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- W1997469484 abstract "Recently a label-free FLIM imaging method has been proposed to distinguish metabolic states of germ cells (Stringari et al, 2011). This method, based on the phasor analysis of autofluorescence lifetime (AF-phasorFLIM), avoids some problems of state-of-art approaches due to multi-exponential fitting. The capability of phasorFLIM analysis to separate the different molecular species in a pixel lies on the rule of phasor addition. PhasorFLIM identifies molecules by their position in the phasor plot because every molecular species has a specific phasor that can be calibrated in reference samples. We applied phasorFLIM to detect retinoids autofluorescence in live P19 embryonal carcinoma cells that undergo differentiation when treated with retinoic acid (RA). RA and retinol are essential for cell-cell signaling during vertebrate organogenesis. RA is involved in stem cell differentiation and it is a morphogen. It is a diffusible signal that generates patterns in embryo because it induces different cellular responses depending on its local concentration. The presence of RA in tissues has been inferred through gene activation, and the RA average tissular concentration measured by HPLC. However, the spatial distribution in tissues and cells of the active molecule has never been observed or quantified directly. We built a database of phasorFLIM standards of the major molecules that contribute to autofluorescence. The database allows assigning the contribution of fluorescent molecules to P19 autofluorescence. P19 cells were treated with retinol to follow in time the effect of the molecule on cellular metabolism. PhasorFLIM analysis was performed using SimFCS and showed an increase of retinoids fluorescence after treatment. In cells treated with retinol, we observed retinol in plasma and nuclear membrane and an increase of RA in the nucleus. AF-phasorFLIM allowed determining, distinguishing and localizing the contribution of retinoids to autofluorescence in live P19 cells." @default.
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- W1997469484 date "2012-01-01" @default.
- W1997469484 modified "2023-10-18" @default.
- W1997469484 title "Mapping Retinoids in Live P19 Cells with Autofluorescence Phasorflim Imaging" @default.
- W1997469484 doi "https://doi.org/10.1016/j.bpj.2011.11.1053" @default.
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