1. Microscopie holographique à contraste de phase quantitative dans les cellules vivantes P. Marquet 1, E. Cuche 2, J.-Y. Chatton 1, C. Depeursinge 2, P. J. Magistretti 1 1 Institut de physiologie, faculté de médecine, Université de Lausanne, CH-1005 Lausanne, Switzerland 2 Institut d’Optique appliquée, EPFL, CH-1015 Lausanne

2. Electromagnetic waves Mathematical expression: d  ->  (d, n, )

3. Light coherence A) coherent source B) non coherent source “random phase” d d

4. Sample illumination Microscope Objective Specimen Detector (CCD camera) Incident wave Diffracted wave Dimension detected: Intensity[J/Sm 2 ] (Intensity contrast microscopy)

5. Origin of the Phase Shift n ext < n int  -->  (d, n ext, n int )

6. Young’s interference Phase and amplitude are recorded

7. Principe of phase recording CCD

8. hologram

9. Hologram reconstruction Digital reconstruction Numerical simulation of reference wave diffraction by the hologram: 1) intensity contrast images 2) Quantitative phase contrast images

10. HeNe CCD NF /2 PBS B.E. /2 Mirror Microscope Objective Perfusion chamber with cultured cells Mirror Reference Wave Holography set up Characteristics Lateral resolution :  400 nm (0.75 /NA) z-Axis resolution :  1..10 nm

11. Applications 3D morphology and volume measurements in living cells a) b) a) Amplitude contrast b) Phase contrast Astrocyte in culture