Optical Coherence Tomography (OCT): Principles and Applications

Cross-Sectional Imaging

• Involves imaging the inside of a thick specimen.
• Achieved by various techniques:
  • Non-optical techniques: CT, MRI, Ultrasound, etc.
  • Optical techniques:
    • Physical Sectioning: Invasive (dissection and imaging using widefield microscope).
    • Optical sectioning: Non-invasive (OCT, Confocal microscope, etc.).

Physical Sectioning vs. Optical Sectioning

• Widefield Microscopy:
  • Light emitted from the entire volume of the sample is collected by the objective when imaging thick samples.
  • Images often contain a high level of background signal, obscuring specimen detail and reducing contrast.
  • Requires dissecting the specimen into thin slices using a medical knife.
  • Intensive specimen preparation (biopsy, fixation, slicing, etc.) is required.
  • Not applicable for in-vivo imaging.
• Optical Sectioning:
  • Ability to image thin sections without mechanically slicing a thick specimen.

Physical Sectioning Process

• Biopsy: A fresh, unfixed specimen after surgical removal. Should be fixed as soon as possible to prevent degeneration or drying-out.
• Fixation: A surgical specimen fixed in formalin, creating a “life-like” state and ready for grossing.
• Grossing: Slices about 4mm thick are taken from appropriate areas and placed in labeled cassettes for processing.
• Microtomy: A ribbon of sections is cut from a paraffin block using a rotary microtome.
• Mounting: Sectioned slice is mounted on a microscope slide after being floated out on warm water to flatten it.
• Imaging: All the ribbon sections are imaged one by one (with staining when necessary).

Optical Coherence Tomography (OCT) and Confocal Scanning Microscopy (CSM)

• Use gating mechanisms to eliminate out-of-focus light.
• Can generate cross-sectional images of thick specimens (3D) without physical cutting (optical sectioning).
• Perform imaging point-by-point.
  • CSM uses spatial gating.
  • OCT uses temporal gating.
• Can be used for in-vivo imaging.
• May not require intensive specimen preparation.

Learning Outcomes

• Compare OCT imaging with other imaging modalities.
• Understand the principle and modalities of OCT.
• Review the medical applications of OCT.

Introduction to OCT

• Modern biomedical optical imaging technology invented in the 1990s by Prof J. Fujimoto, MIT.
• Performs cross-sectional imaging in real-time at micrometer-scale resolution.
• Applications:
  • In situ and in vivo biomedical imaging.
  • Material characterization.
• Significant impact in ophthalmology for retinal imaging and measurement of the dimensions of the anterior segment of the eye.

OCT Citations in Web of Science

• Graph showing the number of OCT citations in Web of Science from 1991 to 2007, with a significant increase over time.
• Applications include Ophthalmology, Surgery, Technical, Cardiovascular, Neurology + Neuroscience, Gastroenterology, and Dermatology.

Penetration Depth and Resolution of Imaging Modalities

• Confocal Microscopy: Penetration depth of $100 \mu m$, resolution of $1\mu m$
• Optical Coherence Tomography (OCT): Penetration depth of $1-2-3 mm$, resolution of $2-10 \mu m$
• Ultrasound: Penetration depth of $1cm$, resolution of $150 \mu m$
• High-resolution Computed Tomography (CT): Penetration depth of $10cm$, resolution of $300 \mu m$
• Magnetic Resonance Imaging (MRI): Penetration depth of Entire body, resolution of $1mm$

OCT Characteristics

• For topographic imaging of biological tissues in situ & real-time.
• Penetration depth: 2-3 mm.
• Spatial resolution: 1-15 μm.
• Non-invasive.
• Inherits some characteristics of Ultrasound and Microscopy.

OCT Principle

• Analogous to Ultrasound with light.
• Measures the amplitude and echo time delay of light reflected/backscattered by the sample.
• Travel times of light cannot be measured by direct electronic detection because SpeedLight  1,000,000  SpeedUltrasound.
• Measurements are based on correlation techniques using low-coherence interferometry (LCI).

Low-Coherence Interferometry (LCI)

• Uses a broadband light source.
• Coherence length is defined as: $L_c = \frac{2 \ln 2}{\pi} \frac{\lambda^2}{\Delta \lambda}$
• Visible interference is observed only when the path difference between the sample and reference arms is within the coherence length.

Interferometry with Monochromatic Light

• $L_c = \frac{2 \ln 2}{\pi} \frac{\lambda^2}{\Delta \lambda}$ (same formula for coherence length, but for monochromatic light)

Low-Coherence Interferometry with Broadband Light Source

• Fringes (interference patterns) are observed within the envelope of the signal.

