Arnaud Dubois

Ultrahigh-resolution full-field optical coherence tomography.

We have developed a white-light interference microscope for ultrahigh-resolution full-field optical coherence tomography of biological media. The experimental setup is based on a Linnik-type interferometer illuminated by a tungsten halogen lamp. En face tomographic images are calculated by a combination of interferometric images recorded by a high-speed CCD camera. Spatial resolution of 1.8 microm x 0.9 microm (transverse x axial) is achieved owing to the extremely short coherence length of the source, the compensation of dispersion mismatch in the interferometer arms, and the use of relatively high-numerical-aperture microscope objectives. A shot-noise-limited detection sensitivity of 90 dB is obtained in an acquisition time per image of 4 s. Subcellular-level images of plant, animal, and human tissues are presented.

Thermal-light full-field optical coherence tomography.

We have built a high-resolution optical coherence tomography (OCT) system, based on a Linnik-type interference microscope, illuminated by a white-light thermal lamp. The extremely short coherence length of the illumination source and the large aperture of the objectives permit resolution close to 1 microm in three dimensions. A parallel detection scheme with a CCD camera provides cross-section (x-y) image acquisition without scanning at a rate of up to 50 Hz. To our knowledge, our system has the highest resolution demonstrated to date for OCT imaging. With identical resolution in three dimensions, realistic volume rendering of structures inside biological tissues is possible.

Three-dimensional cellular-level imaging using full-field optical coherence tomography

An ultrahigh-resolution full-field optical coherence tomography (OCT) system has been developed for cellular-level imaging of biological media. The system is based on a Linnik interference microscope illuminated with a tungsten halogen lamp, associated with a high-resolution CCD camera. En face tomographic images are produced in real time, with the best spatial resolution ever achieved in OCT (0.7 microm x 0.9 microm, axial x transverse). A shot-noise limited detection sensitivity of 80 dB can be reached with an acquisition time per image of 1 s. Images of animal ophthalmic biopsies and vegetal tissues are shown.

Photostability of dye molecules trapped in solid matrices

The photostability of dye molecules trapped in transparent solid matrices synthesized by the solgel technique was studied both experimentally and theoretically using a model with numerical and approximate analytical solutions. The model is based on a one-photon photodestruction process with the creation of an absorbing bleached molecule. We give the number of photons that different trapped dye molecules can absorb on average before they are bleached. Dyes such as Perylene Red, Perylene Orange, Pyrromethenes 567 and 597, Rhodamines 6G and B, DCM, a Xanthylium salt, and Neon Red were investigated; significant differences were observed. Some dye molecules in solvents were also studied; increased stability resulted when the molecules were trapped in solid matrices.

In vivo anterior segment imaging in the rat eye with high speed white light full-field optical coherence tomography

We present a new high speed full-field optical coherence tomography (OCT) instrument, the first full-field OCT system that is capable of in vivo ocular imaging. An isotropic resolution of ~ 1 mum is achieved thanks to the use of a xenon arc lamp source and relatively high numerical aperture microscope objectives in a Linnik-type interferometer. Full-field illumination allows the capture of two-dimensional en face images in parallel, using a fast CMOS camera as detector array. Each en face image is acquired in a 4 ms period, at a maximum repetition rate of 250 Hz. Detection sensitivity per en face image is 71 dB. Higher sensitivity can be achieved by image correlation and averaging, although frame rate is reduced. We present the first preliminary results of in vivo imaging in the anterior segment of the rat eye, which reveal some cellular features in the corneal layers.

Initiation of gastrulation in the mouse embryo is preceded by an apparent shift in the orientation of the anterior-posterior axis.

BACKGROUND It is generally assumed that the migration of anterior visceral endoderm (AVE) cells from a distal to a proximal position at embryonic day (E)5.5 breaks the radial symmetry of the mouse embryo, marks anterior, and conditions the formation of the primitive streak on the opposite side at E6.5. Transverse sections of a gastrulating mouse embryo fit within the outline of an ellipse, with the primitive streak positioned at one end of its long axis. How the establishment of anterior-posterior (AP) polarity relates to the morphology of the postimplantation embryo is, however, unclear. RESULTS Transverse sections of prestreak E6.0 embryos also reveal an elliptical outline, but the AP axis, defined by molecular markers, tends to be perpendicular to the long axis of the ellipse. Subsequently, the relative orientations of the AP axis and of the long axis change so that when gastrulation begins, they are closer to being parallel, albeit not exactly aligned. As a result, most embryos briefly lose their bilateral symmetry when the primitive streak starts forming in the epiblast. CONCLUSIONS The change in the orientation of the AP axis is only apparent and results from a dramatic remodeling of the whole epiblast, in which cell migrations take no part. These results reveal a level of regulation and plasticity so far unsuspected in the mouse gastrula.

Investigation of optical coherence tomography as an imaging modality in tissue engineering

Monitoring cell profiles in 3D porous scaffolds presents a major challenge in tissue engineering. In this study, we investigate optical coherence tomography (OCT) as an imaging modality to monitor non-invasively both structures and cells in engineered tissue constructs. We employ time-domain OCT to visualize macro-structural morphology, and whole-field optical coherence microscopy to delineate the morphology of cells and constructs in a developing in vitro engineered bone tissue. The results show great potential for the use of OCT in non-invasive monitoring of cellular activities in 3D developing engineered tissues.

Phase-map measurements by interferometry with sinusoidal phase modulation and four integrating buckets

Phase-shifting interferometry based on the integrating-bucket technique with sinusoidal phase modulation is studied theoretically and demonstrated experimentally to obtain phase maps from double-beam interferometers. The method uses four frames obtained by integration of the time-varying intensity in an interference pattern during the four quarters of the modulation period. An optimum sinusoidal phase modulation is found to minimize the effect of the additive noise. The absolute accuracy of the phase measurements is discussed. Possible applications of the method are demonstrated with two interference microscopes with which the phase modulation is achieved by sinusoidal oscillation of a mirror attached to a piezoelectric transducer and by sinusoidal birefringence modulation with a photoelastic modulator. In both experimental arrangements, phase images can be produced in real time at a rate of several hertz. Noise measurements are reported and compared with theory.

Simultaneous dual-band ultra-high resolution full-field optical coherence tomography

Ultrahigh-resolution full-field optical coherence tomography (FF-OCT) is demonstrated in the 800 nm and 1200 nm wavelength regions simultaneously using a Silicon-based (Si) CCD camera and an Indium Gallium Arsenide (InGaAs) camera as area detectors and a halogen lamp as illumination source. The FF-OCT setup is optimized to support the two broad spectral bands in parallel, achieving a detection sensitivity of approximately 90 dB and a micrometer-scale resolution in the three directions. Images of ex vivo biological tissues are presented (rabbit trachea and Xenopus laevis tadpole) with an increase in penetration depth at 1200 nm. A color image representation is applied to fuse both images and enhance spectroscopic property visualization.

Stroboscopic ultrahigh-resolution full-field optical coherence tomography

We present a new technique that produces en face tomographic images with a 10-micros acquisition time per image. The setup consists of an interference microscope with stroboscopic illumination provided by a xenon arc flash lamp (10-micros flashes at 15 Hz). The tomographic images are obtained from two phase-opposed interferometric images recorded simultaneously by two synchronized CCD cameras. Transverse resolution better than 1.0 microm is achieved by use of high-numerical-aperture microscope objectives. The short coherence length of the source yields an axial resolution of 0.9 microm. 3 x 3 pixel binning leads to a detection sensitivity of 71 dB. Our system is suitable for various applications, particularly in biology for in vivo cellular-level imaging.