Ries Jonas, Ruckstuhl Thomas, Verdes Dorinel, Schwille Petra
Biotechnologisches Zentrum, Technical University of Dresden, Dresden, Germany.
Biophys J. 2008 Jan 1;94(1):221-9. doi: 10.1529/biophysj.107.115998. Epub 2007 Sep 7.
We explore the potential of a supercritical angle (SA) objective for fluorescence correlation spectroscopy (FCS). This novel microscope objective combines tight focusing by an aspheric lens with strong axial confinement of supercritical angle fluorescence collection by a parabolic mirror lens, resulting in a small detection volume. The tiny axial extent of the detection volume features an excellent surface sensitivity, as is demonstrated by diffusion measurements in model membranes with an excess of free dye in solution. All SA-FCS measurements are directly compared to standard confocal FCS, demonstrating a clear advantage of SA-FCS, especially for diffusion measurements in membranes. We present an extensive theoretical framework that allows for accurate and quantitative evaluation of the SA-FCS correlation curves.
我们探索了用于荧光相关光谱法（FCS）的超临界角（SA）物镜的潜力。这种新型显微镜物镜将非球面透镜的紧密聚焦与抛物面镜透镜对超临界角荧光收集的强轴向限制相结合，从而产生一个小的检测体积。检测体积在轴向的微小范围具有出色的表面灵敏度，这在溶液中含有过量游离染料的模型膜的扩散测量中得到了证明。所有SA - FCS测量都直接与标准共聚焦FCS进行比较，证明了SA - FCS的明显优势，特别是在膜的扩散测量方面。我们提出了一个广泛的理论框架，可对SA - FCS相关曲线进行准确和定量的评估。
