Two-Photon Fluorescence Lifetime Imaging (2P-FLIM) for Ion Sensing in Living Cells

This application note describes the powerful fusion of fluorescence lifetime imaging microscopy with two-photon excitation (2P-FLIM) by using the PicoQuant time-resolved confocal fluorescence microscope MicroTime 200. The intracellular ion homeostasis in living cells is one requirement for optimal physiological functionality in living systems. One of the most prominent features in living organisms is the intracellular pH (pHi) since it affects many physiological processes such as cellular metabolism, ion channel conductivity, contractility, ion transport and cell cycle control [1]. On the other hand, chloride, together with bicarbonate, is the major physiological anion in living cells and it is involved in the regulation of pH i, cell volume, fluid secretion and signalling processes [2]. Thus, the adequate quantitative monitoring of intracellular ion concentrations can improve our knowledge about physiological functions in living systems. Fluorescence microscopy has become one of the most powerful tools for investigating physiological processes on cellular scales providing higher contrast than classical microscopic methods. Moreover, specific fluorescent dyes offer the unique possibility to detect the distribution of analytes even within single cells. A favourably approach for the detection of fluorescent dyes is to measure their fluorescence decay time by using the fluorescence lifetime imaging microscopy (FLIM) technique. In contrast to the fluorescence intensity, the fluorescence decay time is mostly independent of variations in dye concentration, illumination intensity or processes such as photobleaching and dye leakage [3]. Thus, absolute quantification of ion concentrations in biological preparations by using FLIM for ionsensitive fluorescent dyes can be more reliable. Among FLIM, additional improvement can be achieved by two-photon (2P) excitation, which is nowadays a well-established technique in modern biological research [4]. Because of the nonlinear process of 2P excitation the generated fluorescence is restricted only to the small focal volume. As a consequence, there is no necessity of a pinhole before the detector to reject out-of focus fluorescence light for confocal detection, increasing the detectable fluorescence signal. Furthermore, most biological tissues strongly scatter visible light making high-resolution deep imaging impossible for classical confocal microscopy. The less-scattered near infrared (NIR) excitation light as used in 2P microscopy can overcome this limitation allowing deep tissue imaging and penetration depths up to 1 mm. In addition, low-energy NIR excitation light and the highly localised excitation strongly reduce global photobleaching of the fluorescent dye as well as tissue damages.