Dispersion, aberration and deconvolution in multi-wavelength fluorescence images.
Dispersion, aberration and deconvolution in multi-wavelength fluorescence images.
复制标题
多波长荧光图像中的色散、像差和反卷积。
DOI：
10.1046/j.1365-2818.1996.122402.x
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发表时间：
1996
影响因子：
2
通讯作者：
Agard,DA
中科院分区：
文献类型：
--
作者：
Scalettar,BA;Swedlow,JR;Sedat,JW;Agard,DA
实验研究了广视场显微镜中非相干点扩散函数的波长依赖性。当波长在荧光显微镜中常用的范围内变化时，样品和光学元件中的色散会导致点扩散函数的显著变化。对于给定的样品，通常可以优化光学条件以产生在给定波长下基本上没有球差的点扩散函数。不幸的是，波长偏离该值将导致球差的点扩展函数。因此，当使用多个荧光团来定位同一样品中的不同组分时，至少一个荧光团的分布图像将发生球差。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分量的定位和分析变得复杂。我们证明，只要用于反卷积的点扩散函数中的球差与图像中的像差匹配得很好，就可以通过约束迭代反卷积来恢复球差图像的最佳分辨率。这种方法的成功基本上与图像中球差的初始程度无关。许多生物图像的反卷积可以通过收集球差和无像差的点扩展函数的小库，然后选择适合于对每幅图像进行反卷积的点扩展函数来实现。这样就可以在多波长图像中准确地研究多个组分的共局域化和相对强度。
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.