Lec3_Microscopy_ML - Light Microscopy Object eye Image Eye...

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Biophysical Methods Slide 1 Microscopy-1 Light Microscopy Object Image Objective eye Eye piece Biophysical Methods Slide 2 Microscopy-1 Fluorescence Microscopy Object Image Objective Eye piece CCD camera
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Biophysical Methods Slide 3 Microscopy-1 Fluorescence Microscopy Object Image Objective Eye piece CCD camera FITC Filter Set Biophysical Methods Slide 4 Microscopy-1 Image processing is based on understanding the microscope Images acquired using a 63 x 1.4 NA lens on a Zeiss IM35 with Photometrics nitrogen- cooled CCD. Even with an excellent objective the images are is blurred. Before (left) and after (right) image restoration. Two optical sections 0.75 μm apart from a series of 46 optical sections at 0.25 μm increments of a Newt eosinophil with rhodamine
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Biophysical Methods Slide 5 Light Microscopy Object Image Objective eye Eye piece Through no microscope can parts be separated (or the features in a really existing structure discerned), if the same ones stand so close to each other, that even the first light bundle produced by diffraction cannot enter the objective together with the undiffracted light beam. Thus results, for every size of the opening angle, a particular smallest distance of the distinguishable, the numerical value of which remains uncertain only because, due to the unequal wavelength, it is unequal for light of the different colors, the relative weight of the individual colors, however, changes much at the observation. Based on any particular color the appropriate minimal value for purely central illumination results from dividing the wavelength with the sine of the half opening angle , for the highest permissible degree of inclined illumination, however, exactly half as large - assuming otherwise equal circumstances. Unauthorized translation from: Ernst Abbe (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung, Arch. f. mikroskop. Anat. 9: 413-468. Title translation: Contributions to the Theory of the Microscope and the Microscopical Perception. . ! = light wavelength In mathematical form we may write this as: NA=n sin " Today we usually express the minimal distance of two points, which are recognized as separate using the formula: D = 1.22 ! " 2 ! NA " D = ! 2 " sin # Biophysical Methods Slide 6 Microscopy-1 Object Image Objective CCD camera The reason for the limited resolution is that the light coming from a point source is diffracted by the aperture of the objective producing what is called an Airy disc. The image of a point has finite dimensions and is called the P oint S pread F unction. PSF =s (x,y)
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Biophysical Methods Slide 7 Microscopy-1 Biophysical Methods Slide 8 Microscopy-1 The imaging properties of a microscope depend on its point spread function (PSF).
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