Time-resolved fluorescence imaging
( Imaging and Visualisation Series )
Professor Hemmila Ilkka, Perkin-Elmer Life Sciences
Date and time: Friday 20th March 2009 at 13:00
Location: UG40, School of Computer Science
Host: Professor Claridge
Time-resolved fluorescence imaging
Time-resolution with long decay-time probes has gained established position as a tool in diagnostics and drug discovery to enable both highly sensitive assays (DELFIA) or simple homogeneous assays (TR-FRET). The potential of TR detection has created interest also in the attempt to gain the same improvements in gated imaging on cells and tissues by means of eliminating all ambient interferences through temporal filtering.
In imaging decay times can be exploited in different ways. In lifetime imaging (FLIM) regular fluorescent probes are used and decay times or decay time changes are employed as the assay response. Lanthanide chelates, having millisecond decay times, allow the use of very simple millisecond time-domain gating and complete temporal discrimination of autofluorescence background. This allows robust background-free images of stained tissues or fixed cellular samples having normally very high autofluorescence interference.
This has been achieved through highly fluorescent, stable and biocompatible lanthanide chelates.
Lanthanide chelates, due to their exceptional Stoke´s shift, are also perfect for various signal amplification technologies, such as nanobeads, clusters or enzymatic amplifications (tyramine reaction). Lack of inner-filter quenching allows efficient and queantitative signal amplification
The applications published so far are mostly based on fluorescent europium chelates as the labels and image acquisition taken after appropriate delay with CCD camera. The overall goal, improving sensitivity, is not achieved in all applications, mainly due to the slow photon production rate of lanthanides making their use slow and cumbersome in confocal imaging system requiring very high resolution. On the other hand, lanthanides have provided clear advantages in imaging applications where accurate quantification is needed (lack of inner filter quenching), samples and conditions where background is problematic (fixed cells, and samples with environmental contaminants), and applications where multiple colors are needed (e.g. multi-label differential cell counting).