Fluorescence lifetime imaging system with nm-resolution and single-molecule sensitivity

Abstract

Fluorescence lifetime measurements of organic fluorophores (upon laser excitation) provide a powerful additional method for distinguishing molecules of interest from the background or other species. This is of particular interest for ultra sensitive analysis and most notably for Single Molecule Detection (SMD). Another demand, in many of these applications, is to provide 2-D information (imaging) together with lifetime information. The method of choice for collection of such data is Time-Correlated Single Photon Counting (TCSPC). We have developed a compact TCSPC system on a single PC board that can perform TCSPC at high throughput, while synchronously driving a piezo scanner table that holds the sample immobilized on a surface. The TCSPC board allows measurement rates up to 3 MHz and provides a time bin resolution down to 30 ps. The detector input has a programmable Constant Fraction Discriminator (CFD). These features qualify the system for use with all common single photon detectors such as Photomultiplier Tubes and Single Photon Avalanche Photodiodes. An overall Instrument Response Function (IRF) width as low as 250 ps FWHM can be achieved with inexpensive PMTs and fast diode lasers. The board is designed for the Peripheral Component Interconnect (PCI) bus, permitting data throughput without loss of counts, sustained over a virtually unlimited measurement time. The board is software reconfigurable to operate in different modes. Here we emphasize the Time-Tagged Time-Resolved (TTTR) measurement mode, which permits recording all photon events with a realtime tag, to allow single molecule photon burst detection and subsequent offline data analysis with unlimited flexibility, e.g. for burst detection and selective histogramming. The Time-Tag clock is used to trigger an external piezo scanner driver that moves the sample. Since the clock source is common for both scanning and time tagging, the individual photons can be matched to scan pixels. We have studied single molecule solutions of Ja167 on a cover slide, to demonstrate the capabilities of the system. Fluorescence Lifetime Imaging can be performed at high resolution with as few as 100 photons per pixel.