A wealth of data point to Delayed Luminescence (DL) as a good candidate for early and reliable detection technique in neoplastic cells and tissues sorting. Aiming at a DL experimental set up for such a kind of information, a testing technique for morphological analysis should be provided. This could certify the early identification of pathologies and abnormalities in cells and tissues by DL. DL technique may be coupled with FIB (Focused Ion Beam) imaging
analysis to give a correlated, both spectroscopic and morphological investigation, at the submicron scale.
A strong link among others has been reported to exist between DL signal characteristics and cytoskeleton structure and dynamics: FIB (Focused Ion Beam) imaging is for the moment being the best non invasive check at all and it can detect morphological alterations as early as possible since its resolution can go down to 2-5 nm.
The cells, that can be highlighted by the fast DL and slow and efficient FIB, can be in parallel analysed by a metabolic manometric technique that uses differential pressure sensors: the different cellular activity of normal and abnormal cells can be recorded and this allows fast and non-invasive investigations, although requiring a minimal number of cells.
In addition it’s possible to study, by the confocal microscopy spectroscopic analysis, DNA fragments, exploiting the optical characteristics of a dye, like ethidium bromide, to detect dynamic and conformational changes in DNA chains. These changes can be artificially induced in cells (e.g. by irradiation) or found in neoplastic cells. The acquired experience allows an independent check of spectroscopic, morphologic and metabolic testing by a control on nucleic acid defects.
These four techniques may be used together creating a "protocol" in order to permit an early and reliable alterations diagnosis of cells and tissues, guaranteeing an high accuracy standard.
Confocal microscopy is one of the most widely used and non-invasive tool for the investigation of biological matter. It improves the performances of the optical microscope by reducing the excitation volume and enhancing the axial resolution due to the use of high numerical aperture lenses. We have adapted an inverted confocal optical microscope to the measurement of fluorescence emission dynamics (lifetime and fluorescence polarization anisotropy). The dynamic spectroscopy measurements are obtained with phase fluorometry and are based on a modulated linearly polarized laser beam which is fed to the epifluorescence port of the microscope. We report the
test of the microscope by comparing the lifetime and fluorescence polarization anisotropy decay obtained in cuvettes in the standard phase modulation fluorometer and on tiny drops on the microscope stage. We show that once a correction factor is introduced in the best fit functions used in the data analysis of the decays, the results obtained on microliters volumes is comparable to those obtained in cuvettes in the standard phase fluorometer spectrometer. An example of application is reported on short DNA fragments.
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