Olivier Thouvenin

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.

Rapid evaluation of fresh ex vivo kidney tissue with full-field optical coherence tomography.

Background: Full-field optical coherence tomography (FFOCT) is a real-time imaging technique that rapidly generates images reminiscent of histology without any tissue processing, warranting its exploration for evaluation of ex vivo kidney tissue. Methods: Fresh tissue sections from tumor and adjacent nonneoplastic kidney (n = 25 nephrectomy specimens; clear cell renal cell carcinoma (CCRCC) = 12, papillary RCC (PRCC) = 4, chromophobe RCC (ChRCC) = 4, papillary urothelial carcinoma (PUC) = 1, angiomyolipoma (AML) = 2 and cystic nephroma = 2) were imaged with a commercial FFOCT device. Sections were submitted for routine histopathological diagnosis. Results: Glomeruli, tubules, interstitium, and blood vessels were identified in nonneoplastic tissue. In tumor sections, the normal architecture was completely replaced by either sheets of cells/trabeculae or papillary structures. The former pattern was seen predominantly in CCRCC/ChRCC and the latter in PRCC/PUC (as confirmed on H&E). Although the cellular details were not very prominent at this resolution, we could identify unique cytoplasmic signatures in some kidney tumors. For example, the hyper-intense punctate signal in the cytoplasm of CRCC represents glycogen/lipid, large cells with abundant hyper-intense cytoplasm representing histiocytes in PRCC, and signal-void large polygonal cell representing adipocytes in AML. According to a blinded analysis was performed by an uropathologist, all nonneoplastic tissues were differentiated from neoplastic tissues. Further, all benign tumors were called benign and malignant were called malignant. A diagnostic accuracy of 80% was obtained in subtyping the tumors. Conclusion: The ability of FFOCT to reliably differentiate nonneoplastic from neoplastic tissue and identify some tumor types makes it a valuable tool for rapid evaluation of ex vivo kidney tissue e.g. for intraoperative margin assessment and kidney biopsy adequacy. Recently, higher resolution images were achieved using an experimental FFOCT setup. This setup has the potential to further increase the diagnostic accuracy of FFOCT.

Appearance of the Retina With Full-Field Optical Coherence Tomography

PURPOSE To interpret full-field optical coherence tomography (FFOCT) images of ex vivo retina. METHODS Flatmounted retinas of human, primate, pig, sheep, rat, and mouse were imaged using FFOCT. To identify retinal ganglion and amacrine cells, fixed samples immunolabeled against Tuj1 and Brn3a or live samples labeled in vitro with green fluorescent protein (GFP) were analyzed by combining FFOCT, fluorescence confocal microscopy (FCM), and fluorescence-FFOCT. To investigate postmortem tissue changes, time series were acquired over 48 hours and on fresh versus fixed tissue. RESULTS With FFOCT, cell types and features such as nerve fiber bundles and RGC somas were resolved without use of contrast agents at 1-μm xyz resolution. Cell somas in the ganglion cell layer (GCL) in large mammals appeared predominantly bright with dark contours, while in rodents, GCL somas appeared dark with bright contours. RGC axon to soma junctions could be traced in the three-dimensional (3D) image stacks. Time series revealed undulation of retinal tissue samples over 48 hours, though no degradation of individual cells was detected, while paraformaldehyde fixation caused increased scattering and shrinkage. CONCLUSIONS Full-field OCT reveals micrometric morphologic detail in the retina without the use of contrast agents. We observed interspecies differences in optical properties of GCL somas. Fixation significantly alters retinal transparency hence reducing the visibility of microscopic features.

Dynamic multimodal full-field optical coherence tomography and fluorescence structured illumination microscopy

Abstract. We report on the development of a configuration of a multimodal full-field optical coherence tomography (FF-OCT) and fluorescence microscope. Our system can simultaneously acquire FF-OCT and structured illumination microscopy images. Dynamic parallel evolution of tissue microstructures and biochemical environments can be visualized. We use high numerical aperture objectives to optimize the combination of the two modalities. We imaged the propagation of mechanical waves initiated by calcium waves in a heart wall to illustrate the interest of simultaneous recording of mechanical and biochemical information.

Intracellular dynamics measurements with full field optical coherence tomography suggest hindering effect of actomyosin contractility on organelle transport

Intracellular motion can be quantitatively monitored in tissues using coherence-gated microscopic techniques. With full-field optical coherence tomography (FFOCT), the use of high numerical aperture microscope objectives provides a high resolution mapping of intracellular dynamics that are probed with subwavelength sensitivity. In the upper temporal bandwidth that we have used (1-6 Hz) the main contribution to the dynamic signal arises from the overall dynamical, optically heterogeneous cytoplasm. We propose a method to specifically study the impact of actomyosin contractility on the intracellular dynamic signal by performing high throughput, comparative measurements of multicellular aggregates with and without blebbistatin action, a selective inhibitor of class-II myosins that disrupts actomyosin contractile activity. Our results indicate a significant increase in the fraction of the signal that decorrelates within 1 second after inhibition of contractility. This observation mitigates the anticipated importance of actomyosin contractile forces to directly move organelles, but highlights their role in hindering organelle transport via their stiffening effect of the viscoelastic cytoplasm.

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.

