Full-field spatially incoherent illumination interferometry: a spatial resolution almost insensitive to aberrations.

We show that with spatially incoherent illumination, the point spread function (PSF) width/spatial resolution of an imaging interferometer like that used in full-field optical coherence tomography (OCT) is almost insensitive to aberrations. In these systems, aberrations mostly induce a reduction of the signal level that leads to a loss of the signal-to-noise ratio without broadening the system PSF. This is demonstrated by comparison with traditional scanning OCT and wide-field OCT with spatially coherent illuminations. Theoretical analysis and numerical calculation as well as experimental results are provided to show this specific merit of incoherent illumination in full-field OCT. To the best of our knowledge, this is the first time that such a result has been demonstrated.

Dark-field full-field optical coherence tomography.

Full-field optical coherence tomography (FF-OCT) provides en face images from deep in the tissue with high spatial resolution. Specular reflections, however, may reduce image contrast as it can be much stronger than the backscattered signal from a specimen. To this end, we demonstrate dark-field FF-OCT (d-FF-OCT) that can block specular reflections by the help of an opaque disk in the pupil-conjugated plane. The reference mirror is replaced by a blazed grating, which eliminates a walk-off between the sample and the reference beams on a camera that otherwise limits the imaging field-of-view (FOV). We show that d-FF-OCT can suppress specular reflections efficiently from the glass-specimen interface by at least two orders of magnitude and yield higher contrast images compared to the conventional FF-OCT.

Optical phase modulation by natural eye movements: application to time-domain FF-OCT image retrieval.

Eye movements are commonly seen as an obstacle to high-resolution ophthalmic imaging. In this context we study the natural axial movements of the in vivo human eye and show that they can be used to modulate the optical phase and retrieve tomographic images via time-domain full-field optical coherence tomography (TD-FF-OCT). This approach opens a path to a simplified ophthalmic TD-FF-OCT device, operating without the usual piezo motor-camera synchronization. The device demonstrates in vivo human corneal images under the different image retrieval schemes (2-phase and 4-phase) and different exposure times (3.5 ms, 10 ms, 20 ms). Data on eye movements, acquired with a spectral-domain OCT with axial eye tracking (180 B-scans/s), are used to study the influence of ocular motion on the probability of capturing high-signal tomographic images without phase washout. The optimal combinations of camera acquisition speed and amplitude of piezo modulation are proposed and discussed.

Distortion matrix concept for deep optical imaging in scattering media.

In optical imaging, light propagation is affected by the inhomogeneities of the medium. Sample-induced aberrations and multiple scattering can strongly degrade the image resolution and contrast. On the basis of a dynamic correction of the incident and/or reflected wavefronts, adaptive optics has been used to compensate for those aberrations. However, it only applies to spatially invariant aberrations or to thin aberrating layers. Here, we propose a global and noninvasive approach based on the distortion matrix concept. This matrix basically connects any focusing point of the image with the distorted part of its wavefront in reflection. A singular value decomposition of the distortion matrix allows to correct for high-order aberrations and forward multiple scattering over multiple isoplanatic modes. Proof-of-concept experiments are performed through biological tissues including a turbid cornea. We demonstrate a Strehl ratio enhancement up to 2500 and recover a diffraction-limited resolution until a depth of 10 scattering mean free paths.

Real-time non-contact cellular imaging and angiography of human cornea and limbus with common-path full-field/SD OCT.

In today's clinics, a cell-resolution view of the cornea can be achieved only with a confocal microscope (IVCM) in contact with the eye. Here, we present a common-path full-field/spectral-domain OCT microscope (FF/SD OCT), which enables cell-detail imaging of the entire ocular surface in humans (central and peripheral cornea, limbus, sclera, tear film) without contact and in real-time. Real-time performance is achieved through rapid axial eye tracking and simultaneous defocusing correction. Images contain cells and nerves, which can be quantified over a millimetric field-of-view, beyond the capability of IVCM and conventional OCT. In the limbus, palisades of Vogt, vessels, and blood flow can be resolved with high contrast without contrast agent injection. The fast imaging speed of 275 frames/s (0.6 billion pixels/s) allows direct monitoring of blood flow dynamics, enabling creation of high-resolution velocity maps. Tear flow velocity and evaporation time can be measured without fluorescein administration.

In vivo high resolution human corneal imaging using full-field optical coherence tomography.

We present the first full-field optical coherence tomography (FFOCT) device capable of in vivo imaging of the human cornea. We obtained images of the epithelial structures, Bowman's layer, sub-basal nerve plexus (SNP), anterior and posterior stromal keratocytes, stromal nerves, Descemet's membrane and endothelial cells with visible nuclei. Images were acquired with a high lateral resolution of 1.7 µm and relatively large field-of-view of 1.26 mm x 1.26 mm - a combination, which, to the best of our knowledge, has not been possible with other in vivo human eye imaging methods. The latter together with a contactless operation, make FFOCT a promising candidate for becoming a new tool in ophthalmic diagnostics.

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 grid's 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.

Fast subsurface fingerprint imaging with full-field optical coherence tomography system equipped with a silicon camera.

Images recorded below the surface of a finger can have more details and be of higher quality than the conventional surface fingerprint images. This is particularly true when the quality of the surface fingerprints is compromised by, for example, moisture or surface damage. However, there is an unmet need for an inexpensive fingerprint sensor that is able to acquire high-quality images deep below the surface in short time. To this end, we report on a cost-effective full-field optical coherent tomography system comprised of a silicon camera and a powerful near-infrared LED light source. The system, for example, is able to record 1.7 cm×1.7 cmen face images in 0.12 s with the spatial sampling rate of 2116 dots per inch and the sensitivity of 93 dB. We show that the system can be used to image internal fingerprints and sweat ducts with good contrast. Finally, to demonstrate its biometric performance, we acquired subsurface fingerprint images from 240 individual fingers and estimated the equal-error-rate to be ∼0.8%. The developed instrument could also be used in other en face deep-tissue imaging applications because of its high sensitivity, such as in vivo skin imaging.

En face coherence microscopy [Invited].

En face coherence microscopy or flying spot or full field optical coherence tomography or microscopy (FF-OCT/FF-OCM) belongs to the OCT family because the sectioning ability is mostly linked to the source coherence length. In this article we will focus our attention on the advantages and the drawbacks of the following approaches: en face versus B scan tomography in terms of resolution, coherent versus incoherent illumination and influence of aberrations, and scanning versus full field imaging. We then show some examples to illustrate the diverse applications of en face coherent microscopy and show that endogenous or exogenous contrasts can add valuable information to the standard morphological image. To conclude we discuss a few domains that appear promising for future development of en face coherence microscopy.

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.

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

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.

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.

3D static elastography at the micrometer scale using Full Field OCT.

Full-Field OCT (FF-OCT) is able to image biological tissues in 3D with micrometer resolution. In this study we add elastographic contrast to the FF-OCT modality. By combining FF-OCT with elastography, we create a virtual palpation map at the micrometer scale. We present here a proof of concept on multi-layer phantoms and preliminary results on ex vivo biological samples such as porcine cornea, human breast tissues and rat heart. The 3D digital volume correlation that is used in connection with the 3D stack of images allows to access to the full 3D strain tensor and to reveal stiffness anisotropy.

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.

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.