A high sensitivity time-resolved microfluorimeter for real-time cell biology

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Abstract
We describe an instrument based on the novel combination of synchrotron radiation, a high sensitivity time‐resolved microfluorimeter, and a multiframe single photon counting data acquisition system. This instrument 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 number of fluorophores. The low irradiance on the samples (2) greatly reduces probe photobleaching and specimen photodamage during prolonged exposures. The Daresbury Synchrotron Radiation Source provides 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 temporal stability required for continuous measurements over periods of hours. A very low limit of detection (4 molecules/mW/cm2) is accomplished by combining a high‐gain single photon counting detection system with a low fluorescence background optical layout. The latter is achieved by the inclusion of collimating optics, a reflecting objective, and a specially designed beam stop situated in the epi‐fluorescence light‐path. A typical irradiance of 8 mW/cm2 on a sample of ∼105 fluorescein molecules gives, in under 20 s, a fluorescence decay profile with a peak height of 104 counts, over 400 channels, and a signal to background ratio better than 40. The data acquisition system has been developed to have a real‐time time‐resolved fluorescence collection capability (denoted as TR2) so that fluorescence lifetime data can be continually collected throughout a changing process. To illustrate the potential of this instrument, we present the results of a TR2 experiment in which lifetime measurements of fluorescence resonance energy transfer are used to monitor the degree of clustering of epidermal growth factor receptors during endocytosis, over a period of about 1 h, with a 5 s resolution.