M. Fato

Three-dimensional computer-aided reconstruction of FMRFamide immunopositive neuron distribution in the ventral ganglion of the barnacle Balanus amphitrite (Cirripedia, Crustacea)

We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec) of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCD-camera-equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide-like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90 degrees rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

Imaging of Endocytosis in Paramecium by Confocal Microscopy

Endocytosis is the general term for internalization of fluid, solutes, macromolecules, plasma membrane components, and particles by the invagination of the plasma membrane and the formation of vesicles and vacuoles through membrane fusion. The means by which food material enters the body is to a great extent dependent on the size of the particles involved [1, 2]. One type of endocytosis is phagocytosis where large (>250 nm) particles are taken up by cells. In protozoa, phagocytosis is a feeding mechanism. Particles are brought into the cell in large endocytic vesicles called phagosomes (food vacuoles). The phagosomes fuse with lysosomes and digestion of the ingested particles occurs. In multicellular organisms, phagocytosis is a behavior seen only in certain specialized cells (for example, macrophages). The process is essentially the same whether it is phagocytosis of particles or other organisms or pinocytosis of molecules. Both endocytic processes are affected identically by inhibitors of aerobic metabolism and by low temperatures.

Structure analysis of Eledone cirrhosa by CLSM and SFM

CLSM (Confocal Laser Scanning Microscopy) and SFM (Scanning Force Microscopy) can be considered in use as high-powered biophysical tools for investigating the very delicate and complex way in which structure and function are related in biological systems. Organisational motifs underlying the structure of important biostructures can be imaged in a hydrated state in living samples or under conditions that are very close to living or physiological states. Among these biostructures, helically organised ones, such as chromatin-DNA, play a fundamental role in the way in which a cell functions. We are studying the structure of the sperm head of the octopus Eledone cirrhosa by means of CSLM and SFM. In particular we aim to study at different resolution levels the sperm head and its constituents and to use the very same sample as a large copy of chromatin itself. Earlier studies concluded that the rigid helical structure of the sperm head is due to its chromatin; probably this is true also for the shape of the head itself as found for several animal species. These studies reported that DNA begins to form 10 nm fibers during spermiogenesis and the fibers condenses further as the process evolves, but no model was obtained to describe the final arrangement of DNA. The possibility of performing such high resolution experiments could give useful hints to solve this problem.

Optical sectioning microscopy and bioimage-oriented interfaces for 2D/3D and time-variant characterization of biostructures

Bioimage-oriented interfaces are linked to a 3D image formation system, i.e. the optical microscope, for a new approach to image manipulation in biology and medicine. Stereo monitors and 3D head-tracking and sensor devices allow the operator to interact with 3D data sets representing biostructures and to navigate through the object space. Image restoration algorithms have been implemented in order to gather quantitative information and to study the delicate and interesting relationship between structure and function in biological systems.

Spatio-temporal analysis of single-celled organisms (protozoa) structures via confocal microscopy

This work aims to study in vivo vesicle formation, transport and fusion during the digestive process of the ciliated protozoan Paramecium primaurelia.

Three-dimensional reconstruction of paramecium primaurelia oral apparatus through confocal laser scanning optical microscopy

Studies on the complementary mating types of Paramecium primaurelia (Protozoa, Ciliates) have shown that cell lines which differ from each other in mating type expression are characterized by different cell contents, organization, and physiology. Referring to these differences and to the differential rates of food vacuole formation, oral apparatuses of the two mating type cells are assumed to possibly differ from each other in some traits, such as, for instance, in their lengths. In our work, the highly organized oral structures are analyzed by means of a laser scanning confocal optical microscope (CLSM), which provides their 3-D visualization and measurement. The extraction of the 3-D intrinsic information related to the biological objects under investigation can be in turn related to their functional state, according to the classical paradigm of structure to function relationships identification. In our experiments, we acquired different data sets. These are optical slices of the biological sample under investigation, acquired in a confocal situation, through epi-illumination, in reflection, and, for comparison with conventional microscopy, 2-D images acquired via a standard TV camera coupled to the microscope itself. Our CLSM system is equipped with a laser beam at 488 and 514 nm and the data have been acquired with various steps of optical slicing, ranging from .04 to .25 micrometers. The volumes obtained by piling-up the slices are rendered through different techniques, some of them directly implemented on the workstation controlling the CLSM system, some of them on a SUN SPARC station 1, where the original data were transferred via an Ethernet link. In this last instance, original software has been developed for the visualization and animation of the 3-D structures, running under UNIX and X-Window, according to a ray-tracing algorithm.