Albrecht B, Failla A V, Heintzmann R, Cremer C
Kirchhoff Institute for Physics (KIP), University of Heidelberg, Applied Optics & Information Processing, D-69120 Heidelberg, Germany.
J Biomed Opt. 2001 Jul;6(3):292-9. doi: 10.1117/1.1383293.
During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.
在过去几年中，通过几种光学显微镜方法实现了远超远场光学显微镜传统“阿贝极限”的测量。点扩散函数（PSF）工程、光谱精确距离显微镜（SPDM）及相关方法被用于证明此类测量的可行性。SPDM能够测量具有不同光谱特征的点状荧光物体之间的位置和多个距离，这些距离远低于由PSF半高宽定义的光学分辨率标准。在此，我们报告一种软件方法，用于在线可视化空间调制照明（SMI）显微镜中检测到的任何物体在横向和轴向上的光分布。这极大地促进了SMI显微镜的常规应用。该软件使用运行在Windows NT上的Microsoft Visual C++开发。此外，通过虚拟显微镜研究了SPDM方法的一些理论极限方面。对于SMI显微镜的情况，考虑到光子统计和图像分析程序，研究了轴向距离测量的精度。结果表明，即使在生物结构研究典型的低荧光强度条件下，也能确定纳米范围内的精确距离测量，并且能够以这种精度检测到约40纳米量级的轴向距离。
