White-light TIRF combined with FLIM

Leutingewolde, The Netherlands - August 10, 2006…Lambert Instrument news. TIRF (Total Internal Reflection Fluorescence) microscopy facilitates extremely high-sensitivity and high-contrast visualisation of small spots near the cover glass, without disturbing cellular activity, thereby enabling the tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. This new combination of TIRF and frequency domain FLIM makes it possible to measure lifetimes of small adhesion points near the cover glass.

The images shown here were taken with a Nikon TE2000-U wide-field microscope and a white-TIRF illuminator combined with the Lambert Instruments Fluorescence lifetime imaging Attachment (LIFA). As light source the modulated LED of 468nm 3W was used and as demonstrated here enough intensity was generated to obtain fluorescence lifetime images with TIRF. The same images were generated with TIRF (top panel) and widefield fluorescence microscopy (bottom panel). In the right panel the intensity images are shown and the corresponding fluorescence lifetime images are shown in the left panel. The color scales on the left indicate the fluorescence ratio or FI values.

The cells expressing dSH2-GFP (kindly provided by Ms. S.E. Ledevedec, Leiden University, The Netherlands) in small adhesion dots as well as in the nuclei are shown by wide-field microscopy. However, by use of TIRF only the fluorescence close to the coverslip is obtained and thus specifically just the adhesion dots are excited. The fluorescence lifetime images therefore give a more accurate measurement in the TIRF mode, as out of focus light is emitted from the average lifetime in the adhesion dots.

Contact Advanced Labs for more information or a quotation.

Tags: microscopy, TIRF, FLIM, Nikon