Failla Antonio Virgilio, Cavallo Antonio, Cremer Christoph
Kirchhoff Institute for Physics, University of Heidelberg, Germany.
Appl Opt. 2002 Nov 1;41(31):6651-9. doi: 10.1364/ao.41.006651.
A new approach for determining the sizes of individual, small fluorescent objects with diameters considerably below the optical resolution limit is described in which spatially modulated illumination (SMI) microscopy and 360-647-nm excitation wavelengths are used. The results of SMI virtual microscopy computer simulations indicate that, in this wavelength range, reliable measurements of sizes as small as approximately 20 nm are feasible if the low numbers of fluorescence photons that are usually detected from such small objects are taken into account. This method is based on the well-known fact that the modulation of the diffraction image in a SMI microscope is disturbed by the size of the object. Using appropriately calculated calibration functions, one can use this disturbance of the modulation to determine the size of the original object.
本文描述了一种用于确定直径远低于光学分辨率极限的单个小荧光物体尺寸的新方法，该方法使用空间调制照明（SMI）显微镜和360 - 647纳米的激发波长。SMI虚拟显微镜计算机模拟结果表明，在此波长范围内，如果考虑到通常从此类小物体检测到的少量荧光光子，那么可靠测量低至约20纳米的尺寸是可行的。该方法基于一个众所周知的事实，即SMI显微镜中衍射图像的调制会受到物体尺寸的干扰。通过使用适当计算的校准函数，可以利用这种调制干扰来确定原始物体的尺寸。
