The intrinsic autofluorescence properties of biological tissues can be affected by the occurrence of histological and biochemical alterations induced by pathological processes. In this study the potential of autofluorescence to distinguish tumor from normal tissues was investigated with the view of a real-time diagnostic application in neurosurgery to delineate glioblastoma resection margins. The autofluorescence properties of nonneoplastic and neoplastic tissues were analyzed on tissue sections and homogenates by means of a microspectrofluorometer, and directly on patients affected by glioblastoma multiforme, during surgery, with a fiber-optic probe. Scan-microspectrofluorometric analysis on tissue sections evidenced a reduction of emission intensity and a broadening of the main emission band, along with a redshift of the peak position, from peritumoral nonneoplastic to neoplastic tissues. Differences in both spectral shape and signal amplitude were found in patients when the glioblastoma lesion autofluorescence was compared with those of cortex and white matter taken as healthy tissues. Both biochemical composition and histological organization contribute to modify the autofluorescence emission of neoplastic, with respect to nonneoplastic, brain tissues. The differences found in the in vivo analysis confirm the prospects for improving the efficacy of tumor resection margin delineation in neurosurgery.

The nature and the extent of the autofluorescence modification between normal and tumor tissue in sections explain at least partly the evidence of the "in vivo" analysis and highlight the importance of excitation for full exploitation of the potentials of autofluorescence in diagnosis.

Rose bengal, a xanthene derivative among the most efficient producer of singlet oxygen, was submitted to a chemical modification consisting in the introduction of an acetate group into the aromatic ring fluorophore structure. The acetate group acts as a quencher, thus inactivating both fluorescence and photosensitization properties of the molecule. In the modified structure, rose bengal acts as a fluorogenic substrate giving rise to the cellular reaction termed fluorochromasia. The acetate group is recognized by a carboxylic esterase activity that splits it. Removal of the quencher group results in restoring the native structure of photosensitizer inside the cells. The intracellular turnover of rose bengal acetate was studied in rat glioma-derived cultures cells, in terms of the balance of the processes of influx and enzyme hydrolysis of the fluorogenic substrate, and of the efflux of the fluorescent product. A large intracellular accumulation of photosensitizer is obtained when treatments are performed with the fluorogenic substrate, even at the drug concentration at which rose bengal does not enter the cells. The intracellular localization allows rose bengal to exert a more effective photosensitization effect. Provided that the quencher group is selected according to the metabolic properties of the tumor cells, the use of fluorogenic substrates as photosensitizer precursors could improve fluorescence diagnosis and the photodynamic therapy of tumors, exploiting the biological properties that distinguish pathological from normal conditions.

The intrinsic autofluorescence properties of biological tissues can change depending on alterations induced by pathological processes. Evidence has been reported concerning the application of autofluorescence as a parameter for in situ cancer detection in several organs. In this paper, autofluorescence properties of normal and tumor tissue in the brain are described, suitable for a real-time diagnostic application. Data were obtained both on ex vivo resected samples, by microspectrofluorometric techniques, and in vivo, during surgical operation, by means of fiberoptic probe. Significant differences were found in autofluorescence emission properties between normal and tumor tissues, in terms of both spectral shape and signal amplitude, that confirm the potential of autofluorescence as a parameter to distinguish neoplastic from normal condition. The noninvasiveness of the technique opens up interesting prospects for improving the efficacy of neurosurgical operations, by allowing an intraoperative delineation of tumor resection margins.

The dependence of autofluorescence properties on the metabolic and functional engagement and on the transformation condition was studied on single cells. Normal Galliera rat fibroblasts at low subculture passage (cell strain), at high subculture passage (stabilized cell line J, and transformed cell line derived from a rat sarcoma were used as a cell model. The study was performed by microspectrofluorometric and fluorescence imaging technique. The autofluorescence properties of cells were studied by excitation at two wavelengths, namely 366 nm and 436 nm, that are known to favor the emission of different fluorophores. Spectral shape analysis indicated that under excitation at 366 nm autofluorescence is ascribable mainly to coenzyme molecules, particularly to reduced pyridine nucleotides, while under excitation at 436 nm, flavin and lipopigment emission is favored. The energetic metabolic engagement of the different cell lines was analyzed in terms both of parameters related to anaerobicaerobic pathways (biochemical assay) and of mitochondrial features (supravital cytometry). The results showed that the cell strain and the stabilized and transformed cell lines can be distinguished from one another on the basis of both overall fluorescence intensity and the relative contributions of spectral components. These findings indicated a relationship between autofluorescence properties and energetic metabolism engagement of the cells that, in turn, is dependent on the proliferative activity and the transformed condition of the cells. In that it is a direct expression of the energetic metabolic engagement, autofluorescence can be assumed as an intrinsic parameter of the cell biological condition, suitable for diagnostic purposes. DiOC6, 3,3'-dihexyloxacarbocyanine iodide; FAD, flavin adenine dinucleotide; FI, fluorescence intensity: FWHM, full width at half maximum; FG, Galliera fihrohlast; HBw, half band width; HO 33342, Hoechst 33342: LDH, lactate dehydrogenase; NAD. nicotinamide adenine dinucleotide; NADP. nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; SDH, succinate dehydrogenase; S G S . sarcoma galliera strain; T. transmittance: TRIS, tris(hydroxymethy1)aminornethane. tAbbr-eriarions: