Olivier Monnom

Digital holographic microscopy for the three-dimensional dynamic analysis of in vitro cancer cell migration.

Cancer cell motility and invasion are critical targets for anticancer therapeutics. Whereas in vitro models could be designed for rapid screening with a view to investigate these targets, careful consideration must be given to the construction of appropriate model systems. Most investigations focus on two-dimensional (2-D) assays despite the fact that increasing evidence suggests that migration across rigid and planar substrates fails to recapitulate in vivo behavior. In contrast, few systems enable three-dimensional (3-D) cell migration to be quantitatively analyzed. We previously developed a digital holographic microscope (DHM) working in transmission with a partially spatial coherence source. This configuration avoids the noise artifacts of laser illumination and makes possible the direct recording of information on the 3-D structure of samples consisting of multiple objects embedded in scattering media, such as cell cultures in matrix gels. The software driving our DHM system is equipped with a time-lapse ability that enables the 3-D trajectories of living cells to be reconstituted and quantitatively analyzed.

Partial spatial coherence effects in digital holographic microscopy with a laser source

We investigate a digital holographic microscope that permits us to modify the spatial coherence state of the sample illumination by changing the spot size of a laser beam on a rotating ground glass. Out-of-focus planes are refocused by digital holographic reconstruction with numerical implementation of the Kirchhoff-Fresnel integral. The partial coherence nature of the illumination reduces the coherent artifact noise with respect to fully coherent illumination. The investigated configuration allows the spatial coherence state to be changed without modifying the illumination level of the sample. The effect of the coherence state on the digital holographic reconstruction is theoretically and experimentally evaluated. We also show how multiple reflection interferences are limited by the use of reduced spatial coherent illumination.

Digital holographic microscopy with reduced spatial coherence for three-dimensional particle flow analysis

We investigate the use of a digital holographic microscope working in partially coherent illumination to study in three dimensions a micrometer-size particle flow. The phenomenon under investigation rapidly varies in such a way that it is necessary to record, for every camera frame, the complete holographic information for further processing. For this purpose, we implement the Fourier-transform method for optical amplitude extraction. The suspension of particles is flowing in a split-flow lateral-transport thin separation cell that is usually used to separate the species by their sizes. Details of the optical implementation are provided. Examples of reconstructed images of different particle sizes are shown, and a particle-velocity measurement technique that is based on the blurred holographic image is exploited.

Pattern recognition with a digital holographic microscope working in partially coherent illumination

We describe the implementation of the automatic spatial-frequency-selection filter for recognition of patterns obtained with a digital holographic microscope working with a partially coherent source. The microscope provides the complex-optical-amplitude field that allows a refocusing plane-by-plane of the sample under investigation by numerical computation of the optical propagation. By inserting a correlation filter in the propagation equation, the correlation between the filter and the propagated optical field is obtained. In this way, the pattern is located in the direction of the optical axis. Owing to the very weak noise level generated by the partially coherent source, the correlation process is shift invariant. Therefore the samples can be located in the three dimensions. To have a robust recognition process, a generalized version of the automatic spatial-frequency-selection filters has been implemented. The method is experimentally demonstrated in a two-class problem for the recognition of protein crystals.

Border processing in digital holography by extension of the digital hologram and reduction of the higher spatial frequencies.

When a digital holographic reconstruction is performed, digital diffraction effects occur at the borders when the hologram amplitudes at the two opposite border points are different on each vertical or horizontal line. We propose a method of digital hologram extension to reduce such diffraction effects. The method consists of extending the size of the digital hologram and of filling the extended part by complex values that minimize, according to a numerical criterion, the highest spatial frequencies. The theoretical aspects of the method are given and the results from a demonstration are provided.

Improvement in visibility of an in-focus reconstructed image in digital holography by reduction of the influence of out-of-focus objects

When a reconstruction is performed on a digital holographic image that contains small objects at different depths, diffraction that is due to out-of-focus objects disrupts the visibility of the nearby focused objects. We propose a method to substitute for focused object amplitudes other amplitudes that will reduce propagation diffraction effects when other depths are investigated. The replacement amplitudes are computed by use of an algorithm that reduces the highest spatial frequencies of the resultant image. The theoretical aspects of the method are presented, and results for simulated and experimental examples are shown.

Toward Prevention of Cowdriosis

Mass production of Ehrlichia ruminantium variants from different regions of sub‐Saharan Africa is one of the difficulties that must be overcome in producing a heartwater vaccine. Vaccine productivity can be limited by endogenous induction of interferon (IFN), which inhibits the propagation of Ehrlichia ruminantium (ER) in cell culture. Different kinds of endothelial cells, in which ER multiply efficiently, could be grown in a scalable way in VueLife Teflon bags on Cytodex 3 microcarriers where bead‐to‐bead transfer of cells occurs. The digital holographic microscope designed at the Université Libre de Bruxelles allows detection of the most appropriate time to harvest intracellular microorganisms for vaccine production.