Kate Grieve

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.

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.

Assessment of Sentinel Node Biopsies With Full-Field Optical Coherence Tomography.

Current techniques for the intraoperative analysis of sentinel lymph nodes during breast cancer surgery present drawbacks such as time and tissue consumption. Full-field optical coherence tomography is a novel noninvasive, high-resolution, fast imaging technique. This study investigated the use of full-field optical coherence tomography as an alternative technique for the intraoperative analysis of sentinel lymph nodes. Seventy-one axillary lymph nodes from 38 patients at Tenon Hospital were imaged minutes after excision with full-field optical coherence tomography in the pathology laboratory, before being handled for histological analysis. A pathologist performed a blind diagnosis (benign/malignant), based on the full-field optical coherence tomography images alone, which resulted in a sensitivity of 92% and a specificity of 83% (n = 65 samples). Regular feedback was given during the blind diagnosis, with thorough analysis of the images, such that features of normal and suspect nodes were identified in the images and compared with histology. A nonmedically trained imaging expert also performed a blind diagnosis aided by the reading criteria defined by the pathologist, which resulted in 85% sensitivity and 90% specificity (n = 71 samples). The number of false positives of the pathologist was reduced by 3 in a second blind reading a few months later. These results indicate that following adequate training, full-field optical coherence tomography can be an effective noninvasive diagnostic tool for extemporaneous sentinel node biopsy qualification.

Imaging Microscopic Features of Keratoconic Corneal Morphology.

Purpose: To search for gold-standard histology indicators using alternative imaging modalities in keratoconic corneas. Methods: Prospective observational case–control study. Fourteen keratoconic corneas and 20 normal corneas (10 in vivo healthy subjects and 10 ex vivo donor corneas) were examined. Images of corneas were taken by spectral domain optical coherence tomography (SD-OCT) and in vivo confocal microscopy (IVCM) before keratoplasty. The same removed corneal buttons were imaged after keratoplasty with full-field optical coherence microscopy (FFOCM) and then fixed and sent for histology. Controls consisted of normal subjects imaged in vivo with IVCM and donor corneas imaged ex vivo with FFOCM. Corneal structural changes related to pathology were noted with each imaging modality. Cell density was quantified by manual cell counting. Results: Keratoconus indicators (ie, epithelial thinning/thickening, cell shape changes, ferritin deposits, basement membrane anomalies, Bowman layer thinning, ruptures, interruptions, scarring, stromal modifications, and appearance of Vogt striae) were generally visible with all modalities. Additional features could be seen with FFOCM in comparison with gold-standard histology, particularly in the Bowman layer region, whereas the combination of SD-OCT plus IVCM detected 76% of those features detected in histology. Three-dimensional FFOCM imaging aided interpretation of two-dimensional IVCM and SD-OCT data. Basal epithelial cell and keratocyte densities were significantly lower in patients with keratoconus than those in normals (P < 0.0001). Conclusions: Structural and cellular assessment of the keratoconic cornea by means of either in vivo SD-OCT combined with IVCM or ex vivo FFOCM in both cross-sectional and en face views can detect as many keratoconus indicators as gold-standard histology.

High-resolution three-dimensional imaging inside biological media using white-light interference microscopy

We present a white-light interference microscope designed to produce high-resolution three-dimensional images of biological media. This technique is an alternative to conventional optical coherence tomography (OCT). The experimental setup is based on a Linnik interferometer illuminated with a tungsten halogen lamp. En face tomographic images are obtained in real-time without scanning by computing the difference of two phase-opposed interferometric images recorded by a high-resolution CCD camera. The short coherence length of the source and the compensation of dispersion mismatch in the interferometer arms yield an optical sectioning ability with 0.8 μm resolution in water. Transverse resolution of 1.0 μm is achieved by using high numerical aperture microscope objectives. A shot-noise limited detection sensitivity of 86 dB can be reached with 2 s acquisition time. High-resolution images of the Xenopus Laevis tadpole are shown.

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.

High resolution in-vivo imaging of skin with full field optical coherence tomography

Full-field OCT (FFOCT) has the ability to provide en-face images with a very good axial sectioning as well as a very high transverse resolution (about 1 microns in all directions). Therefore it offers the possibility to visualize biological tissues with very high resolution both on the axial native view, and on vertical reconstructed sections. Here we investigated the potential dermatological applications of in-vivo skin imaging with FFOCT. A commercial FFOCT device was adapted for the in-vivo acquisition of stacks of images on the arm, hand and finger. Several subjects of different benign and pathological skin conditions were tested. The images allowed measurement of the stratum corneum and epidermis thicknesses, measurement of the stratum corneum refractive index, size measurement and count of the keratinocytes, visualization of the dermal-epidermal junction, and visualization of the melanin granules and of the melanocytes. Skins with different pigmentations could be discriminated and skin pathologies such as eczema could be identified. The very high resolution offered by FFOCT both on axial native images and vertical reconstructed sections allows for the visualization and measurement of a set of parameters useful for cosmetology and dermatology. In particular, FFOCT is a potential tool for the understanding and monitoring of skin hydration and pigmentation, as well as skin inflammation.

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, Bowmans layer, sub-basal nerve plexus (SNP), anterior and posterior stromal keratocytes, stromal nerves, Descemets 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.

Full-field OCT technique for high speed event-based optical flow and particle tracking

This article introduces a method to extract the speed and density of microparticles in real time at several kHz using an asynchronous event-based camera mounted on a full-field optical coherence tomography (FF-OCT) setup. These cameras detect significant amplitude changes, allowing scene-driven acquisitions. They are composed of an array of autonomously operating pixels. Events are triggered when an illuminance change at the pixel level is significant at 1μs time precision. The event-driven FF-OCT algorithm relies on a time-based optical flow computation to operate directly on incoming events and updates the estimation of velocity, direction and density while reducing both computation and data load. We show that for fast moving microparticles in a range of 0.4 - 6.5mm/s, the method performs faster and more efficiently than existing techniques in real time. The target application of this work is to evaluate erythrocyte dynamics at the microvascular level in vivo with a high temporal resolution.

Adaptive optics for in-vivo exploration of human retinal structures

Adaptive optics (AO)-enhanced imaging of the retina is now reaching a level of technical maturity which fosters its expanding use in research and clinical centers in the world. By achieving wavelength-limited resolution it did not only allow a better observation of retinal substructures already visible by other means, it also broke anatomical frontiers such as individual photoreceptors or vessel walls. The clinical applications of AO-enhanced imaging has been slower than that of optical coherence tomography because of the combination of technical complexity, costs and the paucity of interpretative scheme of complex data. In several diseases, AO-enhanced imaging has already proven to provide added clinical value and quantitative biomarkers. Here, we will review some of the clinical applications of AO-enhanced en face imaging, and trace perspectives to improve its clinical pertinence in these applications. An interesting perspective is to document cell motion through time-lapse imaging such as during agerelated macular degeneration. In arterial hypertension, the possibility to measure parietal thickness and perform fine morphometric analysis is of interest for monitoring patients. In the near future, implementation of novel approaches and multimodal imaging, including in particular optical coherence tomography, will undoubtedly expand our imaging capabilities. Tackling the technical, scientific and medical challenges offered by high resolution imaging are likely to contribute to our rethinking of many retinal diseases, and, most importantly, may find applications in other areas of medicine.