Single-Cell Mass Spectrometry Analysis of Metabolites Facilitated by Cell Electro-Migration and Electroporation.

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.

Depth of field multiplexing in microscopy.

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.

Spectro-temporal encoded multiphoton microscopy and fluorescence lifetime imaging at kilohertz frame-rates.

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.

Internal cellular details of Euglena gracilis visualized by scanning electron microscopy.

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.

Persisting circadian rhythm of cell division in a photosynthetic mutant of Euglena.

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.

Galactosyl diglycerides: their possible function in Euglena chloroplasts.

Illumination of euglenas grown in the dark induces the formation of chloroplasts characterized by the simultaneous appearance of chlorophyll and galactosyl diglycerides in a relatively fixed ratio. The fatty acyl chains of the galactosyl diglycerides are constructed so that they can provide a stable lock-and-key fit with the phytol chains of chlorophyll in such a way as to localize the porphyrin structures of chlorophyll and space them for efficient photoreception. Light-starved photobiotic euglenas show chloroplast shrinkage with a concurrent partial loss of galactosyl diglycerides.

A multiple origin for plastids and mitochondria.

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.

The dialogue between protozoa and bacteria in a microfluidic device.

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.

Anthracene phytotoxicity in the freshwater flagellate alga Euglena agilis Carter.

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 (EC50) of 4.28 mg L-1. Between 5 and 15 mg L-1, anthracene inhibited the maximum quantum yield (Fv/Fm) of photosystem II (PSII) and the maximum photosynthetic electron transport rate through PSII (rETRmax) with EC50 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 EC50 values for both overlap with anthracene levels (8.3 mg L-1) permitted by the US Environmental Protection Agency (USEPA).

Phytochrome-like responses in Euglena: A low fluence response that reorganizes the spectral dependence of the high irradiance response in long-day photoperiodic induction of cell division.

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.

High irradiance responses involving photoreversible multiple photoreceptors as related to photoperiodic induction of cell division in Euglena.

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.

Algae through the looking glass.

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.

Fluorescence behavior of Euglena photoreceptor.

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.

Identification of a functional Gs protein in Euglena gracilis.

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.

Gas/liquid sensing via chemotaxis of Euglena cells confined in an isolated micro-aquarium.

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.