Biophotonics Imaging Laboratory - University of Illinois at Urbana-Champaign : OCT Technology

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OCT Technology

Multimodality Microscopy It is becoming increasingly important to visualize the dynamics of cells in three-dimensions, as it is clear that their behavior in 3-D and in vivo differs dramatically from behavior under 2-D or in vitro culture conditions. We have developed a new integrated microscope that can perform structural and functional imaging of cells in 3-D and for extended periods of time. This integrated microscope combines optical coherence microscopy (OCM), multi-photon microscopy (MPM), and second harmonic generation (SHG) microscopy in a single instrument using a single titanium:sapphire laser source. Spectral-domain OCM data provides structural information based on the optical scattering from the sample, without the need for exogenous contrast agents to label structures. MPM provides functional information by using exogenous fluorophores or genetically-expressed fluorescent proteins that are linked to cell function or physiology. With simultaneous acquisition, perfect image registration is possible, providing spatiotemporal relationships between cell and tissue structure and function.

Experimental setup for the multimodality microscope. Colored regions correspond to: blue – optical source, red – OCM, green – MPM, yellow – dispersion compensation, purple – main optical axis of the microscope. Inset shows the optical hardware for the upright microscope configuration corresponding to the purple-colored region in the schematic.

Multimodal microscopy of fibroblasts on a planar substrate. (a-d) Fibroblasts from a GFP-mouse, and (e-h) 3T3 fibroblasts with GFP-vinculin are imaged in this 2-D culture. (a,e) MPM and (b,f) OCM image channels are combined (c,g) for multimodal visualization to demonstrate the relationship between cell morphology, cell adhesion activity, and nuclei (blue channel). (d,h) Phase contrast microscopy of similar cultures is shown for comparison. The light-dark banding observed in (b,f) is due coherent interference effects from reflections off of the planar substrate and the cell membrane. The scale bars represent 20 µm.

Functional interactions between GFP-vinculin fibroblasts and microtextured substrate following mechanical stretching of the elastic polymer substrate. Substrate was stretched in the directions indicated by the arrows. (a) Multimodal image combining OCM and MPM data. Inset shows the MPM channel. (b,c) OCM and multimodal images of boxed region in (a) at en face planes 6 µm and 12 µm, respectively, above the plane in (a). Note the increase in GFP-vinculin signal-expression, which is present over a larger area of the cells. The images in (c) illustrate the depth-dependent optical sectioning of this instrument. At this plane, signal from the planar substrate is decreased (darker), while the upper peg surface reflection is increased. MPM signals from the cells are decreased at the upper en face imaging planes of these cells. The scale bar represents 20 µm.

Three-dimensional multimodal images of fibroblasts from a transgenic GFP mouse cultured in Matrigel under mechanical stimulation. 3-D culture was subjected to 5% cyclic uniaxial stretching for 18 hours, in the directions indicated by the arrows. (a) 3-D multimodal images of cells, with corresponding projections in three orthogonal planes. Color channels correspond to: Red – OCM, Blue – nuclei, Green – GFP. (b) Phase-contrast and (c) fluorescence microscopy images of the same 3-D cultures are shown for comparison. The scale bars represent 20 µm.

Tan W, Vinegoni C, Norman JJ, Desai TA, Boppart SA. Imaging cellular responses to mechanical stimuli within three-dimensional tissue constructs. Microsc. Res. Tech. in Press, 2006.

Fiber and Laser Sources Axial resolution of OCT images is primarily dependent on the bandwidth of the illumination source. There is much interest in continuum generation techniques, which are used to generate a single-mode, high bandwidth light needed for point illumination. We have devised an inexpensive and easy-to-implement augmentation to a Ti-sapphire laser that widens the bandwidth from 20 to over 200 nm using commercially available ultrahigh numerical aperture fiber. This technique provides a readily available broad-bandwidth source that can easily enhance a fiber-optic OCT system.

Spectra of Ultra-high numerical apperture 3 with various fiber lengths. Dashed curves, original input spectra; solid curves, output spectra.

Marks DL, Oldenburg AL, Reynolds JJ, Boppart SA. Study of an ultrahigh numerical aperture fiber continuum generation source for optical coherence tomography. Opt. Ltr. 27:2010-2012, 2002.

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Portable OCT Systems In order to introduce and fully utilize OCT in the clinical environment, significant engineering challenges must be addressed, including making the OCT system portable, making the acquisition software user-friendly, and making the beam-delivery systems reliable. Essential to portable OCT systems is a compact optical source that enables high-resolution imaging and an acquisition system rate that enables real-time collection of 1-D, 2-D, or 3-D OCT data sets. Recent advances in source technology, coupled with the development of spectral-domain OCT system, have made these portable systems feasible and more practical. We have and continue to develop portable OCT systems that are now routinely used in clinical settings such as the operating room, the procedure room, and the physician’s office.

A prototype portable OCT system currently being used in operating rooms for the intraoperative assessment of tumor tissue during surgical resection.

Beam Delivery Systems The successful implementation and use of OCT in clinical settings requires novel beam delivery systems that can address the wide range of applications for imaging different tissues. Laboratory-based beam-delivery systems include technology such as the Multimodality Microscope described above. More clinically-based systems include hand-held surgical imaging probes, resembling a laser pointer except with transverse-scanning capabilities, OCT catheters for imaging deep within internal body lumens and cavities, and OCT imaging needles which can be inserted into solid tissues (tumors, organs). Considerable engineering challenges are faced in the design, assembly, and performance of these systems, frequently requiring the assembly and alignment of micro-optics.

Modified surgical microscope incorporating scanners for simultaneous white-light and OCT imaging of tissue in the open surgical field.

Schematic and photograph of an OCT catheter. OCT catheters can be up to several meters in length, and less than a millimeter in diameter. Micro-optics at the distal end focus and re-direct the light out at a right angle. The inner fiber and micro-optics are rotated or translated within a stationary transparent sheath to perform radial or linear OCT imaging, respectively.

OCT needle probe designs for (A) forward-directed axial-scan acquisition and (B) side-directed OCT imaging performed by either rotating or translating the needle within the tissue. Similar fiber designs can be incorporated directly into needle-biopsy devices.

Prototype OCT needle control unit featuring both rotational and translational mechanical scanning. A representative radial-OCT image from a needle probe is shown.

Refractive index needle probe designed to measure the refractive index of in vivo tissue using low-coherence interferometry.

Boppart SA, Luo W, Marks DL, Singletary KW. Optical coherence tomography: feasibility for basic research and image-guided surgery of breast cancer. Breast Cancer Research and Treatment, 84:85-97 2004.

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Zysk AM, Adie SG, Armstrong JJ, Leigh MS, Paduch A, Sampson DD, Nguyen FT, Boppart SA. Needle-based refractive index measurement using low coherence interferometry. Optics Letters, In press, 2006.

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