Lambert Instruments

TIRF (Total Internal Reflection Fluorescence) microscopy facilitates extremely high-contrast visualisation and thereby high sensitivity of fluorescence near the cover glass. Typically the optical section adjacent to the cover glass is about 100 nm. TIRF does not disturb cellular activity, thereby enabling tracking of biomolecules, and the study of their dynamic activity and interactions at the molecular level. TIRF enables the selective visualisation of processes and structures of the cell membrane and pre-membrane space like vesicle release and transport, cell adhesion, secretion, membrane protein dynamics and distribution or receptor-ligand interactions. The unique combination of TIRF and frequency domain FLIM makes it possible to measure lifetimes of for instance small focal adhesions near the cover glass.

Lifetime (pseudo colours)	Intensity (grey scale)
		TIRF
		Widefield

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

The images shown here are taken with the Nikon TE2000-U widefield microscope with 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.
For more information on white-TIRF, please visit the technology page or contact Nikon (Mr. R. Stad).

 

Lifetime (pseudo colours)	Intensity (grey scale)
		TIRF
		Widefield

Cells expressing dSH2-GFP (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) in small focal adhesions as well as in the nuclei.

The images shown here are taken with the Olympus TIRFM (laser-TIRF), combined with the Lambert Instruments Fluorescence lifetime imaging Attachment (LIFA). As light source the modulated diode laser of 473nm 20mW was used and as demonstrated here enough intensity was generated to obtain fluorescence lifetime images with TIRF.
For more information on laser-TIRF, please visit the technology page or contact Olympus (Mr. G. Bouw).

 

Lifetime (pseudo colours)	Intensity (grey scale)
		TIRF

Cells expressing dSH2-GFP (kindly provided by Ms. S.E. Le Devedec, Leiden University, The Netherlands) in small focal adhesions as well as in the nuclei.

The images shown here are taken with the Nikon laser-TIRF, combined with the Lambert Instruments Fluorescence lifetime imaging Attachment (LIFA). As light source the modulated diode laser of 473nm 20mW was used and as demonstrated here enough intensity was generated to obtain fluorescence lifetime images with TIRF.
For more information on laser-TIRF, please visit the technology page or contact Nikon (Mr. R. Stad).

More details on this TIRF-FLIM set-up are described at the product page.