FLIM Measurement Examples - all images were created by the SymPhoTime software

On the right: Interactions of protein partners in their natural environment inside living cells can be studied with time-resolved FRET microscopy. The technique was used to characterize intra-nuclear dimer formation for the transcription factor C/EBP α in living pituitary GHFT1-5 cells of mice. Members of the C/EBP family of transcription factors are critical determinants of cell differentiation. Dimerization of CFP-YFP-C/EBP Δ 154 protein molecules in the cell nucleus could be detected with FRET. Excitation was performed at 440 nm and the fluorescence was detected around 470 nm. Left image: intensity image; right image: calculated fluorescence lifetime image from the time-resolved measurement in false coloring.

(Sample courtesy of Ye Chen and Ammasi Periasamy, FRET workshop, W.M. Keck Center for Cellular Imaging, University of Virginia)

On the left: For this lifetime image a cancer cell line of liver cells was cultivated. The cells were stained with phospholipids labeled with NBD, a dye which lifetime is depending on the hydrophobicity of its surrounding environment. The lifetime allows to gain information about the molecular structure of cellular compartments. The fluroescence was excitated with a wavelength of 470 nm and detected using a 500 nm longpass filter. Left image: intensity image; right image: calculated fluorescence lifetime image from the time-resolved measurement in false coloring.

(Sample courtesy of Astrid Tannert and Thomas Korte, Humboldt University Berlin, Molecular Biophysics, Germany)

On the right: FLIM measurement of a mouse kidney sample, stained with Alexa-Fluor 488, Alexa-Fluor 568 and DAPI, as fluorescence lables. The excitation wavelength was 438 nm at a repetition rate of 20 MHz and a 510 nm longpass filter was used to discriminate the fluorescence against scattered excitation light. The pinhole size of the confocal setup was kept constant at 150 µm. The left image shows the non-time resolved LSM result which was accumulated over 5 frames and the right image shows the calculated fluorescence lifetime in false colouring. This FLIM image was recorded in 8 minutes and 170 Frames were accumulated. In this case three different lifetimes could be recovered (blue 0.8 ns, green 2.1 ns, red 4.5 ns).

The lifetime image clearly shows a strong contrast enhancement between the different cells and organelles. From the intensity image only it is not possible to distinguish between different types of cells. The lifetime image even allows distinction of the nucleus.

On the left: Autofluorescence from an unstained sample of apple slice. The excitation wavelength was 405 nm at a repetition rate of 20 MHz, and the emission was observed through a 510 nm longpass filter. The pinhole was set to 60 µm. The recording time was 8 minutes and 170 frames where accumulated. The left image shows the results from the non-time resolved LSM measurement and the right image shows the result from the time-resolved measurement. In this case three different lifetimes could be recovered (blue 0.2 ns, green 0.9 ns, red 3.8 ns).

The lifetime image shows a strong contrast enhancement between the different parts of the apple slice. A clear difference is visible between functionally distinct regions of the sample, which is not visible in the pure intensity image.