Table 2.
Fluorescence-Based Method | Time Scale | Advantages | Disadvantages | |
---|---|---|---|---|
Confocal fluorescence spectroscopy | in solution | ns–ms | - molecule sorting for donor- and/or acceptor-labeled species via alternating laser excitation (ALEX) possible - measurements on freely diffusing and immobilized molecules possible |
- limited concentration range of labeled species (5–200 pM) - time resolution is limited by the diffusion-time in the focus |
immobilized | ns–min | - fluorescence lifetimes measurements possible - kinetic information |
- interactions with the surface due to immobilization might negatively influence activity of the biomolecule - sequential measurements |
|
Total internal reflection fluorescence microscopy (TIRF) | ms–min | - molecule sorting for donor- and/or acceptor-labeled species via ALEX possible - parallel measurement of hundreds of individual molecules - spatial information on the observed molecules - able to monitor not synchronized or rare events |
- interactions with the surface due to immobilization might negatively influence activity of the biomolecule - no fluorescence lifetime measurements possible |