Upon entering skin, light undergoes wavelength-dependent absorption and scattering events that, while limiting penetration depth, can provide contrast for optical imaging. Furthermore, skin contains a range of intrinsic fluorophores that can provide fluorescence contrast for optical imaging. We are developing optical imaging systems that utilize absorption and fluorescence contrast to study skin.
|Figure 1. Left: Absorption and scattering spectra of skin constituents (from http://omlc.ogi.edu/spectra/ and Hillman 2007.Righ: Optical absorption, scattering and fluorescnece processes ocurring in the skin.|
Laminar optical tomography (LOT) is an imaging technique capable of providing depth-sensitive measurements in skin to depths exceeding 1 mm. LOT can detect both absorption and fluorescence contrast, making it distinct from optical coherence tomography (OCT) which predominantly detects back-scattering structures. Absorption contrast is generally provided by melanin and blood, with melanin responsible for much of the epidermal absorption and the blood chromophores, oxy and deoxy-hemoglobin, responsible for much of the dermal absorption. LOT utilizes simultaneous imaging at 3 wavelengths to extract depth resolved measurements that are sensitive to these chromophores. LOT also has the ability to measure fluorescence. Intrinsic fluorophores such as collagen, keratin, elastin, NADH, FAD and tryptophan offer additional sources of optical contrast that can be exploited in skin, without the need to add exogenous contrast agents.
We are currently evaluating the ability of LOT to assist in skin cancer margin detection and depth of invasion determination. Excisional surgery is a common treatment for skin cancers during which the lesion is removed with a margin of surrounding apparently healthy skin. The surrounding border size varies with tumor type and level. Since skin cancer lesions are often found on the face, careful surgical planning is required to preserve function of facial features such as the eyes and nose. Tumor thickness is an important prognostic risk factor for skin cancer. Invasion into the dermis can result in poorer prognosis, since lymph node and visceral metastases may result. We are developing LOT with the aim of measuring low contrast, subsurface margins of cancerous lesions and noninvasively measuring the thickness of the lesion.
Hillman E. M. C, Burgess S. A. "Sub-millimeter Resolution 3D Optical Imaging of Living Tissue using Laminar Optical Tomography", Laser & Photonics Reviews, 3 (1-2), 159-180, (2009).
Burgess S. A, Bouchard M. B, Yuan B, Hillman E. M. C. “Simultaneous Multi-Wavelength Laminar Optical Tomography"Optics Letters, 33 (22), 2710-2712 (2008).
Burgess S. A, Yuan B, Bouchard M. B, Ratner D, Hillman E. M. C. Simultaneous Multi-Wavelength Laminar Optical Tomography Imaging of dermal lesions. In: OSA Biomedical Topical Meetings, OSA Technical Digest, Optical Society of America, Washington DC, 2008. March 16-19; St Petersberg FL.
Burgess S. A, Yuan B, Pease E, Iranmahboob A, Hillman E. M. C. Laminar Optical Tomography for Dermal and Cardiac Imaging.Engineering Conferences International: Advances in Optics for Biotechnology, Medicine and Surgery, Naples, June 2007.
Hillman E. M. C, Devor A, Dunn AK, Boas DA“Laminar Optical Tomography: High-resolution 3D functional imaging of superficial tissues“ [Invited] In: Medical Imaging, SPIE, San Diego, CA (2006)
Hillman E. M. C, Devor A, Boas DA “High-resolution functional optical imaging of living tissues“ [Invited] In: IEEE International Symposium of Biomedical Imaging Medical Imaging (ISBI) Arlington, VA (2006)
Hillman, E. M. C, Boas, D. A, Dale, A. M, Dunn, A. K, "Laminar Optical Tomography: demonstration of millimeter-scale depth-resolved imaging in turbid media", Optics Letters, 29, (14), 1650-1652 (2004).
We have constructed a two-photon microscope for high-speed in-vivo imaging. While the system was originally constructed for brain imaging, we have found that the intrinsic fluorophores and sources of second harmonic generation in the skin can allow in-vivo imaging of 3D dermal structures that rival conventional histology. Furthermore, we have developed a novel hyperspectral imaging approach that allows us to clearly delineate the different constituents of skin allowing segmentation and quantitative analysis of different structures.What is Hyperspectral Two-Photon Microscopy?
Our new technique exploits the tunability of most Ti:Sapphire lasers to acquire detailed spectroscopic information about every voxel in an image. Our system uses a Spectra Physics MaiTai Ti:Sapphire laser which can scan from 710 to 920nm and also incorporates 3 spectrally resolved detectors for collection of emitted light. Therefore, by sequentially acquiring images while scanning the excitation wavelength of our system, we can collect image data in which each pixel is an excitation-emission map of the fluorescent (or SHG-generating) substance in that pixel.
We can take two approaches to analyzing this data: 1) We can use basic spectral unmixing to delineate all of the objects within the image that have the same spectral signature. With intrinsic signal imaging of skin, this means that sebaceous glands can be distinguished from hair follicles, dermal collagen, keratinocytes etc. In stained samples, this would allow rapid segmentation of a large number of dyes. 2) We can fit the excitation / emission spectra of each voxel to the spectra of known constituents of skin, such as collagen SHG, NADH, FAD, keratin, elastin etc. When combined with 3D scanning, we can generate detailed volumetric renderings of living tissue that provide significant advantages over conventional ex-vivo histology. While detailed analysis of intrinsic emission spectra has previously been explored, excitation spectra a much more robust for unmixing. This is because emitted (usually visible) light is strongly attenuated by absorbers in tissue such as hemoglobin, meaning that emission spectra can exhibit depth-dependent distortion of their spectra making unmixing and spectral fitting challenging. Excitation spectra are much more tolerant of this effect because there are few absorbers in tissue between 710 and 920nm, so spectral signatures are better maintained. We are utilizing this technique to study diseased and health models of skin, as well as exploring the potential of hyperspectral two-photon microscopy for imaging other intact tissues.
Radosevich A. J, Bouchard M. B, Burgess S. A, Chen B. R, Hillman E. M. C. “Hyper-spectral in-vivo two-photon microscopy of intrinsic fluorophores”, Optics Letters, 33 (18), 2164-2166, (2008)
Radosevich AJ, Bouchard M. B, Burgess S. A, Stolper R, Chen B, Hillman E. M. C. Hyperspectral in-vivo two-photon microscopy. In: OSA Biomedical Topical Meetings, OSA Technical Digest, Optical Society of America, Washington DC, 2008. March 16-19; St Petersberg FL.
Grosberg LE, Radosevich AJ, Asfaha S, Yang X, Wang TJ, Hillman EMC, "Spectral characterization and unmixing of intrinsic contrast in intact normal and diseased gastric tissues using hyperspectral two-photon microscopy" PlosOne, 6(5): e19925 (2011).