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.

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.

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.

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.

MICROSPECTROPHOTOMETRY AS A METHOD TO IDENTIFY KLEPTOPLASTIDS IN THE NAKED FRESHWATER DINOFLAGELLATE GYMNODINIUM ACIDOTUM(1).

A relatively small number of freshwater dinoflagellates are involved in symbiotic association with cryptophytes. The chloroplasts of the cryptophytes are retained by the dinoflagellate and give it the characteristic phycobilin pigmentation, either phycoerythrin or phycocyanin. The pigment characterization of the retained chloroplasts can give precise and accurate information about the type of cryptophyte preyed upon by the dinoflagellate. For this purpose, we performed microspectrophotometric evaluation of the pigments of Gymnodinium acidotum Nygaard and three different cryptophytes present in samples collected from a tributary of the river Arno, in Tuscany (Italy). The comparison of the different spectroscopic data allowed us to discriminate effectively among the cryptophytes preyed upon by the dinoflagellate.

Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor.

This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90A). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor.