Image Restoration in Fluorescence Microscopy

Dit proefschrift is goedgekeurd door de promotor(en): This work was carried out in graduate school ASCI. ASCI dissertation series number 39. This thesis presents image restoration techniques for applications in (confocal) fluorescence microscopy. We have gained a better understanding of the behavior of non-linear image restoration algorithms and we have developed novel methods to improve their performance in such a way that more accurate measurements can be performed on three-dimensional fluorescence images. In this first chapter we introduce the principles of fluorescence microscopy and discuss the properties of the three-dimensional image formation in a fluorescence microscope. An image is blurred and distorted by noise during its formation and acquisition. These distortions hide fine details in the image hampering both the visual and the quantitative analysis of the image. The purpose of image restoration is to invert this and to suppress the noise restoring the fine details in the image which results in an improved analysis of the image. The principles of image restoration are discussed in the second part of this chapter. We give an overview of various restoration techniques used in fluorescence microscopy. We discuss the influence of regularization and the background on the performance of non-linear image restoration algorithms. Since the invention of the first compound light microscope by Zacharias Jansen in 1595 (Jones, 1995), the instrument has evolved enormously. The modern light microscope is a versatile instrument for microscopic analysis. The construction of the first epi-illuminated fluorescence microscope by Ploem (Ploem, 1967), made the light microscope a useful instrument for fluorescence microscopy. In epi-illumination, the illumination of the sample and the detection of its emitted fluorescence light are done using the same objective lens (Figure 1.1). This strongly reduces the penetration of illumination light in the detection light path, which makes the detection of the weak fluorescence light feasible 1. light source sample plane of focus dichroic mirror epi-illumination objective C CD camera out of focus light Figure 1.1 Schematic diagram of a conventional wide-field fluorescence microscope showing its inability of to discriminate the out-of-focus light from the in-focus light. A strong characteristic of the epi-fluorescence microscope is its wide-field illumination, which enables the simultaneous imaging of the entire focal plane. Modern scientific grade fluorescence microscopes are excellent tools for acquiring microscopic images of two-dimensional samples with a discriminating power of well below one micrometer. The performance of a wide-field microscope in acquiring three-dimensional data is not …

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