Spectrally resolved fluorescence lifetime imaging microscopy (SFLIM)—an appropriate method for imaging single molecules in living cells

Abstract

Ever since the first examination of cellular structures, scientists have been fascinated by investigations of single cells. Over the last decade, many methods (e.g., capillary electrophoresis, electrochemical detection, mass spectroscopy, and optical spectroscopy) have been proposed for analyzing single cells [1]. Many of these methods are appropriate for in vitro experiments, which provide only a snapshot view of cells. However, some optical methods, like fluorescence spectroscopy, are also advanced enough for investigations of living cells. This is due to their simple application without the need for fixation or lysis of the respective cell. A variety of light microscopy techniques for imaging living cells, such as widefield excitation, confocal scanning and total internal reflection excitation, have been reviewed by D. J. Stephens and V. J. Allan [2]. The fluorescently labeled components required (e.g., proteins or DNA) can be added to the cell culture medium, from where they are taken up by the cells if the components are able to pass through the cell membrane. Alternatively, they can be directly injected into living cells via micropipets. Due to their toxicity, the concentration of the fluorescent dye should be as low as possible. This can be achieved by using single-molecule detection techniques that enable the measurement of low concentrations (approximately 10 M) of chromophores in homogeneous solutions and of individual immobilized molecules. In 2001, Sauer at al. established spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) [3], which uses a standard confocal microscope set-up (Fig. 1a). For this technique, a pulsed laser diode with a wavelength of usually 635 nm and a repetition rate of 40–80 MHz is used. The laser beam is coupled into an oil immersion objective and focused onto the sample surface on a scanning table. Generally, the average excitation power is adjusted to 0.25–5 kW/cm. The fluorescence light emitted by the sample is collected by the same objective and focused onto a pinhole with a diameter of 50–100 μm. Fluorescence light passing through the pinhole is spectrally separated by a dichroic beam splitter and detected by two avalanche photodiodes. To generate fluorescence lifetime images, the signals from the two avalanche photodiodes are recorded by a timecorrelated single-photon counting interface card and the data are analyzed by applying a maximum likelihood estimator (MLE) algorithm, which is an appropriate method of determining monoexponential fluorescence lifetimes from low photon count statistics. Figures 1b–d show images of an untreated living cell (3T3 mouse fibroblast) in RPMI 1640 medium recorded by SFLIM [4]. The first image (Fig. 1b) shows the overall fluorescence intensity. The cell culture medium exhibits a constant background with a count rate of approximately 4 kHz, which is due to autofluorescence of the fetal calf serum (which is in the medium), whereas the cell itself shows lower autofluorescence. Some more intense signals occur in the cytoplasm arising from the Anal Bioanal Chem (2007) 387:37–40 DOI 10.1007/s00216-006-0762-1