Using LCI for High Axial Resolution

• High axial resolution can be obtained by using broadband light.
• $\Delta z = 2 \ln 2 \times 2 \frac{\lambda_o^2}{\pi \Delta \lambda}$, where $\Delta z$ is the axial resolution.
• Example:
  • $\Delta \lambda = 150 nm$, $\Delta z \approx 1 \mu m$
  • $\Delta \lambda = 50 nm$, $\Delta z \approx 3 \mu m$

Principle of OCT Imaging

• Interference fringe envelope represents the axial profile of the biological sample (A-Scan).

Detection of OCT Signal

• Optical heterodyne detection: $I(t) \propto A<em>{sample} A</em>{ref} \cos(\omega t)$, where $\omega = \frac{4 \pi \nu}{\lambda}$ and $\nu$ is the mirror velocity.

Detection Sensitivity of OCT

• Detectable reflectivity ~ $10^{-9} - 10^{-10}$.
• OCT signal ~ Heterodyne gain $A<em>{sample} A</em>{ref}$.
• High detection sensitivity is of great interest.

Attenuation of Useful Photons in Tissues

• Scattering is the predominant attenuation mechanism.
• Penetration > 1 mm (> microscopy).
• No contrast agent, no fluorescence (≠ microscopy).
• $I = I<em>0 \exp(-\mu</em>s z)$, where $\mu_s$ is the scattering coefficient.
  • When $\mu_s = 100 mm^{-1}$,
    • at $z = 0.1 mm$, $\frac{I}{I_0} = \frac{1}{e^{10}} \approx 4.5 \times 10^{-5}$
    • at $z = 0.2 mm$, $\frac{I}{I_0} = \frac{1}{e^{20}} \approx 2 \times 10^{-9}$

Resolution in OCT

• Transverse Resolution:
  • Determined by the size of the focused laser spot.
  • $\Delta r = \frac{2 \lambda f'}{\pi D}$, where $\Delta r$ is the transverse resolution, $\lambda$ is the wavelength, $f'$ is the focal length, and $D$ is the beam diameter.
• Axial Resolution:
  • Determined by the coherence length of illuminating light.
  • $\Delta z = \frac{2 \ln 2}{\pi} \frac{\lambda^2}{\Delta \lambda}$, where $\Delta z$ is the axial resolution, $\lambda$ is the wavelength, and $\Delta \lambda$ is the bandwidth.

Factors Affecting Resolution

• Transverse Resolution: Focusing properties.
• Axial Resolution: Spectral properties of the source.
• Axial resolution does not depend on focusing properties.
• Important for applications where the available numerical aperture may be limited (e.g., Ophthalmology, endoscopic imaging).

Depth of Field (DOF)

• The largest distance d along the optical axis for which the small details are still well resolved.
• Objective
  • $\Delta r = \frac{1.22 \lambda}{2 NA}$
  • $DOF = \frac{2 \lambda n}{NA^2}$
• Both resolutions depend on the NA of the microscope.
• High NA: high resolution, small DOF.
• Low NA: low resolution, large DOF.

OCT Modalities

• TD-Domain OCT (TD-OCT).
• Fourier-Domain OCT (FD-OCT).
• Full-Field OCT (FF-OCT).

TD-OCT, FD-OCT, and FF-OCT Imaging

• TD-OCT, FD-OCT: Cross-sectional (XZ section) 3D image.
• FF-OCT: En face (XY section) 3D image.

OCT Modalities Overview

• TD-Domain OCT (TD-OCT):
  • Axial sample profile is acquired by scanning the reference mirror.
  • Transversal scanning required for 2D/3D imaging.
• Fourier-Domain OCT (FD-OCT):
  • No reference mirror scanning is required to acquire axial sample profile.
  • Transversal scanning required for 2D/3D imaging.
• Full-Field OCT (FF-OCT):
  • Axial scanning is required to acquire axial sample profile.
  • No transversal scanning is required.

TD-OCT Setup

• Fibre-optic Michelson interferometer.
• Depth information is obtained sequentially in time by scanning the reference mirror.

TD-OCT Drawbacks

• Requires reference mirror scanning.
• For a limited power, there is a trade-off between sensitivity, axial resolution and speed.

Fourier-Domain OCT (FD-OCT) Advantages

• In FD-OCT, depth information is obtained by spectroscopic measurements without scanning of the reference mirror.
• Faster and more sensitive compared to TD-OCT.
• Types:
  • Swept-Source OCT (SS-OCT): Tunable laser source, single detector.
  • Spectral-Domain OCT (SD-OCT): Spectrometer-based.
• High axial resolution is limited by the availability of broadband tunable lasers in SS-OCT. High axial resolution can be achieved more easily in SD-OCT.

