1. 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. 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. 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)

4. 4 Double immunolabelling antigens antibody (rabbit IgG), antibody (mouse IgG) fluorochromes antibody (anti-rabbit IgG), antibody (anti-mouse IgG)

5. 5 Microscopes inverted (living cells) up-right (subcellular particles or fixed cells) transillumination : halogen lamp epi-fluorescence epi-fluorescence : mercury lamp transillumination

6. 6 Laser scanning confocal microscope : basic design

7. 7 Confocal microscopy : excitation (band), emission (filter)

8. 8 Confocal microscopy : double labelling Confocal microscopy : triple labelling

9. 9 Example of double labelling anti - FAK (rabbit polyclonal)F-actin (phalloïdin - alexa 568)merge courtesy of M. Mettlen 10 µm

10. 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. 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)