A novel fluorescence lifetime imaging system that optimizes photon efficiency

Abstract

Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost digital frequency domain FLIM (DFD-FLIM) system, which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. On the basis of the mathematical model, we present an error analysis that shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination. Microsc. Res. Tech. 71:201-213, 2008. V V C 2007 Wiley-Liss, Inc. INTRODUCTIONWe have developed a physical implementation and statistical model of a new method for fluorescence lifetime imaging (FLIM) data collection and analysis. Our approach falls in the category of the ''frequency domain'' approach to lifetime acquisition, yet uses a detector operating in the photon counting mode. This digital frequency domain (DFD) method overcomes the problems of duty cycle, modulation of the detector gain and expensive radio frequency synthesizers used in the classical analog frequency domain approach. In our approach we implemented all the operations performed in a frequency-domain lifetime instrument in a digital form using a single field programmable chip. Since all operations including the generation of the light modulation frequency, the generation of the cross-correlation sampling frequency and the assignment of the time of arrival of a photon to a bin are digital, there are no calibrations or adjustments to be performed. The mathematical model presented below fully accounts for all the elements of the DFD method. In addition, the mathematical model reproduces, as a limiting case, the principle of the time-correlated single photon counting (TCSPC) approach. Therefore, on a common statistical basis we can compare the two approaches and derive some general conclusions about the relative precision of the two methods. We found that with proper system design the two methods can be made to have comparable precision. More importantly, the mathematical model was used to maximize the precision of the DFD implementation and to determine which parameters are crucial to reach optimal performance. In terms of precision of the lifetime measurement, we were able to fully quantify the effect of the instrument response including the jitter of the detection system.Fluorescence lifetime is a fundamental spectroscopic quantity that allows quantitative analysis through several approaches, including the identification of molecu-

In vivo measurements of diffuse reflectance and time‐resolved autofluorescence emission spectra of basal cell carcinomas

“…Additionally, the recent demonstration of low cost fluorescence lifetime measurement circuitry, e.g. [65], suggests that the cost of spectrometer B could be significantly reduced.…”

Spectrally resolved fluorescent lifetime imaging

“…Pure, single-component lifetimes lie on a half-circle with a radius of 0.5 centred at (0.5, 0). From a set of measurements of m and f, an AB-plot may be constructed directly in polar coordinates or in Cartesian coordinates after a simple transformation (Clayton et al 2004;Hanley & Clayton 2005;Redford & Clegg 2005;Digman et al 2008;Colyer et al 2008). 2 Aðx; y; z; lÞ Z mðx; y; z; lÞsin fðx; y; z; lÞ ð2:7Þ and Bðx; y; z; lÞ Z mðx; y; z; lÞcos fðx; y; z; lÞ: ð2:8Þ…”

Development of new photon-counting detectors for single-molecule fluorescence microscopy

“…Indeed, in the same way it is possible to determine the fluorescence lifetime of a single molecule with N 100 photons, using a maximum-likelihood estimation approach [18,176]; such a small number of photons is sufficient to obtain a phasor value with reasonably small variance [139,177]. Therefore, not only does it appear feasible to track individual molecules with approximately 10 nm resolution and kHz frame rate, but it is simultaneously possible to measure their lifetime at the same rate, thus monitoring their electronic environment as they explore it (e.g.…”

“…Although this resolution is lower than what the detector is intrinsically capable of, it is sufficient to extract quantitative information about fluorescence decay using very few photons [177], provided a phasor analysis is used, Table 1. The H33D series of wide field photon-counting detectors.…”