Microscopie holographique à contraste de phase quantitative dans les cellules vivantes P. Marquet...
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Transcript of Microscopie holographique à contraste de phase quantitative dans les cellules vivantes P. Marquet...
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