Presentation on theme: "1. Resolution (R) : separation of close objects, light wavelength NA, numerical aperture 2. Contrast : distinction of objects from background “light field”"— Presentation transcript:
1. Resolution (R) : separation of close objects, light wavelength NA, numerical aperture 2. Contrast : distinction of objects from background “light field” illumination - bright field(basic illumination) - phase contrast(good for thin sections) - differential interference contrast(good for thick objects : 3D) example : with an immersion-oil lens of NA = 1.40, at = 550 nm R = 550 nm / (2 x 1.40) = 196 nm = ~ 0.2 m “dark field” illumination, e.g. fluorescence - conventional microscopy - confocal laser scanning microscopy Microscopy : two basic criteria R = 2 x NA 1
2 Fluorescence microscopy 1. What is fluorescence ? a fluorochrome absorbs light at short wavelengths and emits light of longer wavelengths; R = ~ 0.2 m 2. Why fluorescence ? - high sensitivity and contrast (molecular level) - localization of specific probes in fixed or living biological samples - monitoring dynamic processes (e.g. Ca i 2+, pH i, diffusion, cytoskeletal reorganisation, vesicular transport...)
3 Immunolocalization 1. Principe : antigen identification in a biological sample by antibodies 2. Methods : antigen antibody (rabbit IgG) fluorochrome directindirect antibody (goat) anti-IgG (rabbit Fc)
9 Example of double labelling anti - FAK (rabbit polyclonal)F-actin (phalloïdin - alexa 568)merge courtesy of M. Mettlen 10 µm
10 Example of triple labelling double immunolabelling + nuclear staining anti - pan Src (rabbit polyclonal) + anti - rabbit IgG - Alexa 488 anti - v-Src (mouse monoclonal) + anti - mouse IgG - Alexa 568 merge nuclear staining by TO-PRO-3 courtesy of M. Mettlen 10 µm
11 Advantages of confocal microscopy (Triple labelling of a mouse intestine section) Confocal imageNon-confocal image In this non-confocal view, specimen images outside the focal plane degrade the information of interest from the focal plane (blurring). In this confocal image, specimen details blurred in non-confocal imaging become distinctly visible : the image is also greatly improved in contrast. courtesy of Zeiss (Germany)
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