A HIGH-SENSITIVITY TIME-RESOLVED MICROFLUOROMETER FOR REAL-TIME CELL BIOLOGY

We describe an instrument based on the novel combination of synchrotro n radiation, a high sensitivity time-resolved microfluorimeter, and a multiframe single photon counting data acquisition system. This instru ment has been designed specifically to measure kinetic events in live cells using fluorescence resonance energy transfer, and is capable of rapidly collecting multiple consecutive decay profiles from a small nu mber of fluorophores. The low irradiance on the samples (< 10 mW/cm(2) ) greatly reduces probe photobleaching and specimen photodamage during prolonged exposures. The Daresbury Synchrotron Radiation Source provi des fully wavelength tunable light pulses that have a full width half- maximum of 160 ps at a repetition rate of 3.125 MHz, with the high tem poral stability required for continuous measurements over periods of h ours. A very low limit of detection (< 10(4) molecules/mW/cm(2)) is ac complished by combining a high-gain single photon counting detection s ystem with a low fluorescence background optical layout. The latter is achieved by the inclusion of collimating optics, a reflecting objecti ve, and a specially designed beam stop situated in the epi-fluorescenc e light-path. A typical irradiance of 8 mW/cm(2) on a sample of simila r to 10(5) fluorescein molecules gives, in under 20 a, a fluorescence decay profile with a peak height of 10(4) counts, over 400 channels, a nd a signal to background ratio better than 40. The data acquisition s ystem has been developed to have a real-time time-resolved fluorescenc e collection capability (denoted as TR(2)) so that fluorescence lifeti me data can be continually collected throughout a changing process. To illustrate the potential of this instrument, we present the results o f a TR(2) experiment in which lifetime measurements of fluorescence re sonance energy transfer are used to monitor the degree of clustering o f epidermal growth factor receptors during endocytosis, over a period of about 1 h, with a 5 s resolution. (C) 1996 American Institute of Ph ysics.