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

Abstract

Articles you may be interested inHighly sensitive real-time detection of DNA hybridization by using nanoporous waveguide fluorescence spectroscopy Appl. Phys. Lett. 105, 031103 (2014); 10.1063/1.4890984Time-resolved x-ray photoelectron spectroscopy techniques for real-time studies of interfacial charge transfer dynamics AIP Conf.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 ͑Ͻ10 mW/cm 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 ͑Ͻ10 4 molecules/mW/cm 2 ͒ 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/cm 2 on a sample of ϳ10 5 fluorescein molecules gives, in under 20 s, a fluorescence decay profile with a peak height of 10 4 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 TR 2 ͒ 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 TR 2 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.

Fluorescence lifetime‐resolved pH imaging of living cells

“…FLIM measurements can be performed as time‐domain and frequency‐domain methods, and both methods can be adapted to confocal and multiphoton microscopies. These extensive capabilities of FLIM provide the appropriate lifetime resolutions to track the fluorescent tags in living cells (22–25) and to characterize intramolecular interactions (22, 26–29).…”

Adenovirus Type-5 Entry and Disassembly Followed in Living Cells by FRET, Fluorescence Anisotropy, and FLIM

“…Fluorescence lifetime imaging microscopy (FLIM) and confocal laser scanning microscopy (CLSM) were also used to evaluate the pH of the immediate environment of Ad5 and its rate of uptake via the measurement of the fluorescence lifetime of an Ad5-bound pH probe. Fluorescence lifetime measurements are more challenging than measurements based on fluorescence steadystate intensities, but have the advantage of providing quantitative results largely free from artifacts such as photobleaching and radiative transfer (Day and Piston, 1999;Martin-Fernandez et al, 1996). In these experiments we have demonstrated two distinct phases of Ad5 capsid disassembly that can be attributed to the dissociation of fibers, and to the combined dissociation of penton, hexon, and protein IX.…”

“…Fluorescence decays were recorded using a lifetime microfluorimeter at the synchrotron radiation source (SRS) (Daresbury Laboratory, Warrington, UK) using horizontally polarized pulsed synchrotron radiation as excitation light and an emission polarizer at the magic angle (54.7°) in the emission path as previously described (Martin-Fernandez et al, 1996). Fluorescence anisotropy decays were collected by measuring the components of the fluorescence decay parallel [I par (t)] and perpendicular [I per (t)] to the linearly polarized excitation light: r(t) ¼ (I par ÿ I per ) / (I par 1 2I per ) with a polarizing microscope as described elsewhere (Martin-Fernandez et al, 1998).…”

“…To measure the time course of the efficiency of FRET during Al 4881594 -Ad5 entry in CHO-CAR cells, fluorescence decay profiles of the donor fluorophore were recorded in consecutive time frames using a lifetime microfluorimeter under wide-field illumination conditions (Martin-Fernandez et al, 1996). Photobleaching in the confocal FLIM measurements prevented the continuous collection from the same area of the sample of images and time-course data for the duration of the experiment, which was two hours.…”

“…Photobleaching in the confocal FLIM measurements prevented the continuous collection from the same area of the sample of images and time-course data for the duration of the experiment, which was two hours. Therefore, for FRET time-course measurement the spatial resolution of the microscope was sacrificed to allow continuous measurement on the same area at the required time resolution (Martin-Fernandez et al, 1996). Fluorescence decay data were recorded in time frames from cells infected with Al 4881594 -Ad5, Al 488 -Ad5, and native Ad5.…”