Advanced Microscopy Techniques for Molecular Biophysics.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Glucans, Paramylon and Other Algae Bioactive Molecules.

Algae (macro- and micro-algae) can be defined as light-driven cell factories that synthesize bioactive compounds consisting of primary metabolites (i [...].

Paramylon and Other Bioactive Molecules in Micro and Macroalgae.

Many algae synthesize compounds that have exceptional properties of nutraceutical, pharmacological, and biomedical interest. Pigments, fatty acids, phenols, and polysaccharides are among the main compounds investigated so far. Polysaccharides are the most exploited compounds, widely used in pharmaceutical, food, and chemical industries, which are at present entering into more advanced applications by gaining importance, from a therapeutic point of view, as antioxidant, antimicrobial, antitumor, and immunomodulatory agents. Establishing algae as an alternative supplement would complement the sustainable and environmental requirements in the framework of human health and well-being. This review focuses on the proprieties and uses of the main micro- and macroalgae metabolites, describing their potential for application in the different industrial sectors, from food/feed to chemical and pharmacological. Further, current technologies involved in bioactive molecule extraction strategies are documented.

Water monitoring by means of digital microscopy identification and classification of microalgae.

Marine and freshwater microalgae belong to taxonomically and morphologically diverse groups of organisms spanning many phyla with thousands of species. These organisms play an important role as indicators of water ecosystem conditions since they react quickly and predictably to a broad range of environmental stressors, thus providing early signals of dangerous changes. Traditionally, microscopic analysis has been used to identify and enumerate different types of organisms present within a given environment at a given point in time. However, this approach is both time-consuming and labor intensive, as it relies on manual processing and classification of planktonic organisms present within collected water samples. Furthermore, it requires highly skilled specialists trained to recognize and distinguish one taxa from another on the basis of often subtle morphological differences. Given these restrictions, a considerable amount of effort has been recently funneled into automating different steps of both the sampling and classification processes, making it possible to generate previously unprecedented volumes of plankton image data and obtain an essential database to analyze the composition of plankton assemblages. In this review we report state-of-the-art methods used for automated plankton classification by means of digital microscopy. The computer-microscope system hardware and the image processing techniques used for recognition and classification of planktonic organisms (segmentation, shape feature extraction, pigment signature determination and neural network grouping) will be described. An introduction and overview of the topic, its current state and indications of future directions the field is expected to take will be provided, organizing the review for both experts and researchers new to the field.

Unveiling the Secrets of Escher's Lithographs.

An impossible structure gives us the impression of looking at a three-dimensional object, even though this object cannot exist, since it possesses parts that are spatially non-connectable, and are characterized by misleading geometrical properties not instantly evident. Therefore, impossible artworks appeal to our intellect and challenge our perceptive capacities. We analyzed lithographs containing impossible structures (e.g., the Necker cube), created by the famous Dutch painter Maurits Cornelis Escher (1898-1972), and used one of them (The Belvedere, 1958) to unveil the artist's hidden secrets by means of a discrete model of the human retina based on a non-uniform distribution of receptive fields. We demonstrated that the ability of Escher in composing his lithographs by connecting spatial coherent details into an impossible whole lies in drawing these incoherent fragments just outside the zone in which 3D coherence can be perceived during a single fixation pause. The main aspects of our paper from the point of view of image processing and image understanding are the following: (1) the peculiar and original digital filter to process the image, which simulates the human vision process, by producing a space-variant sampling of the image; (2) the software for the filter, which is homemade and created for our purposes. The filtered images resulting from the processing are used to understand impossible figures. As an example, we demonstrate how the impossible figures hidden in Escher's paintings can be understood.

Paramylon, a Potent Immunomodulator from WZSL Mutant of Euglena gracilis.

ß-glucans, heterogeneous glucose polymers present in many organisms, have the capability to activate the innate immune system. Efficacy of activation depends on purity of the compound, molecular structure, polymerization degree, and source. One of the purest forms of crystallized ß-(1-3)-glucan present in nature is the paramylon extracted from the WZSL non-chloroplastic mutant of Euglena gracilis, which can be processed to produce linear nanofibers capable of interacting with specific receptors present on cell membranes. The effects of these nanofibers, already investigated on plants, animals, and humans, will be analyzed.

