Cells and tissues exposed to light of appropriate wavelenght exhibit fluorescence emission at the UV-visible spectral range. This phenomenon called natural fluorescence (NF) is related to molecules widely present in nature and known as chromophores. Few NF studies at cellular level have been made and little is known about the correlation between photophysical teatures and morpho-functional state of the cell. In this study, the NF properties of undifferenliated and 12-0tetradecanoylphorbol 13-acetate (TPA)-differentiated FLG 29 1 cells have been characterized on single-cell basis. FLG 29.1 cells were cultured in RPMI 1840 medium with PCS 10% and splitted twice weekly. The differentiation was obtained by TPA 0. VM. Cells were studied after 24, 48 and 72 hrs TPA incubation, in compartison with untreated controls. Microspectrofluorometry and fluorescence imaging was performed by a microscope equipped with an oil-immersion CF-UV fluor objective 100x (N.A. 1.30). A high pressure mercury lamp was used as excitation source in combination with 366 nm interference filters. The NF collected by the objective and passed through a dichroic mirror, was analyzed by a multispectral digital CCD camera with 768x512 pixels and lumogen coating or, alternatively, with a multichannel spectral analyzer (Hamamatsu, PMA 11 ). The NF spectra of the cells showed a wide bluegreen band, peaked in the 450-495 nm range and a shoulder over 520 nm, representing the emission of nicotinic coenzymes and flavins respectively. The image analysis of undifferentiated cells showed an intense homogeneously distributed fluorescence. Differentiated cells revealed a less intense total emission, with the NF mostly localized at level of cellular organelles and particularly evident after 72h treatment. Our data indicate a correlation between NF properties and differentiation. A link can be supposed between NF properties and metabolic changes occurring during the differentiation process.
Natural fluorescence imaging of flg 29.1 cells differentiated by phorbol ester
Mazzinghi P;
1997
Abstract
Cells and tissues exposed to light of appropriate wavelenght exhibit fluorescence emission at the UV-visible spectral range. This phenomenon called natural fluorescence (NF) is related to molecules widely present in nature and known as chromophores. Few NF studies at cellular level have been made and little is known about the correlation between photophysical teatures and morpho-functional state of the cell. In this study, the NF properties of undifferenliated and 12-0tetradecanoylphorbol 13-acetate (TPA)-differentiated FLG 29 1 cells have been characterized on single-cell basis. FLG 29.1 cells were cultured in RPMI 1840 medium with PCS 10% and splitted twice weekly. The differentiation was obtained by TPA 0. VM. Cells were studied after 24, 48 and 72 hrs TPA incubation, in compartison with untreated controls. Microspectrofluorometry and fluorescence imaging was performed by a microscope equipped with an oil-immersion CF-UV fluor objective 100x (N.A. 1.30). A high pressure mercury lamp was used as excitation source in combination with 366 nm interference filters. The NF collected by the objective and passed through a dichroic mirror, was analyzed by a multispectral digital CCD camera with 768x512 pixels and lumogen coating or, alternatively, with a multichannel spectral analyzer (Hamamatsu, PMA 11 ). The NF spectra of the cells showed a wide bluegreen band, peaked in the 450-495 nm range and a shoulder over 520 nm, representing the emission of nicotinic coenzymes and flavins respectively. The image analysis of undifferentiated cells showed an intense homogeneously distributed fluorescence. Differentiated cells revealed a less intense total emission, with the NF mostly localized at level of cellular organelles and particularly evident after 72h treatment. Our data indicate a correlation between NF properties and differentiation. A link can be supposed between NF properties and metabolic changes occurring during the differentiation process.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
