Marfan syndrome (MFS) is an inherited disorder of connective tissue due to mutations in FBN1 (90%) and TGFBR1 and TGFBR2 (5 to 10%) genes. Clinical and differential diagnosis is difficult because of the inter- and intrafamiliar marked heterogeneity and the variable onset age of clinical manifestations. Among the disorders, in differential diagnosis, thoracic aortic aneurysm (TAA) and Ullrich scleroatonic muscular dystrophy (UCMD) are reported. We evaluate the possibility of utilizing autofluorescence (AF) analysis as a diagnostic tool in the clinical and/or differential diagnosis of MFS and related disorders and in the investigation of the molecular mechanisms involved. Both multispectral imaging autofluorescence microscopy (MIAM) and autofluorescence microspectroscopy (AMS) have been used to characterize AF emission of fibroblasts from patients affected by inherited connective tissue disorders. Our preliminary results show significant differences in AF emission between normal and pathological fibroblasts, suggesting possible improvement in diagnostics of connective tissue disorders by AF analysis.
Many studies demonstrated that mechanical stress is a key factor for tissue homeostasis, while unloading induce loss of
mass and impairment of function. Because of their physiological function, muscle, connective tissue, bone and cartilage
dynamically interact with mechanical and gravitational stress, modifying their properties through the continuous
modification of their composition. Indeed, it is known that mechanical stress increases the production of extracellular
matrix (ECM) components by cells, but the mechanotransduction mechanisms and the optimal loading conditions
required for an optimal tissue homeostasis are still unknown. Considering the importance of cell activation and ECM
production in tissue regeneration, a proper use of mechanical stimulation could be a powerful tool in tissue repair and
tissue engineering.
Studies exploring advanced modalities for supplying mechanical stimuli are needed to increase our knowledge on
mechanobiology and to develop effective clinical applications.
Here we describe the effect of photomechanical stress, supplied by a pulsed Nd:YAG laser on ECM production by cells
of connective tissues.
Cell morphology, production of ECM molecules (collagens, fibronectin, mucopolysaccharides), cell adhesion and cell
energy metabolism have been studied by using immunofluorescence and autofluorescence microscopy. The results show
that photomechanical stress induces cytoskeleton remodelling, redistribution of membrane integrins, increase in
production of ECM molecules. These results could be of consequence for developing clinical protocols for the treatment
of connective tissue dideases by pulsed Nd:YAG laser.
Laser welding of corneal tissue is an alternative technique to conventional suturing procedures in ophthalmic surgery. The welding effect is achieved after staining the wound with a chromophore (Indocyanine Green, shortly: ICG) and then irradiating it with a low power diode laser. We present a study on the healing process of corneal wounds using Multispectral Imaging Autofluorescence Microscopy (MIAM). This technique is based on the characterization of fluorescence arising from tissue components (autofluorescence): it is particularly useful in studying corneal tissue, because it is mainly composed of type I collagen, one of the most important endogenous fluorophores. Laser welding tests of the cornea were carried out on rabbits in which full thickness corneal cuts of about 5 mm were sutured using a diode laser emitting at 810 nm, with a power of 80 mW. Bioptic sections of rabbit corneas were examined in a follow up study of 90 days after surgery, and the results were complementary to histological analysis performed in previous studies. Autofluorescence images showed a faster healing process and a better reorganization of the architecture of stromal fibers, in comparison with conventional suturing procedures. MIAM technique can represent a new tool to study the morphology of corneal tissue, offering some real advantages with respect to standard histological analysis. In fact, it does not require any chemical manipulation of the samples, providing information on the biological structure by directly monitoring distribution and emission intensity of endogenous fluorophores.
Many cell and tissue components exposed to light of appropriate wavelength exhibit UV-visible emission. This is a spontaneous fluorescence, defined autofluorescence in contrast with that produced by highly fluorescent exogenous probes. This communication deals with the Multispectral Imaging Autofluorescence Microscopy (MIAM) technique, that allows living cell and tissue analysis without use of any reagent or treatment modifying the native conditions of the samples. The technique is suitable for a wide range of applications, practically in all the fields where samples containing fluorescent substances occur. Examples of application in biology and medicine are given, showing the power of the technique in both basic research and diagnostics.
Multispectral imaging autofluorescence microscopy (MIAM) is used here for the analysis of lymphatic tissues. Lymph node biopsies, from patients with lympthoadenopathy of different origin have been examined. Natural fluorescence (NF) images of 3 micrometers sections were obtained using three filters peaked at 450, 550 and 680 nm with 50 nm bandpass. Monochrome images were combined together in a single RGB image. NF images of lymph node tissue sections show intense blue-green fluorescence of the connective stroma. Normal tissue shows follicles with faintly fluorescent lymphocytes, as expected fro the morphologic and functional characteristics of these cells. Other more fluorescent cells (e.g., plasma cells and macrophages) are evidenced. Intense green fluorescence if localized in the inner wall of the vessels. Tissues coming from patients affected by Hodgkin's lymphoma show spread fluorescence due to connective infiltration and no evidence of follicle organization. Brightly fluorescent large cells, presumably Hodgkin cells, are also observed. These results indicate that MIAM can discriminate between normal and pathological tissues on the basis of their natural fluorescence pattern, and, therefore, represent a potentially useful technique for diagnostic applications. Analysis of the fluorescence spectra of both normal and malignant lymphoid tissues resulted much less discriminatory than MIAM.
A new technique for the absorption of cells is presented. A NIR diode laser (810 nm) and an absorption enhancing dye (indocyanine green, ICG) are used. Localized heating of ICG aggregates close to the cell surface generates bubbles inside the cells. Expansion and emergence of these bubbles cause the cells to open, thus creating the condition for successful transfection. Successful application to plant (tomato) cells is reported for the first time. The experimental results are directly linked both to the absorption characteristics of the dye employed and to the nature of the wall. Optoporation was achieved only by the use of the dye. Laser powers were employed comparable to those used for animal cells.
Haematic cells, excited with radiation of suitable wavelength, give rise to a natural fluorescence (NF) emission. This paper investigates NF to develop new techniques for applications in both basic research and medical diagnostics. Results show that the cell populations examined exhibit peculiar emission bands. The intracellular fluorescence pattern reveals that flouresence is mainly located at cytoplasmic-level, thus related to the metabolic processes of the cells. The photophysical properties of cells appear different among the normal populations and between normal and leukaemic ones. Therefore the recognition of the various cellular elements, according to their fluorescence emission, is possible.
Monica Monici, Giovanni Agati, Franco Fusi, Pietro Bernabei, Roberto Caporale, Pierluigi Ferrini, Anna Croce, Giovanni Bottiroli, Stefano Cioncolini, Alberto Innocenti, Riccardo Pratesi
The determination of blood cell composition has been a valuable tool in diagnoses. In particular, both total and differential counts are considered the basic parameters that characterize the leukocyte population. Since 100 years ago, manual techniques were introduced that allow a morphological examination of blood smears. At present, the automated analysis has been proved to be particularly difficult to standardize. In fact, the identification and count of the five leukocyte populations are not completely solved problems in routine methods for hematological analysis. Optoelectronics could have a decisive role in the development of new techniques that can ensure characteristics of automation, reliability, accuracy and rapidity of execution. Fluorescence spectroscopy techniques could represent a valid approach. Recently, the evaluation of tissue and cell autofluorescence has been applied to the diagnosis of solid tissue neoplasies. In this work, we have considered the possibility to develop a reliable method of leukocyte analysis based on their intrinsic fluorescence emission properties. The study has been performed by applying both spectrofluorometric techniques to enriched suspensions of cells and microspectrofluorometric techniques to single leukocytes. The results obtained have shown the possibility to recognize some cell populations on the grounds of the intrinsic fluorescence characteristics.
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