AB-plot assisted determination of fluorophore mixtures in a fluorescence lifetime microscope using spectra or quenchers

Most treatments of frequency domain lifetime measurements indicate that a set of measurements must be made at multiple frequencies in order to determine the lifetimes of the components in a mixture. Although this is the case in general, under special conditions, single-frequency data can resolve multiple lifetimes. Here, data are presented showing several approaches to determining fluorescence lifetimes in two-component mixtures using single-frequency data. Common to all of the procedures presented is exploitation of variations in the relative contributions of a particular fluorophore to the total fluorescence from a mixture of fluorophores. This variation can be produced intentionally by observing a number of samples which contain different relative amounts of the fluorophores. It can be produced fortuitously by observing spatial variations in a mixture of fluorophores in a specimen or set of specimens observed with a lifetime imaging system. It can also be produced by examination of the lifetime spectrum obtained from a fluorophore mixture or by varying the concentration of a quencher in a fluorophore mixture, in which the two fluorophores have different rate constants for quenching. In many instances, the set of approaches presented here will be unsuitable for examination of arbitrary samples of unknown composition for which the multifrequency approach should be used. However, measurements produced using single-frequency methods may be applied to good effect for controlled experiments having defined fluorophores or sets of fluorophores, particularly in the case of biological lifetime imaging studies.     

SPAD array module for multi-dimensional photon timing applications

Multi-dimensional time correlated single-photon counting has reached a prominent position among analytical techniques employed in the medical and biological fields. The development of instruments able to perform temporal and spectral fluorescence analysis (sFLIM) at the same time is limited by the performance of single-photon detectors, since currently available arrays cannot simultaneously satisfy all the requirements. To face this rising quest, a fully-parallel eight-channel module, based on a monolithic single-photon avalanche diode (SPAD) array with great temporal resolution, high photon detection efficiency and low dark count rate, has been designed and fabricated. The system relies on a novel architecture of the single pixel, based on the integration of the timing pick-up circuit next to the photodetector, making the negative effects of electrical and optical crosstalk on photon timing performance negligible. To this end, the custom technological process used to fabricate the SPAD has been modified, allowing the integration of MOS transistors without impairing the structure and the performance of the detector. The single channel is complemented by an external active quenching circuit, fabricated in a standard CMOS technology, that ensures high maximum counting rate (>5?MHz) and low after-pulsing (<2%). Finally, the output timing signals are read and conditioned by proper CMOS electronics. The complete system shows a very good temporal resolution of about 45?ps (FWHM).     

Imaging lifetime and anisotropy spectra in the frequency domain

We report the development of a system combining the capabilities of fluorescence imaging spectroscopy ( x , λ, I ), fluorescence lifetime (τ) and static and dynamic fluorescence anisotropy ( r ), enabling the wide-field measurement of the spectroscopic parameters of fluorophores: ( x , λ, I , τ, r ). The system employs a frequency domain data collection strategy with a modulated light emitting diode as the light source. A polarization rotator placed in the excitation path after a polarizer allows alternating parallel and perpendicular images to be collected without moving parts. A second polarizer on the emission side serves as the analyzer, leading to estimations of the wavelength-dependent dynamic anisotropies. The spectrograph has a nominal range of 365–920 nm; however, the light-emitting diodes and filter sets used in this study restricted the usable range from about 510 to 700 nm. The system was tested on rhodamine 6G (R6G) solutions containing 0, 15, 37, 45, 59, 74 and 91 glycerol. These experiments gave rotational diffusion results comparing favourably with literature values while also demonstrating a trend towards shorter measured lifetimes at high refractive index. The ability of the system to resolve mixtures was tested on mixtures of anti-human IgG-FITC (γ-chain-specific) and R6G. These fluorophores have similar lifetimes but could be separated using anisotropy parameters. The imaging capabilities of the system were tested on mixtures of fluorescent beads with glycerol solutions of R6G.