Catherine Yourassowsky

Focus plane detection criteria in digital holography microscopy by amplitude analysis

We propose and test a focus plane determination method that computes the digital refocus distance of an object investigated by digital holographic microscopy working in transmission. For this purpose we analyze the integrated amplitude modulus as a function of the digital holographic reconstruction distance. It is shown that when the focus distance is reached, the integrated amplitude is minimum for pure amplitude object and maximum for pure phase object. After a theoretical analysis, the method is demonstrated on actual digital holograms for the refocusing of pure amplitude and of pure phase microscopic samples.

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.

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.

Extended focused imaging of a microparticle field with digital holographic microscopy

We present a numerical technique for extended focused imaging and three-dimensional analysis of a microparticle field observed in a digital holographic microscope working in transmission. The three-dimensional localization of objects is performed using the local focus plane determination method based on the integrated amplitude modulus. We apply the refocusing criterion locally for each pixel, using small overlapping windows, to obtain the depth map and a synthetic image in which all objects are refocused independent from their refocusing distance. A successful application of this technique in the analysis of the microgravity particle flow experiment is presented.

Full off-axis red-green-blue digital holographic microscope with LED illumination

We developed a new full off-axis red-green-blue (RGB) digital holographic microscope with an LED illumination. A decisive advantage of the use of LED illumination is a large image quality improvement due to its partially coherent nature. The off-axis configuration enables the fast recording of the holographic data in each spectral channel. The digital holographic refocusing and the optical phase map computation are successfully demonstrated. The multiwavelength operation provides a significant improvement of the collected information for colored samples.

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.

High throughput holographic imaging-in-flow for the analysis of a wide plankton size range

We developed a Digital Holographic Microscope (DHM) working with a partial coherent source specifically adapted to perform high throughput recording of holograms of plankton organisms in-flow, in a size range of 3 µm-300 µm, which is of importance for this kind of applications. This wide size range is achieved with the same flow cell and with the same microscope magnification. The DHM configuration combines a high magnification with a large field of view and provides high-resolution intensity and quantitative phase images refocusing on high sample flow rate. Specific algorithms were developed to detect and extract automatically the particles and organisms present in the samples in order to build holograms of each one that are used for holographic refocusing and quantitative phase contrast imaging. Experimental results are shown and discussed.

Refocus criterion for both phase and amplitude objects in digital holographic microscopy.

For digital holographic microscopy applications, we modify the focus criterion based on the integration of the amplitude modulus to make possible its use regardless of the phase or amplitude nature of the objects under test. When applied on holographic data, the original criterion gives, at the focus plane, a minimum or a maximum, for amplitude or phase objects. The criterion we propose here operates on high-pass filtered complex amplitudes. It is shown that the proposed criterion gives a minimum for both types of objects when the focus plane is reached. Experimental results on real samples and simulations are provided, illustrating the efficiency and the potential of the method.

Applications of digital holographic microscopes with partially spatial coherence sources

We implemented partially spatial coherent illuminations in digital holographic microscopes (DHM) working in transmission. The benefits gained with those sources are outlined. A major advantage is the drastic reduction of the speckle noise making it possible high image quality comparable to the best classical transmission microscopes. Several implementations of biomedical applications, where digital holography provides significant information, are described. With a rapid DHM permitting the analysis of dynamical phenomena, applications in microfluidics are also provided.