SD-OCT Principle

• Spectral interferogram is obtained by a spectrometer.
• Axial profile is reconstructed by Fourier transform (FT).

A-Scan Reconstruction by FT

• The entire depth profile is obtained in one shot without scanning the reference mirror.

Medical Applications of OCT

• TD-OCT: historically the first OCT to be invented.

OCT in Ophthalmology

• The most dramatic impact of OCT.
• Applications: Retina, Anterior segment (Cornea, Iris, Lens).

Retina OCT Imaging

• Axial (depth) resolution: 10 µm (1024 pixels).
• Transverse resolution: 20 µm (128 to 768 pixels).
• Acquisition time: 0.3 to 1.9 seconds.
• First commercial system: Stratus OCT, Zeiss, 2002.

Diagnosing and Monitoring Retinal Diseases with OCT

• Diabetic Macular Edema (Glaucoma).
• Macular Hole.
• Age-Related Macular Degeneration.

High-Resolution Retinal OCT

• Light source: femtosecond laser (5 fs !).
• Axial resolution = 1 µm.

Cellular-Level OCT

• Xenopus laevis.

Anterior Segment Imaging by OCT

• Cornea, Pupil, Lens, Iris, etc.

Biometry of the Anterior Segment

• Irido-corneal angle.

Social Context and Interest of OCT

• Age-related macular degeneration: 30 million people.
• Macular edema : 135 million people with diabetes.
• Glaucoma: 110 million people.
• OCT is a unique technique for diagnosing retinal diseases at early stages, when treatments can still be applied before loss of vision occurs.
• OCT is rapidly becoming a standard in ophthalmology.

Detection of Neoplastic Changes

• OCT functions as a type of “optical biopsy”.
• Screening and identification of neoplastic changes in situ without excision.

OCT vs. Biopsy and Histopathology

• OCT can detect epithelial cancers.
• Histopathology is standard for the diagnosis of dysplasia and carcinoma but suffers from sampling errors and is cumbersome for screening.

OCT Studies

• In vitro studies to establish the correspondence between OCT and histology of various tracks, including gastrointestinal, urinary, respiratory, reproductive, and skin.

Gastrointestinal Tissues (Rabbit Colon)

• Good correlation with histology.
• Reveal changes in tissue morphology associated with cancer development.

Endoscopic OCT

• Based on fiber-optic technology, allowing for small catheters to be designed for endoscopic imaging.

Endoscopic OCT Imaging of the Human Esophagus

• Barrett's epithelium.

Human Esophagus (Endoscopic OCT)

• Demonstrates the ability of OCT to differentiate changes in architectural morphology in Barrett’s (associated with a highly increased risk of developing adenocarcinoma).
• Normal: Layered structure of squamous epithelium, with well-differentiated epithelium, lamina propria, muscularis mucosa, submucosa and muscularis propria.
• Barrett’s: Loss of squamous epithelial organization, formation of glandular structures.

Human Skin OCT in Dermatology

• Imaging of highly scattering tissues (in situ).
• Features such as the stratum corneum (SC), the dermal epidermal junction (DEJ), the epidermis (ED), sweat ducts (SD) and the nail (N) can clearly be identified.

Potential of OCT in Cancer Diagnosis

• OCT could remove the current need for large numbers of biopsies to be taken blindly when cancer is suspected.
• OCT can provide a real-time “first look” prior to excision.
• OCT could reduce sampling errors and improve sensitivity of diagnosis.

Medical Applications of OCT

• FD-OCT: appropriate for fast imaging.

FD-OCT and Real-Time Imaging

• The latest advancements in speed make real-time imaging possible.
• Potential application: guiding the femtosecond laser surgery of glaucoma.

Glaucoma

• Refers to a group of eye disorders characterized by the damage of the retinal nerve.
• When this damage occurs, blind spots develop.
• Often painless and can go undetected for a long time until noticeable irreversible vision loss occurs.
• One of the main causes of blindness: ~ 110 million people affected (~ 1 million in France), ~ 6 million people blind.

Risk Factors of Glaucoma

• The major risk factor is an increased intraocular pressure (IOP).
• Increased IOP occurs when aqueous humor is accumulated in the eye.
• Open-angle glaucoma occurs when the resistance of trabecular meshwork increases.

