Frequency-domain fluorescence microscopy with the LED as a light source

We describe a frequency-domain lifetime fluorometer based on a microscope a nd a modulated light-emitting diode (LED) excitation source (370/460 nm), w hich operates in the frequency range 120 Hz-250 MHz. We collected multifreq uency phase and modulation fluorescence responses from cellular areas as sm all as 10-15 mum in diameter. We also collected fluorescence lifetime data from cells stained by a lipophilic coumarin sensitized europium fluorophore , Coum-Eu, with a millisecond lifetime, and Ru(bpy)(2)phe-C-12, with micros econd lifetime. Nanosecond lifetimes from native nuclei stained with SYTO 1 4 and SYTO 16 probes were measured as well. We demonstrate that a simple LE D excitation source can, for many applications, successfully replace comple x and expensive laser systems, which have been used for cellular frequency- domain lifetime measurements. As the LEDs are very stable with low noise, i t will be possible to image even smaller sample areas using brighter LEDs. With availability of modulated LEDs emitting at several wavelengths coverin g almost the entire visible spectrum it is easy to assemble a system for th e fluorophore of choice. The ability to select an excitation source for a g iven fluorophore and low price make such an excitation source even more pra ctical.