Line-Scanning Microscopy for Time-Gated and Spectrally Resolved Fluorescence Imaging

Laser-scanning fluorescence microscopy for efficient acquisition of time-gated and spectrally resolved fluorescence images was developed based on line illumination of the laser beam and detection of the fluorescence image through a slit. In this optical arrangement, the fluorescence image was obtained by scanning only one axis perpendicular to the excitation line, and the acquisition time was significantly reduced compared with conventional laser-scanning confocal microscopy. A multidimensional fluorescence dataset consisting of fluorescence intensities as a function of x-position, y-position, fluorescence wavelength, and delay time after photoexcitation was analyzed and decomposed based on the parallel factor analysis model. The performance of the line-scanning microscopy was examined by applying it to the analysis of one of the plant defense responses, accumulation of antimicrobial compounds of phytoalexin in oat (Avena sativa), induced by the elicitor treatment.