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.

 

Lifetime (pseudocolors)

Intensity (grayscale)

 

 

 

Spectrally resolved

 

2D image

 

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).

 

Lifetime (pseudocolors)

Intensity (grayscale)

 

Spectrally resolved GFP transfected cells (donor only)

 

Spectrally resolved GFP-RFP transfected cell (FRET)

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).