Full-field optical coherence tomography (FFOCT) enables in-depth, high-resolution tissue imaging at the cellular level. Pathologists using the technology achieve high sensitivity scores, offering possible future improvements in healthcare quality and decreases in costs, especially for cancer care.
Optical coherence tomography (OCT), on which the technique is based, undertakes interferometric selection of singly backscattered photons using superposition of waves to decipher their characteristics). It is a powerful technique for imaging biological tissues, particularly for eye examination.1 FFOCT, in contrast with most OCT approaches (time domain- or Fourier domain OCT, for example), directly takes 2D ‘ en face' images using megapixel cameras.2 Since the large depth-of-field needed in other OCT approaches is not required here, the setup provides 2D or 3D tomographic images with a 3D resolution of 1μm. This is typically one order of magnitude higher than for standard commercially available OCT systems, and matches the cellular resolution required for tissue examination.
Figure 1. (Top) The experimental full-field optical coherence tomography (FFOCT) setup. (Bottom) The commercial Light-CT scanner. Light reflected by each pixel of the reference mirror interferes with that backscattered from the corresponding volumetric pixel. The camera simultaneously records millions of pixels, and the piezoelectric modulation of the path difference and synchronous detection enables rejection of unwanted scattered light.
The FFOCT typically comprises a Michelson interferometer with identical microscope objectives in both arms, known as a Linnik interferometer (see Figure 1). The setup appears simple in design, but can be difficult to align. Getting the zero path difference means balancing centimeter paths to less than one micrometer. For this reason the Light-CT scanner3 we used for the studies described here is a ‘plug and play’ system with fast automatic alignment. The user can choose to register 2D large field images on a few cm2 (about 5min/cm2) with a limited number of depths or a stack of hundreds of 1mm2 images in order to display 3D views.
Figure 2. FFOCT image of breast sample and corresponding histological slide showing invasive adenocarcinoma (stellar tumor).
Figure 3. FFOCT image of normal skin and corresponding histological slide. We distinguish the three layers of the skin: epidermis (E), dermis (D), hypodermis (H), as well as a solar elastosis region (SE) and blood vessels (BV). SC: stratum corneum; SS: stratum spinosum; SE: superficial epidermis; HF: hair follicle; SG: sebaceous glands.
We used a filtered halogen lamp (about 600 to 900nm) because of its broad spectrum, enabling sectioning at a sub-micrometer level and ensuring that the sample can keep its integrity. We confirmed this through various tests, finding no alteration in any of the parameters assessed (histopathology and immunohistochemistry) following the FFOCT procedure on breast tissue.4
Comparing morphological features revealed by FFOCT with histological or frozen sections, pathologists achieved high levels of sensitivity in their results. Two pathologists could distinguish between in situ normal and benign tissue and invasive carcinomas, with a sensitivity of 97% and 90%, respectively.4 For this reason, we anticipate that FFOCT should be a valuable tool for intraoperative diagnosis, as well as tissue selection. Indeed, we expect FFOCT to help reduce the rates of re-operation and multiple biopsies, as well as saving time and space in refrigeration for tissue conservation.
Figure 4. FFOCT image of a normal brain. A: Cortex appears gray. B, C: Neuronal cell bodies (see arrows). D, E: Myelinated axon bundles (arrow) leading to white matter. F, G: vasculature (arrow). B, F: Hemalun and phloxin stainings. D: Luxol blue staining.
The selection of images below of normal and pathological tissues illustrates the comparisons we have performed in close collaboration with various hospitals. So far, we have been able to work successfully on fresh tissue of breast tumors4 and sentinel nodes, on ex vivo and in vivo skin,5 brain,6 kidney,7 lungs, and prostate. Our next goal is to implement FFOCT in vivo for rigid endoscopy7 in order to perform intraoperative surgery guidance as well as in situ biopsy guidance using a fine needle.
Langevin Institute, ESPCI ParisTech
Claude Boccara is Dean of Research and previously contributed to the advance of optical sciences as director of the optics laboratory. Co-inventor of the full-field optical coherence tomography (FFOCT) technique, he is chief science officer and co-founder of LLTech. He has received 10 scientific awards, including the NIH Bench to Bedside Pioneer Award.
Fabrice Harms, Anne Latrive
Fabrice Harms is head of product development, and led the development of the first generation of FFOCT microscopes. He has filed six patents, and is currently the intellectual property manager for LLTech. He obtained his MSc in optics from the Institute of Optics Graduate School in France in 2000 and previously worked as R&D manager at Imagine Eyes on ophthalmic applications of adaptive optics.
Anne Latrive is a project manager developing novel endoscopic imaging devices. She worked with Prof. Claude Boccara during her PhD at the Langevin Institute, ESPCI, on FFOCT for endoscopy. She also has an degree in engineering from the Ecole Centrale Paris Graduate School and an MSc in biophysics from the Pierre and Marie Curie University in Paris.
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2. A. Dubois, L. Vabre, A. C. Boccara, E. Beaurepaire, High-resolution full-field optical coherence tomography with a Linnik microscope, Appl. Opt. 41, p. 805-812, 2002.
4. O. Assayag, M. Antoine, B. Sigal-Zafrani, M. Riben, F. Harms, A. Burcheri, K. Grieve, E. Dalimier, B. Le Conte de Poly, C. Boccara, Large field, high resolution full-field optical coherence tomography: a pre-clinical study of human breast tissue and cancer assessment, Technol. in Cancer Res. and Treatment Express
1(1), p. 21-34, 2013. http://www.tcrtexpress.org/mc_files/1/3-Assayag_tcrtex_2013_3.pdf
5. E. Dalimier, D. Salomon, Full-field optical coherence tomography: a new technology for 3D high-resolution skin imaging, Dermatology
224, p. 84-92, 2012. doi:10.1159/000337423
6. O. Assayag, K. Grieve, B. Devaux, F. Harms, J. Pallud, F. Chretien, C. Boccara, P. Varlet, Imaging of non tumorous and tumorous human brain tissue with full-field optical coherence tomography, NeuroImage: Clinical, p. 549-557, 2013.
7. A. Latrive, C. Boccara, In vivo and in situ cellular imaging full-field optical coherence tomography with a rigid endoscopic probe, Biomed. Opt. Express
2(10), p. 2897-2904, 2011. doi:10.1364/BOE.2.002897