The role of Euglena gracilis paramylon in modulating xylem hormone levels, photosynthesis and water-use efficiency in Solanum lycopersicum L.

ß-1,3-glucans such as paramylon act as elicitors in plants, modifying the hormonal levels and the physiological responses. Plant hormones affect all phases of the plant life cycle and their responses to environmental stresses, both biotic and abiotic. The aim of this study was to investigate the effects of a root treatment with Euglena gracilis paramylon on xylem hormonal levels, photosynthetic performance and dehydration stress in tomato (Solanum lycopersicum). Paramylon granules were processed to obtain the linear fibrous structures capable to interact with tomato cell membrane. Modulation of hormone levels (abscisic acid, jasmonic acid and salicylic acid) and related physiological responses such as CO2 assimilation rate, stomatal and mesophyll conductance, intercellular CO2 concentration, transpiration rate, water-use efficiency, quantum yield of photosystem II and leaf water potential were investigated. The results indicate a clear dose-dependent effect of paramylon on the hormonal content of xylem sap, photosynthetic performance and dehydration tolerance. Paramylon has the capability to enhance plant defense capacity against abiotic stress, such as drought, by modulating the conductance to CO2 diffusion from air to the carboxylation sites and improving the water-use efficiency.

Euglena gracilis paramylon activates human lymphocytes by upregulating pro-inflammatory factors.

The aim of this study was to verify the activation details and products of human lymphomonocytes, stimulated by different ß-glucans, that is Euglena paramylon, MacroGard®, and lipopolysaccharide. We investigated the gene expression of inflammation-related cytokines and mediators, transactivation of relevant transcription factors, and phagocytosis role in cell-glucan interactions, by means of RT-PCR, immunocytochemistry, and colorimetric assay. Our results show that sonicated and alkalized paramylon upregulates pro-inflammatory factors (NO, TNF-α, IL-6, and COX-2) in lymphomonocytes. A clear demonstration of this upregulation is the increased transactivation of NF-kB visualized by immunofluorescence microscopy. Phagocytosis assay showed that internalization is not a mandatory step for signaling cascade to be triggered, since immune activity is not present in the lymphomonocytes that have internalized paramylon granules and particulate MacroGard®. Moreover, the response of Euglena ß-glucan-activated lymphomonocytes is much greater than that induced by commercially used ß-glucans such as MacroGard®. Our in vitro results indicate that linear fibrous Euglena ß-glucan, obtained by sonication and alkaline treatment can act as safe and effective coadjutant of the innate immune system response.

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.

Reconstruction of the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its digital image: the challenge of algal colours.

A novel procedure for deriving the absorption spectrum of an object spot from the colour values of the corresponding pixel(s) in its image is presented. Any digital image acquired by a microscope can be used; typical applications are the analysis of cellular/subcellular metabolic processes under physiological conditions and in response to environmental stressors (e.g. heavy metals), and the measurement of chromophore composition, distribution and concentration in cells. In this paper, we challenged the procedure with images of algae, acquired by means of a CCD camera mounted onto a microscope. The many colours algae display result from the combinations of chromophores whose spectroscopic information is limited to organic solvents extracts that suffers from displacements, amplifications, and contraction/dilatation respect to spectra recorded inside the cell. Hence, preliminary processing is necessary, which consists of in vivo measurement of the absorption spectra of photosynthetic compartments of algal cells and determination of spectra of the single chromophores inside the cell. The final step of the procedure consists in the reconstruction of the absorption spectrum of the cell spot from the colour values of the corresponding pixel(s) in its digital image by minimization of a system of transcendental equations based on the absorption spectra of the chromophores under physiological conditions.

Swimming patterns of the quadriflagellate Tetraflagellochloris mauritanica (Chlamydomonadales, Chlorophyceae).

Chlamydomonadales are elective subjects for the investigation of the problems related to locomotion and transport in biological fluid dynamics, whose resolution could enhance searching efficiency and assist in the avoidance of dangerous environments. In this paper, we elucidate the swimming behavior of Tetraflagellochloris mauritanica, a unicellular-multicellular alga belonging to the order Chlamydomonadales. This quadriflagellate alga has a complex swimming motion consisting of alternating swimming phases connected by in-place random reorientations and resting phases. It is capable of both forward and backward swimming, both being normal modes of swimming. The complex swimming behavior resembles the run-and-tumble motion of peritrichous bacteria, with in-place reorientation taking the place of tumbles. In the forward swimming, T. mauritanica shows a very efficient flagellar beat, with undulatory retrograde waves that run along the flagella to their tip. In the backward swimming, the flagella show a nonstereotypical synchronization mode, with a pattern that does not fit any of the modes present in the other Chlamydomonadales so far investigated.

