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.

Microscopie holographique à contraste de phase quantitative dans les cellules vivantes

P. Marquet1, E. Cuche2, J.-Y. Chatton1, C. Depeursinge2, P. J. Magistretti1

1Institut de physiologie, faculté de médecine, Université de Lausanne, CH-1005 Lausanne, Switzerland2Institut d’Optique appliquée, EPFL, CH-1015 Lausanne

Electromagnetic waves

Mathematical expression:

d

)cos(),( 0 oEtxE

-> (d, n, )

Light coherence

A) coherent source

B) non coherent source

x

X

“random phase”

d

d

Sample illumination

Microscope Objective

Specimen

iE

sE

Detector(CCD camera)

Incident wave

Diffracted wave

)),(cos()( nrraEs

Dimension detected: Intensity[J/Sm2]

(Intensity contrast microscopy)

)()( 22raErI ss

Origin of the Phase Shift

next< nint

--> (d, next, nint)

Young’s interference

Phase and amplitude are recorded

refsd EEE

Principe of phase recording

sE

refE

CCD

iE

hologram

Hologram reconstruction

Digital reconstruction

Numerical simulation of reference wave diffraction by the hologram:

1) intensity contrast images 2) Quantitative phase contrast images

HeNe

CCD

NF

/2 PBS B.E.

B.E.

/2

Mirror

MicroscopeObjective

Perfusion chamberwith cultured cells

Mirror

Reference Wave

Holography set up

CharacteristicsLateral resolution : 400 nm (0.75/NA) z-Axis resolution : 1..10 nm

Applications

3D morphology and volume measurements inliving cells

a) b)

a) Amplitude contrast b) Phase contrast

Astrocyte in culture