Conventional Treatments for Glaucoma

• Eyedrops:
  • First choice of treatment.
  • Decrease rate of production or facilitate removal of aqueous humor.
  • Drawbacks: Poor patient compliance, cost, side effects, often not effective.
• Surgical methods:
  • Filtration surgery (trabeculectomy).
  • Drawbacks: Post-surgical complications such as infection, leakage, irritation, etc., low global success rate (60%).

Laser Treatments for Glaucoma

• Argon Laser Trabeculoplasty (ALT): Uses visible lasers to burn the base of the trabecular meshwork.
• Our approach: To use IR femtosecond laser to make surgery around Schlemm’s canal directly through the sclera.
• Requires development of 1) An optimized laser source for the surgery 2) An imaging system for monitoring the surgery

Importance of OCT Imaging the Schlemm’s Canal

• We want to image the Schlemm’s canal (SC).
• OCT can also make in situ imaging non-invasively in real-time.

SD-OCT Experimental Setup for Imaging Schlemm’s Canal

• Includes SLD light source, fiber coupler, scan lens, spectrometer, line camera, etc.

Imaging the Schlemm's Canal

• Ex situ, Cross-sectional image of the sclera showing the Schlemm's canal.

3-D Image of Schlemm's Canal

• Showing the structure in three dimensions.

Coupling OCT With Surgical Laser

• To use OCT to view real time laser incisions and surgery.

OCT Imaging of Laser Incisions

• OCT cross-sectional images showing the progression of incised holes.
• Demonstrates the potential of OCT for monitoring the glaucoma laser surgery

SS-OCT 3D Image of African Tadpole

• SS-OCT showing 3d image of tadpole.

Full-Field OCT (FF-OCT)

• En face tomographic images (not cross-sectional).
• Full-field illumination (no transverse light beam scanning).
• High spatial resolution: 1 µm (isotropic).
• FF-OCT is often called Optical Coherence Microscopy (OCM).
• Based on low-coherence interference microscopy.

FF-OCT Principle

• Halogen lamp (extended source, broadband source).
• Microscope objectives and 2D CCD are used.
• Extended source of halogen lamp.

Full-Field OCT Imaging

• Due to the short coherence length (Lc), only the structures located in a thin (Lc/2) slice generate coherent back-scattered photons.
• $L<em>c = \Delta</em>z$

FF-OCT Image Acquisition

• By measuring the interference amplitude, an image of this slice is obtained (en face tomographic image).
• The interference amplitude is measured by phase-shifting interferometry.
• The tomographic image is calculated by difference of the two phase-opposed images.

Applications of Full-Field OCT

• Biology: embryology, neurology.
• Ophthalmology: characterisation of ocular tissues (retina, cornea, lens).
• Clinical diagnosis in oncology - Breast cancers (pre operative), Gastrointestinal cancers (pre-operative), Skin cancers.

FF-OCT in Embryology

• Xenopus Laevis.
• Mouse embryo (in vitro).

FF-OCT in Ophthalmology

• Rat eye (in vitro): CORNEA, Retina, CRYSTALLINE LENS.
• Commercialization started (2011) of an ex-vivo FF-OCT microscope for research applications: Light-CT™ scanner.

FF-OCT Imaging of Human Colon

• Fresh tissue, ex-vivo.
• Normal vs. Tumoral colon.

FF-OCT Imaging of Human Breast Tissue

• Fresh tissue, ex vivo.
• Healthy fibrous tissue, Duct with calcification, Lobule, Fat cells, Vessel.

Imaging Using Infrared Light

• Seeing through haze & fog with SWIR (1.4 - 3 μm).

Dual Band FF-OCT

• Dual-band full-field OCT setup.
• Full-Field OCT in different spectral regions
• Rabbit trachea using differential image

Extensions of FF-OCT

• Fluorescence FF-OCT with acridine orange staining
• Spectroscopic FF-OCT

Potential of FF-OCT

• FF-OCT images are similar to histology images, without preparation of thin slices and without dye.
• Major medical application = very fast anatomopathology of biopsies performed during the operation of cancers (breast, colon, esophagus).
• FF-OCT is limited to biopsy tissues (not fast enough for in vivo and in situ imaging).
• In the future: in vivo, in situ histology-equivalent images without excision?

OCT Modalities: Summary

• TD-OCT, FD-OCT: Cross-sectional (XZ section) 3D image.
• FF-OCT: XY section (en face) 3D image.
• OCT Biomedical Applications
  • Ophthalmology, dermatology, cardiology, oncology, neurology.
• First commercial OCT system:
  • Stratus OCT, Zeiss, 2002