning electron microscopy. Subsequent viewing at magnifications of x 5,000 to x 21,000 reveals organelles, storage granules, endoplasmic reticulum, and nuclear pores, while retaining great depth of focus.

The impressive homologies between mitochondria and plastids, on the one hand, and procaryotic organisms, on the other, make it almost certain that these important cellular organelles had their origin as independent organisms. The vast number of symbiotic relationships of all degrees of evolutionary antiquity which have been found in contemporary organisms point to the ease with which such relationships can be established.

Preillumination, followed by a dark period prior to exposure of dark-grown nondividing cells of Euglena gracilis var. bacillaris to normal lighting conditions for chloroplast development, results in potentiation, or abolishment of the usual lag in chlorophyll accumulation. The degree of potentiation is a function of the length of the preillumination period, the intensity of preilluminating light, and the length of the dark period interposed before re-exposure to continuous light for development. The optimal conditions are found to be: 90 minutes of preillumination with white light at an intensity greater than 30 microwatts per square centimeter (14 foot candles) followed by a dark period of at least 12 hours. Reciprocity is not found between duration and intensity of preilluminating light. Preillumination with blue light and red light was found to be the most effective in promoting potentiation, and the ratio of effectiveness of blue to green to red is consistent with protochlorophyll-(ide) being the photoreceptor. Although red light is effective, there is no reversal by far red light, and these facts, taken together with the effectiveness of blue light, suggest that the phytochrome system is not involved. The amount of chlorophyll formed at the end of preillumination is proportional to the resulting potentiation, suggesting that the amount of protochlorophyll(ide) removed or chlorophyll(ide) formed regulates this phenomenon. Potentiated and nonpotentiated cells show comparable rates of protochlorophyll(ide) resynthesis, suggesting that this is not the limiting factor in nonpotentiated cells. Although light is required for protochlorophyll(ide) conversion in chlorophyll synthesis, a brief preillumination seems also to initiate the production of components in the subsequent dark period which, in nonpotentiated cells, are ordinarily synthesized during the lag period under continuous illumination. These components are necessary to sustain maximal rates of subsequent chlorophyll accumulation.