Water monitoring: automated and real time identification and classification of algae using digital microscopy.

Microalgae are unicellular photoautotrophs that grow in any habitat from fresh and saline water bodies, to hot springs and ice. Microalgae can be used as indicators to monitor water ecosystem conditions. These organisms react quickly and predictably to a broad range of environmental stressors, thus providing early signals of a changing environment. When grown extensively, microalgae may produce harmful effects on marine or freshwater ecology and fishery resources. Rapid and accurate recognition and classification of microalgae is one of the most important issues in water resource management. In this paper, a methodology for automatic and real time identification and enumeration of microalgae by means of image analysis is presented. The methodology is based on segmentation, shape feature extraction, pigment signature determination and neural network grouping; it attained 98.6% accuracy from a set of 53,869 images of 23 different microalgae representing the major algal phyla. In our opinion this methodology partly overcomes the lack of automated identification systems and is on the forefront of developing a computer-based image processing technique to automatically detect, recognize, identify and enumerate microalgae genera and species from all the divisions. This methodology could be useful for an appropriate and effective water resource management.

A second rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein expression in a cell-free system.

Here we report the identification and expression of a second rhodopsin-like protein in the alga Cyanophora paradoxa (Glaucophyta), named Cyanophopsin_2. This new protein was identified due to a serendipity event, since the RACE reaction performed to complete the sequence of Cyanophopsin_1, (the first rhodopsin-like protein of C. paradoxa identified in 2009 by our group), amplified a 619 bp sequence corresponding to a portion of a new gene of the same protein family. The full sequence consists of 1175 bp consisting of 849 bp coding DNA sequence and 4 introns of 326 bp. The protein is characterized by an N-terminal region of 47 amino acids, followed by a region with 7 α-helices of 213 amino acids and a C-terminal region of 22 amino acids. This protein showed high identity with Cyanophopsin_1 and other rhodopsin-like proteins of Archea, Bacteria, Fungi and Algae. Cyanophosin_2 (CpR2) was expressed in a cell-free expression system, and characterized by means of absorption spectroscopy.

Automatic and real time recognition of microalgae by means of pigment signature and shape.

Microalgae are unicellular photoautotrophic organisms that grow in any habitat such as fresh and salt water bodies, hot springs, ice, air, and in or on other organisms and substrates. Massive growth of microalgae may produce harmful effects on the marine and freshwater ecological environment and fishery resources. Therefore, rapid and accurate recognition and classification of microalgae is one of the most important issues in water resource management. In this paper, a new methodology for automatic and real time identification of microalgae by means of microscopy image analysis is presented. This methodology is based on segmentation, shape features extraction, and characteristic colour (i.e. pigment signature) determination. A classifier algorithm based on the minimum distance criterion was used for microalgae grouping according to the measured features. 96.6% accuracy from a set of 3423 images of 24 different microalgae representing the major algal phyla was achieved by this methodology.

Tetraflagellochloris mauritanica gen. et sp. nov. (Chlorophyceae), a New Flagellated Alga from the Mauritanian Desert: Morphology, Ultrastructure, and Phylogenetic Framing.

Morphological, ultrastructural, and molecular-sequence data were used to assess the phylogenetic position of a tetraflagellate green alga isolated from soil samples of a saline dry basin near F'derick, Mauritania. This alga can grow as individual cells or form non-coenobial colonies of up to 12 individuals. It has a parietal chloroplast with an embedded pyrenoid covered by a starch sheath and traversed by single parallel thylakoids, and an eyespot located in a parietal position opposite to the flagellar insertion. Lipid vacuoles are present in the cytoplasm. Microspectroscopy indicated the presence of chlorophylls a and b, with lutein as the major carotenoid in the chloroplast, while the eyespot spectrum has a shape typical of green-algal eyespots. The cell has four flagella, two of them long and two considerably shorter. Sequence data from the 18S rRNA gene and ITS2 were obtained and compared with published sequences for green algae. Results from morphological and ultrastructural examinations and sequence analysis support the placement of this alga in the Chlorophyceae, as Tetraflagellochloris mauritanica L. Barsanti et A. Barsanti, gen. et sp. nov.

