A. Claude Boccara

In vivo and in situ cellular imaging full-field optical coherence tomography with a rigid endoscopic probe

Full-field OCT has proved to be a powerful high-resolution cellular imaging tool for biological tissues. However the standard bulk full-field OCT setup does not match the size requirements for most in situ and in vivo imaging applications. We adapted its principle into a rigid needle-like probe using two coupled interferometers and incoherent illumination: an external processing interferometer is used for in-depth scanning, while a distal common-path interferometer at the tip of the probe collects light backscattered from the tissue. Our experimental setup achieves an axial and transversal resolution in tissue of 1.8 µm and 3.5 µm respectively, for a sensitivity of −80 dB. We present ex vivo images of human breast tissue, and in vivo images of different areas of human skin, which reveal cellular-level structures.

Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

Optimization and characterization of a structured illumination microscope

Structured illumination microscopy provides a simple and cheap mean to obtain optical sections of a sample. It can be implemented easily on a regular fluorescent microscope and is a scanning free alternative to confocal microscopy.We have analyzed theoretically the performances of the technique in terms of sectioning strength, resolution enhancement along the optical axis, and signal to background as a function of the objective used and the grids characteristics (pitch and contrast). We show that under optimized conditions, the axial resolution can be improved by a factor of 1.5 in comparison with an epifluorescence microscope, and that optical cuts with a thickness of less than 400nm can be obtained with a 1.4 numerical aperture objective. We modified the original grid in-step modulation pattern and used a sinusoidal modulation for the grid displacement. Optical sections are computed by combining four images acquired during one modulation period. This algorithm is very stable even for modulations at high frequencies. The speed of the acquisition is thus only limited by the performance of the detector and the signal/background ratio of the sample. Finally, we compared our technique to commercial setups: a confocal microscope, a Spinning Disk Microscope and a Zeiss Apotome.

From supersonic shear wave imaging to full-field optical coherence shear wave elastography

Abstract. Elasticity maps of tissue have proved to be particularly useful in providing complementary contrast to ultrasonic imaging, e.g., for cancer diagnosis at the millimeter scale. Optical coherence tomography (OCT) offers an endogenous contrast based on singly backscattered optical waves. Adding complementary contrast to OCT images by recording elasticity maps could also be valuable in improving OCT-based diagnosis at the microscopic scale. Static elastography has been successfully coupled with full-field OCT (FF-OCT) in order to realize both micrometer-scale sectioning and elasticity maps. Nevertheless, static elastography presents a number of drawbacks, mainly when stiffness quantification is required. Here, we describe the combination of two methods: transient elastography, based on speed measurements of shear waves induced by ultrasonic radiation forces, and FF-OCT, an en face OCT approach using an incoherent light source. The use of an ultrafast ultrasonic scanner and an ultrafast camera working at 10,000 to 30,000  images/s made it possible to follow shear wave propagation with both modalities. As expected, FF-OCT is found to be much more sensitive than ultrafast ultrasound to tiny shear vibrations (a few nanometers and micrometers, respectively). Stiffness assessed in gel phantoms and an ex vivo rat brain by FF-OCT is found to be in good agreement with ultrasound shear wave elastography.

Microscopic measurements of the local heat conduction in polycrystalline diamond films

Abstract It is generally known that the microstructure of polycrystalline CVD diamond samples has a strong impact on their thermal properties. Despite the fact that nowadays layers can be deposited with macroscopic thermal conductivities or diffusivities rivalling those of type II natural diamonds, the samples are highly thermally inhomogeneous and sometimes show local values differing by up to two orders of magnitude. To examine these phenomena more closely, we present a microscopic photothermal measuring method for the local thermal diffusivity. We demonstrate the feasibility of diffusivity measurements at a sample surface of ca. 20 × 20 μm. We show results obtained on a reference sample of known diffusivity and present measurements on a small single crystal diamond, a local measurement at the substrate side of a CVD diamond layer, and a measurement of the diffusivity inside a microcrystal at the growth side of a CVD diamond layer.

Smart optical coherence tomography for ultra-deep imaging through highly scattering media

Iterative time reversal overcomes multiple scattering and breaks the imaging-depth limit in optical coherence tomography. Multiple scattering of waves in disordered media is a nightmare whether it is for detection or imaging purposes. So far, the best approach to get rid of multiple scattering is optical coherence tomography. This basically combines confocal microscopy and coherence time gating to discriminate ballistic photons from a predominant multiple scattering background. Nevertheless, the imaging-depth range remains limited to 1 mm at best in human soft tissues because of aberrations and multiple scattering. We propose a matrix approach of optical imaging to push back this fundamental limit. By combining a matrix discrimination of ballistic waves and iterative time reversal, we show, both theoretically and experimentally, an extension of the imaging-depth limit by at least a factor of 2 compared to optical coherence tomography. In particular, the reported experiment demonstrates imaging through a strongly scattering layer from which only 1 reflected photon out of 1000 billion is ballistic. This approach opens a new route toward ultra-deep tissue imaging.

Dynamic full field optical coherence tomography: subcellular metabolic contrast revealed in tissues by interferometric signals temporal analysis.

We developed a new endogenous approach to reveal subcellular metabolic contrast in fresh ex vivo tissues taking advantage of the time dependence of the full field optical coherence tomography interferometric signals. This method reveals signals linked with local activity of the endogenous scattering elements which can reveal cells where other OCT-based techniques fail or need exogenous contrast agents. We benefit from the micrometric transverse resolution of full field OCT to image intracellular features. We used this time dependence to identify different dynamics at the millisecond scale on a wide range of organs in normal or pathological conditions.

Measuring aberrations in the rat brain by coherence-gated wavefront sensing using a Linnik interferometer

Aberrations limit the resolution, signal intensity and achievable imaging depth in microscopy. Coherence-gated wavefront sensing (CGWS) allows the fast measurement of aberrations in scattering samples and therefore the implementation of adaptive corrections. However, CGWS has been demonstrated so far only in weakly scattering samples. We designed a new CGWS scheme based on a Linnik interferometer and a SLED light source, which is able to compensate dispersion automatically and can be implemented on any microscope. In the highly scattering rat brain tissue, where multiply scattered photons falling within the temporal gate of the CGWS can no longer be neglected, we have measured known defocus and spherical aberrations up to a depth of 400 µm.

Fingerprint imaging from the inside of a finger with full-field optical coherence tomography.

Imaging below fingertip surface might be a useful alternative to the traditional fingerprint sensing since the internal finger features are more reliable than the external ones. One of the most promising subsurface imaging technique is optical coherence tomography (OCT), which, however, has to acquire 3-D data even when a single en face image is required. This makes OCT inherently slow for en face imaging and produce unnecessary large data sets. Here we demonstrate that full-field optical coherence tomography (FF-OCT) can be used to produce en face images of sweat pores and internal fingerprints, which can be used for the identification purposes.

Detection of plasmonic nanoparticles with full field-OCT: optical and photothermal detection.

Detecting the signal backscattered by nanoparticles immersed in highly scattering media such as biological tissue remains a challenge. In this article we report on the use of Full Field OCT (FF-OCT) to slice in depth in phantoms and in tissues in order a) to selectively observe the particles through the backscattered light at suitable wavelengths, and b) to detect the effects of the time-dependent response to full field optical heating through the strong absorption cross-section of these plasmonic nanoparticles. The analysis of the thermal wave behavior leads to the localization of the heat sources even when FF-OCT signals cannot reach the heated area.