Title: Expanded depth of investigation microscope by digital holography in partially coherent illumination
1Bio medical Applications of digital holographic
microscope with partially spatial coherence
sources
Frank Dubois, Catherine Yourassowsky, Natacha
Callens, Brahim Jawad, Christophe Minetti,
Patrick Queeckers Microgravity Research Centre,
ULB, Brussels, Belgium frdubois_at_ulb.ac.be Septembe
r 10, 2008
2Digital holography for microscopy in transmission
- Weak depth of focus when magnification (numerical
aperture) is increased - Full recording of the holographic information in
one step or with phase shift
Investigation depth 100 x Depth of focus
3Digital holographic microscope with partially
coherent source
- Implementation - interferometry Intensity and
phase measurement - Refocusing
- Measurement of the local optical thickness
- Accuracy Few nanometers
- Sources
- LED filtered Phase stepping
- Laser incident on a rotating groundglass phase
stepping or Fouriermethod - Patents
4Effects of the partially spatial coherence (2)
- Multiple reflection elimination
5Effects of the partially spatial coherence (3)
- Reduction of the speckle noise
- Random superposition of coherent contributions
6Partially spatial partial coherent illumination
effect on a scattering sample
- 5µm particles in water
- Partial coherent illumination
- FOV 300µmX300µm
Speckle field
-200µm, 200µm, step 10µm
7Partially spatial partial coherent illumination
effect on a scattering sample
- 5µm particles in water
- Coherent illumination
- FOV 300µmX300µm
-200µm, 200µm, step 10µm
8Advantages of the spatial partial coherence
- PARTIALLY SPATIAL COHERENT ILLUMINATION
- Biocompatible plastic container
- Scattering samples cells culture in gels
- Monitoring applications
- .
9Advantages of the spatial partial coherence
10Cancer cell culture in collagen gell Turbid
media Collaboration avec le Laboratoire de
toxicologie
Journal of Biomedical Optics 11(5), 054032
(September/October 2006)
"Imagerie et Photonique pour les sciences du
vivant et la médecine", 2004, Ed. M. Faupel, P.
Smigielski and R Grzymala, Fontis Media,
Fomartis, 287-302
11Cell fusion application Anticancerous vaccine
RUBIO - MRC
- Monitoring of the cell Fusion
- Express on hybrids antibodies allowing to the
immune system to recognize tumors - Morphologic measurements of the fusions
- Counting
- Optimization of the hybrid production (size,
number of fusion) - Microscope
- Résolution
- Coupling with fluorescence
- Phase shift microscope
12Cell fusion application Anticancerous
vaccineCollaboration Clinique dOncologie
Médicale et Unité de Recherche en Biothérapie et
Oncologie - Dr. A. brandenburger
- Led with phase stepping
- FOV 350µm x 350µm
- Time laps
- Unwrapped optical phase measurement of the local
thickness - Fluorescence to mark the different types of cells
13Cell fusion application Anticancerous vaccine
14Monitoring of cell growing in bioreactorsCollabor
ation Service de Biotechnologie des Cellules
Animales prof. J. Wérenne
- Industrial bioreactors to produce vaccines
- Cell counting
- Determine the right time when to stop the culture
- Phase image
- FOV 330µm x 330µm
15Poursuite des traitements pour lanalyse des
cytodex Augmentation de lépaisseur optique due
à une infection Cytodex (BAE2 ER)
- Témoin
- Cellules infectées
16Monitoring of the protein crystal growth - PromISS
- Digital holography
- Measurement of the growing parameter
- Measurement of the depletion zones interaction
of the growing crystal with its crystallization
solution
I. Zegers, L. Carotenuto, C. Evrard, JM
Garcia-Ruiz, P. De Gieter, L.Gonzales Ramires, E.
Istasse, J-C. Legros, J. Martial, C. Minetti, F.
Otalora, P. Queeckers, C. Chockaert, C.
VandeWeerdt, R. Willaert, L. Wyns,
C.Yourassowsky, F Dubois
17Implementation in the International Space station
PromISS hardware
18Belgian Taxi Flight, Spanish Soyuz Mission, ESA
mission Protein crystal growth monitoring
PromISS 1, 2, 3 4 MRCESA Protein Microscope
for International Space Station
19BIOMICS project Dynamics of cells and biomimetic
systems
- Phospholipidic vesicles flow System that mimics
red blood cells - Dynamical behavior Shear flow, microchannel
- With and without gravity, parabolic flight and
sounding rocket
LSP Grenoble, Sample preparation and flow
chamber design and theoretical modeling, M.
Mader, T. Podgorski, C. Misbah PMMH Paris,
Sample purification techniques, M. Hoyos, P.
Kurowski, C. Ratier MRC Brussels, Optical
techniques and data processing, F. Dubois, N.
Callens, C. Minetti
20Parabolic flight campaigns for BIOMICS (2006 and
2007 ) Sounding Rocket May 2008
- Partially coherent source from a laser
- Fourier method to compute the optical amplitude
from every recorded frame - Refocus criteria
- Altitude 252 kmMicrogravity 6min
May 15, 2008
21Part of the flight movie
- Rate 25 FPS
- FOV 400µm X400µm
22Microfluidic application flow of vesicles in
channels with bifurcations
23Processing
- Refocusing intensity and phase map
- Phase map computation, unwarping
- Phase map correction
- Phase contrast emulation (DIC, PC)
- Border processing
- Automated refocusing
- Amplitude
- Phase
24Summary
- Digital holographic microscopes with reduced
spatial coherence - High imaging quality even in direct
vizualisation - Depth investigation without time distortion
- Emulation of different contrast imaging modes
- Accurate optical thickness measurement (2-3 nm)
- Combination with florescence
- Time laps implementation
- Non invasive real time 3D imaging
- Fast acquisition analysis of fast phenomena
- Reliability and robustness of the
instrumentation