Charles A. DiMarzio

Imaging the body with diffuse optical tomography

Diffuse optical tomography (DOT) is an ongoing medical imaging modality in which tissue is illuminated by near-infrared light from an array of sources, the multiply-scattered light which emerges is observed with an array of detectors, and then a model of the propagation physics is used to infer the localized optical properties of the illuminated tissue. The three primary absorbers at these wavelengths, water and both oxygenated and deoxygenated hemoglobin, all have relatively weak absorption. This fortuitous fact provides a spectral window through which we can attempt to localize absorption (primarily by the two forms of hemoglobin) and scattering in the tissue. The most important current applications of DOT are detecting tumors in the breast and imaging the brain. We introduce the basic idea of DOT and review the history of optical methods in medicine as relevant to the development of DOT. We then detail the concept of DOT, including a review of the tissues optical properties, modes of operation for DOT, and the challenges which the development of DOT must overcome. The basics of modelling the DOT forward problem and some critical issues among the numerous implementations that have been investigated for the DOT inverse problem, with an emphasis on signal processing. We summarize with some specific results as examples of the current state of DOT research.

Deep-tissue focal fluorescence imaging with digitally time-reversed ultrasound-encoded light

Fluorescence imaging is one of the most important research tools in biomedical sciences. However, scattering of light severely impedes imaging of thick biological samples beyond the ballistic regime. Here we directly show focusing and high-resolution fluorescence imaging deep inside biological tissues by digitally time-reversing ultrasound-tagged light with high optical gain (~5×105). We confirm the presence of a time-reversed optical focus along with a diffuse background—a corollary of partial phase conjugation—and develop an approach for dynamic background cancellation. To illustrate the potential of our method, we image complex fluorescent objects and tumour microtissues at an unprecedented depth of 2.5 mm in biological tissues at a lateral resolution of 36 μm×52 μm and an axial resolution of 657 μm. Our results set the stage for a range of deep-tissue imaging applications in biomedical research and medical diagnostics.

Detection of ultrasound-modulated photons in diffuse media using the photorefractive effect.

Ultrasound-modulated optical tomography is a dual-wave sensing technique in which diffusive light in a turbid medium interacts with an imposed acoustic field. A phase-modulated photon field emanates from the interaction region and carries with it information about the optomechanical properties of the medium. We present a technique for detection of ultrasound-induced optical phase modulation using an adaptive, photorefractive-crystal-based interferometry system. Experimental results are presented demonstrating detection of ultrasound-modulated signals in highly scattering media by use of pulsed ultrasound insonation.

Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Background Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain. Methodology/Principal Findings The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded. Conclusions/Significance In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.

Mechanical strain enhances survivability of collagen micronetworks in the presence of collagenase: implications for load-bearing matrix growth and stability

There has been great interest in understanding the methods by which collagen-based load-bearing tissue is constructed, grown and maintained in vertebrate animals. To date, the responsibility for this process has largely been placed with mesenchymal fibroblastic cells that are thought to fully control the morphology of load-bearing extracellular matrix (ECM). However, given clear limitations in the ability of fibroblastic cells to precisely place or remove single collagen molecules to sculpt tissue, we have hypothesized that the material itself must play a critical role in the determination of the form of structural ECM. We here demonstrate directly, using live, dynamic, differential interference contrast imaging, that mechanically strained networks of collagen fibrils, exposed to collagenase (Clostridium histolyticum), degrade preferentially. Specifically, unstrained fibrils are removed ‘quickly’, while strained fibrils persist significantly longer. The demonstration supports the idea that collagen networks are mechanosensitive in that they are stabilized by mechanical strain. Thus, collagen molecules (together with their complement enzymes) may comprise the basis of a smart, load-adaptive, structural material system. This concept has the potential to drastically simplify the assumed role of the fibroblast, which would need only to provide ECM molecules and mechanical force to sculpt collagenous tissue.

Three-dimensional images generated by quadrature interferometry

Quadrature detection techniques have been applied to images obtained from a Mach-Zehnder interferometer with differently polarized beams to yield the real and the imaginary parts of the diffracted fields simultaneously. This approach eliminates the need for phase retrieval by providing complete information on the complex amplitude of the diffracted signal. We present results in which we demonstrate our ability to reconstruct two- and three-dimensional microscopic objects from their complex diffraction patterns.

Confocal reflectance theta line scanning microscope for imaging human skin in vivo.

A confocal reflectance theta line scanning microscope demonstrates imaging of nuclear and cellular detail in human epidermis in vivo. Experimentally measured line-spread functions determine the instrumental optical section thickness to be 1.7±-0.1 μm and the lateral resolution to be 1.0±-0.1 μm. Within human dermis (through full-thickness epidermis), the measured section thickness is 9.2±-1.7 μm and the lateral resolution is 1.7±-0.1 μm. An illumination line is scanned directly in the pupil of the objective lens, and the backscattered descanned light is detected with a linear array, such that the theta line scanner consists of only seven optical components.

Confocal theta line-scanning microscope for imaging human tissues

A confocal reflectance theta line-scanning microscope demonstrates imaging of nuclear and cellular morphology in human skin and oral mucosa in vivo. The illumination and detection are through a divided objective lens pupil, resulting in a theta-microscope configuration. A line is directly scanned in the pupil and descanned onto a linear detector array such that the theta line scanner consists of only seven main optical components. The experimentally measured lateral resolution is 1.0 microm and optical section thickness is 1.7 microm under nominal conditions at 830 nm wavelength. Through full-thickness human epidermis (i.e., in the dermis) the measured lateral resolution is 1.7 microm and the optical section thickness is 9.2 microm. The lateral resolution, sectioning, and image quality in epidermal (epithelial) tissue is comparable to that of point scanning confocal microscopy.

Dual-wedge scanning confocal reflectance microscope

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.

Combining multispectral polarized light imaging and confocal microscopy for localization of nonmelanoma skin cancer.

Multispectral polarized light imaging (MSPLI) enables rapid inspection of a superficial tissue layer over large surfaces, but does not provide information on cellular microstructure. Confocal microscopy (CM) allows imaging within turbid media with resolution comparable to that of histology, but suffers from a small field of view. In practice, pathologists use microscopes at low and high power to view tumor margins and cell features, respectively. Therefore, we study the combination of CM and MSPLI for demarcation of nonmelanoma skin cancers. Freshly excised thick skin samples with nonmelanoma cancers are rapidly stained with either toluidine or methylene blue dyes, rinsed in acetic acid, and imaged using MSPLI and CM. MSPLI is performed at 630, 660, and 750 nm. The same specimens are imaged by reflectance CM at 630, 660, and 830 nm. Results indicate that CM and MSPLI images are in good correlation with histopathology. Cytological features are identified by CM, and tumor margins are delineated by MSPLI. A combination of MSPLI and CM appears to be complementary. This combined in situ technique has potential to guide cancer surgery more rapidly and at lower cost than conventional histopathology.