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Expanded depth of investigation microscope by digital holography in partially coherent illumination

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Title: Expanded depth of investigation microscope by digital holography in partially coherent illumination


1
Bio 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
2
Digital 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
3
Digital 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

4
Effects of the partially spatial coherence (2)
  • Multiple reflection elimination

5
Effects of the partially spatial coherence (3)
  • Reduction of the speckle noise
  • Random superposition of coherent contributions

6
Partially spatial partial coherent illumination
effect on a scattering sample
  • 5µm particles in water
  • Partial coherent illumination
  • FOV 300µmX300µm

Speckle field
-200µm, 200µm, step 10µm
7
Partially spatial partial coherent illumination
effect on a scattering sample
  • 5µm particles in water
  • Coherent illumination
  • FOV 300µmX300µm

-200µm, 200µm, step 10µm
8
Advantages of the spatial partial coherence
  • PARTIALLY SPATIAL COHERENT ILLUMINATION
  • Biocompatible plastic container
  • Scattering samples cells culture in gels
  • Monitoring applications
  • .

9
Advantages of the spatial partial coherence
10
Cancer 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
11
Cell 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

12
Cell 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

13
Cell fusion application Anticancerous vaccine
14
Monitoring 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

15
Poursuite des traitements pour lanalyse des
cytodex Augmentation de lépaisseur optique due
à une infection Cytodex (BAE2 ER)
  • Témoin
  • Cellules infectées

16
Monitoring 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
17
Implementation in the International Space station
PromISS hardware
18
Belgian Taxi Flight, Spanish Soyuz Mission, ESA
mission Protein crystal growth monitoring
PromISS 1, 2, 3 4 MRCESA Protein Microscope
for International Space Station
19
BIOMICS 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
20
Parabolic 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
21
Part of the flight movie
  • Rate 25 FPS
  • FOV 400µm X400µm

22
Microfluidic application flow of vesicles in
channels with bifurcations
23
Processing
  • Refocusing intensity and phase map
  • Phase map computation, unwarping
  • Phase map correction
  • Phase contrast emulation (DIC, PC)
  • Border processing
  • Automated refocusing
  • Amplitude
  • Phase

24
Summary
  • 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
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