Diaspro A, Annunziata S, Robello M
INFM, Biophysical Section, Genoa Research Unit and Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genova, Italy.
Microsc Res Tech. 2000 Dec 1;51(5):464-8. doi: 10.1002/1097-0029(20001201)51:5<464::AID-JEMT9>3.0.CO;2-D.
Sparse fluorescent pointlike subresolution objects have been imaged using a diffraction limited single-pinhole confocal fluorescence microscope. A Maximum likelihood image restoration algorithm has been used in conjunction with a measure of the experimental point spread function for improving the three-dimensional imaging of subresolution sparse objects. The experimental point-spread-function profiles have been improved by a factor of 1.95 in lateral direction and 3.75 in axial direction resulting in full-width half maximum (FWHM) values of 91 +/- 11 nm and 160 +/- 26 nm. This amounts to 1. 43 and 2.15 in optical units, respectively. The lateral and axial FWHM of the sparse pointlike subresolution objects is about 5 and 3 times smaller than the wavelength. This result points to the attractive possibility of utilising a compact confocal architecture for localising punctuate fluorescent objects having subresolution dimensions. The key resides in the utilisation of the measured point spread function coupled to an appropriate image restoration approach, and, of course, in the stability of the confocal system being used.
使用衍射极限单针孔共聚焦荧光显微镜对稀疏的荧光点状亚分辨率物体进行了成像。最大似然图像恢复算法已与实验点扩散函数的一种度量结合使用，以改善亚分辨率稀疏物体的三维成像。实验点扩散函数轮廓在横向方向上提高了1.95倍，在轴向方向上提高了3.75倍，导致半高宽（FWHM）值分别为91±11纳米和160±26纳米。这分别相当于光学单位下的1.43和2.15。稀疏点状亚分辨率物体的横向和轴向FWHM比波长小约5倍和3倍。这一结果表明利用紧凑的共聚焦结构来定位具有亚分辨率尺寸的点状荧光物体具有诱人的可能性。关键在于利用测量的点扩散函数并结合适当的图像恢复方法，当然，还在于所使用的共聚焦系统的稳定性。
