Spectrally Resolved FLIM
The spectrally-resolved Lambert Instruments FLIM Attachment (LIFA) is an imaging system for fluorescence microscopy that preserves the information required to determine the position, spectrum, and lifetime of the observed fluorescence. This is done by combining several modular components. These consist of the typical LIFA (modulated intensified CCD camera, modulated LED excitation) and a prism-based imaging spectrograph.
The data below were obtained with the Lambert Instruments FLIM Attachment (LIFA), either widefield (with LED light; 468nm peak) or confocal (with spinning disk CSU10 and 470nm-diode laser). The fluorescence lifetime images are generated at 2 different z-positions, z1 and z2. Snapshots of several z-positions are shown, as well as movies through even more.
SPECTRALLY RESOLVED
2D IMAGE
CONFOCAL
(SPINNING DISK)
WIDEFIELD
(LED)
The spectrally resolved images show one line (y-axis) out of the 2D intensity image of typical dicot root. The emission wavelengths at the x-axis start at 515 nm. The fluorescence lifetime is shown in pseudo colors. The higher wavelength components have a shorter lifetime (blue) than the short wavelength components (red).
SPECTRALLY RESOLVED GFP TRANSFECTED CELLS (DONOR ONLY)
SPECTRALLY RESOLVED GFP-RFP TRANSFECTED CELL (FRET)
LIFETIME (PSEUDOCOLORS)
INTENSITY (GRAYSCALE)
The spectrally resolved images show one line (y-axis) out of a sample of GFP transfected cells or GFP-RFP transfected cells. The emission wavelengths at the x-axis start at 515 nm; the first peak is the GFP emission peak and the second the RFP emission peak. The fluorescence lifetime is shown in pseudo colors. In the FRET sample (GFP-RFP) the fluorescence lifetime of the GFP has decreased from 2.3 ns (red) to 2.0 ns (yellow).
The differences in lifetime could be due to the fact that the diode laser has one excitation wavelength, exactly 470 nm, while the LED has a range of wavelengths for which we used the emission band pass filter of 465-495 nm.
