Fluorescence Correlation Spectroscopy is widely used in the investigation of the hydrodynamic properties of single molecule. In this measurement, fluctuations of the fluorescence intensity in the small volume, typically in the order of several hundreds nm × a few μm, are recorded as a function of the time. The data obtained by this measurement is converted into the autocorrelation function. Analysis of this autocorrelation function provides the diffusion coefficient of the molecules, the lifetime of the triplet state, and so on. Although this method is a powerful tool for the detection of small area, the experimental conditions, such as the thickness of the cover glass, mismatch of the refractive index and so on, severely affect experimental results and it is usually difficult to obtain the reliable measurements. In the present paper, to overcome these difficulties in the measurements, the authors proposed a new optical configuration for FCS with orthogonally polarized two laser beams, 2fFCS, and they applied this 2fFCS to the measurements of the diffusion coefficient of the solute molecules under various conditions of refractive indices of solutions and excitation laser intensities. By comparing the results by 2fFCS with those by the conventional one beam FCS, they concluded that the 2fFCS is a quite robust method which is affected by optical and photophysical artefacts inherent to the standard FCS with a single laser beam.
蛍光相関分光法（FCS）は溶液中の単分子測定に広く用いられている手法である。この測定法は、蛍光の揺らぎを微小領域（数百nm×数μm）で観測し、シグナルの自己相関関数を計算する。取得したデータを解析することにより、分子の拡散係数や三重項寿命などの情報を知ることができる。本手法は微小領域の検出には有効な手段であるが、カバーガラスの厚さや屈折率の違いなどの測定条件の小さな変化に対しても、測定結果が敏感に影響を受けるという問題を抱えている。これらの問題を解決するため、本論分の著者らは偏光方向が互いに直交する２つのレーザー光を用いた新しいFCS（2fFCS）を紹介している。2fFCSと従来のFCSで、サンプルの屈折率変化、励起強度依存などの比較実験をしたところ、2fFCSは１つのレーザー光を用いた標準的なFCSで発現する光学的なアーティファクトの影響を受けないことが示された。