A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.fluorescence correlation spectroscopy ͉ live cells ͉ sensitivity L imited sensitivity and spatial resolution impede the usage of fluorescent microscopy for quantitative analysis of low copy numbers of biologically relevant molecules in live cells. Therefore, methodological and instrumental advancements are required. The aim of our work is to explore the benefits of integrating confocal laser scanning microscopy (CLSM) with fluorescence correlation spectroscopy (FCS) (1-4) as a platform for quantitative imaging of the spatiotemporal dynamics of cellular processes in real time.Fast scanning was suggested as a possible way to increase signal intensity in CLSM (5-8), but has not been systematically pursued. A contributing factor is that for increased scanning speed the number of detected photons becomes lower. With low photon counts, detector properties become increasingly relevant because the internal noise of the detector may considerably limit the quality of the image. Therefore, our first aim was to build an instrument for CLSM imaging with improved detection efficiency. We achieved this by introducing avalanche photodiodes (APDs) as detectors. Compared with the photomultiplier tubes (PMTs), normally used as detectors in conventional CLSM, the APDs are characterized by higher quantum and collection efficiency-Ϸ70% in APDs compared with 15-25% in PMTs; higher gain, faster response time, and lower dark current (6, 9). The considerably improved signal-tonoise ratio that was achieved by the introduction of APDs enabled the implementation of fast scanning. Fast scanning offers additional advantages: increased fluorescence yield by avoiding intersystem crossing, data collection at higher encountering frequency and from independent volumes, further significantly improving the signal-tobackground ratio (SBR) in imaging.We first demonstrate that improved SBRs enabled us to quantify the average number of molecules in the observation volume element by analyzing the image statistics, without resorting to temporal or...

A magneto-optic Kerr effect polarimeter designed to study the dynamics of magnetization reversal in ultrathin films, multilayer films, and microstructures is described. The polarimeter is integrated into a long focal-length charge coupled device ͑CCD͒ camera based Kerr microscope that permits viewing domain structures and facilitates positioning of the focused polarimeter beam on microstructures in ultrahigh vacuum. Diffraction-limited spatial resolution, based on the f-number of the respective objective lenses, is achieved by the microscope ͑ϳ1 m͒ and polarimeter ͑ϳ5 m͒. The polarimeter is capable of measuring continuous wave or repetitive transient ultrathin film magnetic response at sampling rates of 40 million samples/s ͑MS/s͒ over a micron-scale region defined by the illuminating spot. Hysteresis loops generated by ultrathin ͑monolayer͒ films and microstructures can be measured at high signal-to-noise ratio over a nine-decade range of drive frequencies.