Microscopy deconvolution

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FS Han, HF Fan, FH Li. Image processing in high-resolution electron microscopy using the direct method. II. Image deconvolution, Acta Cryst. A42 353-356,1986. 

JJ Hu, FH Li. Maximum entropy image deconvolution in high resolution electron microscopy. Ultramicroscopy 35 339-350, 1991. 

Figure 12. Metaphase cell hybridized with FISH probes, (a) Widefield fluorescence microscopy without deconvolution and (b) Widefield fluorescence microscopy with deconvolution. The chromosomes are seen in blue. The chromosome that is labeled with FISH probes shows green and red spots which are sharper in the deconvolved image (right) compared to the image without deconvolution (left). Image courtesy, Peter Franklin, Applied Precision Inc., Issaquah, WA, USA.

At the most basic level, the data can be visualized (rendered) as an image volume viewed at varying angles and manually compressed or stretched to fit. Alternatively, a more mathematical approach can be taken if the optical aberrations can be measured. In 3D fluorescence microscopy, this measurement is known as the point spread function (psf), and a process of deconvolution can be used to correct any aberrations with a known psf. Once corrected, the data can be rendered for viewing and measurement with confidence. 

Confocal Scanning and Deconvolution Microscopy Provide Sharp Images of Three-Dimensional Objects... 

Deconvolution microscopy achieves the same Image-sharpening effect as confocal scanning microscopy but through a different process. In this method, Images from consecutive focal planes of the specimen are collected. A separate focal series of images from a test slide of subresolution size (i.e., 0.2 (jim diameter) bead are also collected. Each bead represents a pinpoint of light that becomes an object blurred by the imperfect optics of the microscope. Deconvolution... 

A EXPERIMENTAL FIGURE 5-49 Deconvolution fluorescence microscopy yields high-resolution optical sections that can be reconstructed into one three-dimensional image. A macrophage cell was stained with fluorochrome-labeled reagents specific for DNA (blue), microtubules (green), and actin microfllaments (red). The series of fluorescent Images obtained at consecutive focal planes (optical... 

Confocal microscopy and deconvolution microscopy use different methods to optically section a specimen, thereby reducing the blurring due to out-of-focus fluorescence light. Both methods provide much sharper Images, particularly of thick specimens, than does standard fluorescence microscopy. 

Why are chemical stains required for visualizing cells and tissues with the basic light microscope What advantage does fluorescent microscopy provide In comparison to the chemical dyes used to stain specimens for light microscopy What advantages do confocal scanning microscopy and deconvolution microscopy provide in comparison to conventional fluorescence microscopy ... 

Mathematical Deconvolution/Confocal Microscopy/Digital Image Analysis... 

Third, high resolution is required. Wide-field microscope deconvolution systems routinely provide 0.2-ixm XY resolution and approximately 0.4-/x,m resolution along the optical axis. This approaches or equals the theoretical limits for light microscopy, and FISH experiments frequently require this degree of image quality. 

Finally, some types of experiments require the detection of the FISH signals to be quantitative. Several laboratories have shown that wide-field deconvolution microscopy can detect signals over approximately 2.5-3 orders of magnitude in intensity with excellent linearity. This means that both weak and intense hybridization signals can be reliably detected and quantified in the same sample volume. 

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