Scalettar B A, Swedlow J R, Sedat J W, Agard D A
Howard Hughes Medical Institute, University of California, San Francisco 94143-0448, USA.
J Microsc. 1996 Apr;182(Pt 1):50-60. doi: 10.1046/j.1365-2818.1996.122402.x.
The wavelength dependence of the incoherent point spread function in a wide-field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi-wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co-localization and relative intensities of multiple components can then be accurately studied in a multi-wavelength image.
对宽视场显微镜中非相干点扩散函数的波长依赖性进行了实验研究。在荧光显微镜常用的波长范围内，样品和光学系统中的色散会导致点扩散函数发生显著变化。对于给定的样品，通常可以优化光学条件，以在给定波长下产生基本无球差的点扩散函数。不幸的是，波长偏离该值会导致产生球差的点扩散函数。因此，当使用多种荧光团来定位同一样品中的不同成分时，至少一种荧光团分布的图像将出现球差。这种像差会导致强度和分辨率的损失，从而使多波长图像中多个成分的定位和分析变得复杂。我们表明，只要用于去卷积的点扩散函数中的球差与图像中的像差合理匹配，通过约束迭代去卷积就可以将最佳分辨率恢复到有球差的图像。该方法的成功基本上与图像中初始球差的程度无关。通过收集一小批有球差和无球差的点扩散函数库，然后选择适合对每个图像进行去卷积的点扩散函数，就可以实现对许多生物图像的去卷积。然后可以在多波长图像中准确研究多个成分的共定位和相对强度。