Fundamental questions and concepts about photoreception and the case of Euglena gracilis.

The ability to sense light can be considered the most fundamental and presumably the most ancient property of visual systems. This ability is the basis of phototaxis, one of the most striking behavioral responses of motile photosynthetic microorganisms (i.e. microalgae) to light stimuli, which allows them to move toward or away directional light. In order to fully exploit the information content of light (intensity, direction, distribution) microorganisms need proper perceiving devices, termed photoreceptors, which must act as sensors, to perceive wavelength and direction of light, as transducers, to convert the light signal into chemical and/or electrical information, but also as amplifiers and eventually as transmitters. This review describes the universal structural, behavioral and physiological features necessary for the proper functioning of these devices in algae, and how these features have been investigated by means of different analytical techniques such as for example microspectroscopy, digital fluorescence microscopy, two photons FLIM. The insight of the photoreceptive response mechanism is explained using the unicellular alga Euglena gracilis, in which the different structural, behavioral and physiological features combine to achieve a concerted, efficient response to light stimuli.

Chemistry, physico-chemistry and applications linked to biological activities of ß-glucans.

ß-Glucans is the common name given to a group of chemically heterogeneous polysaccharides. They are long- or short-chain polymers of (1-->3)-ß-linked glucose moieties which may be branched, with the branching chains linked to the backbone by a (1-->6)-ß linkage. ß-(1-->3)-Glucans are widely distributed in bacteria, algae, fungi and plants, where they are involved in cell wall structure and other biological function. ß-Glucans have been shown to provide a remarkable range of health benefits, and are especially important against the two most common conventional causes of death in industrialized countries, i.e. cardiovascular diseases (where they promote healthy cholesterol and blood glucose levels) and cancer (where they enhance immune system functions). This Highlight provides a comprehensive and up-to-date commentary on ß-glucans, their chemistry, physico-chemistry, functional role in immunological responses, and possible applications as therapeutic tools. In addition, we discuss the mechanism behind their health benefits, which are not yet fully understood.

A rhodopsin-like protein in Cyanophora paradoxa: gene sequence and protein immunolocalization.

Here, we report the DNA sequence of the rhodopsin gene in the alga Cyanophora paradoxa (Glaucophyta). The primers were designed according to the conserved regions of prokaryotic and eukaryotic rhodopsin-like proteins deposited in the GenBank. The sequence consists of 1,272 bp comprised of 5 introns. The correspondent protein, named Cyanophopsin, showed high identity to rhodopsin-like proteins of Archea, Bacteria, Fungi, and Algae. At the N-terminal, the protein is characterized by a region with no transmembrane alpha-helices (80 aa), followed by a region with 7alpha-helices (219 aa) and a shorter 35-aa C-terminal region. The DNA sequence of the N-terminal region was expressed in E. coli and the recombinant purified peptide was used as antigen in hens to obtain polyclonal antibodies. Indirect immunofluorescence in C. paradoxa cells showed a marked labeling of the muroplast (aka cyanelle) membrane.

Intramolecular photo-switching and intermolecular energy transfer as primary photoevents in photoreceptive processes: the case of Euglena gracilis.

In this paper we report the results of measurements performed by FLIM on the photoreceptor of Euglenagracilis. This organelle consists of optically bistable proteins, characterized by two thermally stable isomeric forms: A(498,) non fluorescent and B(462), fluorescent. Our data indicate that the primary photoevent of Euglena photoreception upon photon absorption consists of two contemporaneous different phenomena: an intramolecular photo-switch (i.e., A(498) becomes B(462)), and a intermolecular and unidirectional Forster-type energy transfer. During the FRET process, the fluorescent B(462) form acts as donor for the non-fluorescent A(498) form of the protein nearby, which acts as acceptor. We hypothesize that in nature these phenomena follow each other with a domino progression along the orderly organized and closely packed proteins in the photoreceptor layer(s), modulating the isomeric composition of the photoreceptive protein pool. This mechanism guarantees that few photons are sufficient to produce a signal detectable by the cell.

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle.

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.