Ultrahigh resolution imaging of cellular dynamics in explanted corneas and retinas with ocular pathologies using dynamic full-field OCT (Conference Presentation)

Full-Field Optical Coherence Tomography (FF-OCT) reveals submicrometric morphological details in retinal explants without the use of contrast agents. Dynamic FF-OCT (D-FF-OCT) takes advantage of the temporal evolution of the local FF-OCT signal to reveal a movement-dependent contrast inside tissues, mostly relying on cellular motility. Compared to regular FF-OCT images, the relative contrast from stationary structures such as nerve fibers is reduced, and contrast inside cells is enhanced, revealing many more cells, as well as the position of nuclei, and cell metabolism. We used a multimodal FF-OCT, D-FF-OCT and fluorescence microscope to compare and identify the structures observed in D-FF-OCT, which allowed us to reconstruct the full 3-D micrometric organization of corneal and retinal explants. In healthy explants, this multimodal association allows the label-free specific detection of all cell populations, except from the Mueller cells, and of several structural features such as nerve and collagen fibers, and pedicles and spherules. D-FF-OCT also accesses several functional contrasts (relying on metabolism, mechanical and electrical activity) that can be combined to monitor the tissue health over time. It is anticipated that such a combination of static and dynamic OCT information may be used in vivo in future for the early detection of ocular pathologies. To this end, we tried here to foster our understanding of the progression and occurrence of such diseases in animal models. We notably used this optical system to follow the evolution of stem cells injected in the cornea and to assess the concentration of macrophages in retinas with inflammation.

Fluorescence exclusion: A simple versatile technique to calculate cell volumes and local heights (Conference Presentation)

Understanding volume regulation during mitosis is technically challenging. Indeed, a very sensitive non invasive imaging over time scales ranging from seconds to hours and over large fields is required. Therefore, Quantitative Phase Imaging (QPI) would be a perfect tool for such a project. However, because of asymmetric protein segregation during mitosis, an efficient separation of the refractive index and the height in the phase signal is required. Even though many strategies to make such a separation have been developed, they usually are difficult to implement, have poor sensitivity, or cannot be performed in living cells, or in a single shot. In this paper, we will discuss the use of a new technique called fluorescence exclusion to perform volume measurements. By coupling such technique with a simultaneous phase measurement, we were also able to recover the refractive index inside the cells. Fluorescence exclusion is a versatile and powerful technique that allows the volume measurement of many types of cells. A fluorescent dye, which cannot penetrate inside the cells, is mixed with the external medium in a confined environment. Therefore, the fluorescent signal depends on the inverse of the object’s height. We could demonstrate both experimentally and theoretically that fluorescence exclusion can accurately measure cell volumes, even for cells much higher than the depth of focus of the objective. A local accurate height and RI measurement can also be obtained for smaller cells. We will also discuss the way to optimize the confinement of the observation chamber, either mechanically or optically.

High resolution imaging of intracellular dynamics in explanted retinas with dynamic full-field OCT (Conference Presentation)

Full-Field Optical Coherence Tomography (FF-OCT) reveals submicrometric morphological details in retinal explants without the use of contrast agents. Notably, in the nerve fiber and ganglion cell layers, FF-OCT images reveal nerve fibers bundles, single axons, capillaries and even some ganglion cell bodies. Dynamic FF-OCT (D-FF-OCT) takes advantage of the temporal evolution of the local FF-OCT signal to reveal a movement-dependent contrast inside tissues. Notably, the D-FF-OCT signal depends on cellular motility and membrane fluctuations. Compared to regular FF-OCT images, the signal from stationary structures such as nerve fibers is reduced, and contrast inside cells is enhanced, revealing many more cells, as well as the position of nuclei, and cell metabolism. We used a multimodal D-FF-OCT and fluorescence microscope to compare and identify the structures observed in both FF-OCT and D-FF-OCT. In the ganglion cell and inner nuclear layers in both macaque and mouse, two different cell sizes could be measured, which correlated well with ganglion and amacrine cell diameters found in the literature for these two species. We could also detect cell bodies of the photoreceptors in the outer nuclear layer. To our knowledge, this is the first time that an OCT technique can reveal these cell bodies. Finally, to investigate post-mortem tissue changes, time series were acquired over periods of 24 hours and cell contrast was plotted in time to monitor the decrease in intracellular activity over time. It is anticipated that dynamic FF-OCT may be used to non-invasively monitor viability and functional changes in the retina.

Cell Motility as Contrast Agent in Retinal Explant Imaging With Full-Field Optical Coherence Tomography

Purpose To use cell motility as a contrast agent in retinal explants. Methods Macaque and mouse retinal explants were imaged with high resolution full field optical coherence tomography (FFOCT) and dynamic FFOCT, coupled with fluorescence imaging. Results Static and dynamic FFOCT create complementary contrast from different structures within a cell. When imaging in vitro samples, static FFOCT detects steep refractive index gradients to reveal stationary structures including fibers, vessels, collagen, and cell contours, while dynamic FFOCT emphasizes metabolic activity of moving structures that are mainly intracellular, thus creating or enhancing contrast in cells that were previously hidden in noise. Dynamic FFOCT enables detection of most of the retinal cell types in the ganglion cell, inner and outer nuclear layers, where static FFOCT contrast is too low in relation to the noise background. Conclusions Composite static and dynamic FFOCT provides a new kind of FFOCT image containing valuable information for imaging of retinal explants. It provides label-free en face images of living retinas, with a subcellular resolution. Dynamic FFOCT adds information about cell activity, which is of interest in longitudinal explant studies.