BMED 4501 Biophotonics

(Semester 2 – Year 2024 – 2025)

Homework 2 (Full mark: 90)

(Due: May, 21st)

Retinal Imaging and OCT Angiography in Eye-Disease Diagnosis

Retinal imaging plays a central role in modern ophthalmology, allowing detailed visualization of the fundus, or the back part of the eye, for diagnosis and monitoring of retinal diseases (see Fig. 1(A) for an overview of ocular anatomy). One of the most common and vision-threatening retinal  pathologies  is age-related macular degeneration (AMD). AMD exhibits both structural and vascular changes in the macula, which can be effectively assessed using various retinal imaging modalities. Common symptoms of AMD include blurred central vision and metamorphopsia (i.e. distorted version).

Fig. 1 (A) Anatomy of human eye.     (B-C) Images taken by fundus camera: (B) healthy retina (C) age-related macular degeneration. RPE: retinal pigment epithelium

The current gold standard for general retinal imaging is fundus photography, which provides a wide-field, magnified view of the retina, optic disc, choroid, and retinal blood vessels. It is widely used for screening of retinal pathologies. Figures 1(B)–(C) show representative fundus images of both healthy and AMD-affected eyes, highlighting key clinical features such as drusen, pigmentary changes, and retinal pigment epithelium (RPE) irregularities.

However, fundus photography provides only en-face 2D structural information and lacks the ability to image depth or blood flow. To address these limitations, more advanced imaging techniques have been adopted:

(1) Fluorescence angiography  (FA) –  Two extrinsic contrast agents (fluorescence dyes) have been adopted:  sodium  fluorescein and indocyanine green (ICG).  Both fluorescein an  ICG angiography involve dye injection into the systemic circulation (into veins of the forearm) prior to retinal imaging. Few minutes after injection, the retinal and choriodal vasculatures, which are filled with the dye, can be visualized by fluorescence imaging (Fig. 2).

FIG. 2. (A) ICG angiogram and (B) Fluorescein angiogram of the same field-of-view across the fundus.      The vasculature is clearly visualized in both images. Note that the central bright region in (A) reveals choroidal neovascularization (CNV), that is absent in (B), i.e. the creation of new blood vessels in the choroid layer – an indication of AMD.

(2) Fundus  autofluorescence  (FAF) –  A noninvasive imaging  modality  that  captures  the  natural autofluorescence of lipofuscin in the RPE. It is a pigment by-product observed in retinal pigment epithelial   (RPE)   cells.  Normally,   lipofuscin  is constantly being produced by the    RPE   and choriocapillaris   (capillaries   near  choroid).   However, aging process and/or various retinal conditions  could lead to lipofuscin  accumulation.  Excessive  amount  of lipofuscin  interferes with normal cell function and thus results in cell death. Therefore, lipofuscin is a key component related to RPE metabolism (health condition). Distinct patterns of fundus autofluorescence (or absence of autofluorescence)  correlating  with  RPE  death  are  most  strongly  seen  in  age-related  macular degeneration (AMD) (Fig. 3).

Fig. 3 Fundus autofluorescence images. (A) Healthy fundus. (B) Fundus with AMD. Note that banded pattern of increased autofluorescence in the junction.

While typical OCT—which you have already studied in the class —provides high-resolution cross-sectional structural images of retinal layers, it cannot directly visualize blood flow or vascular abnormalities such as choroidal neovascularization (CNV).

To overcome this limitation, Optical Coherence Tomography Angiography (OCTA) has emerged as a noninvasive, dye-free imaging technique that enables depth-resolved visualization of retinal and choroidal vasculature (Fig. 4). In brief, OCTA achieves this by detecting motion contrast from moving erythrocytes (red blood cells) across repeated OCT B-scans at the same location (Fig. 5). This allows the  construction of high-resolution vascular maps without the need for intravenous dyes.

Fig. 4. An example of an active CNV lesion in retina imaged with OCT (left) and OCTA (right). The superficial (ILM to    IPL) and deep retinal plexuses are depicted, along with the outer retina (OPL-BRM), which is normally avascular, and the choriocapillaris. ILM: internal limiting membrane; IPL: Inner Plexiform Layer; OPL: Inner Plexiform Layer; BRM:   Bruch’s membrane (See Fig. 1 for detailed anatomy of the retina).

Fig. 5. Generic workflow of OCTA image construction.

In this homework, you will explore the principles of OCTA, compare it with other imaging modalities, and apply your knowledge of OCT to understand its role in AMD diagnosis. You will also evaluate the advantages and limitations of each modality in visualizing CNV and understand why structural OCT alone is insufficient for vascular imaging.

Questions

1.  (12%) Apart from wide-field fundus photography, confocal scanning laser imaging approach has also been adopted in retinal imaging, termed as confocal scanning laser ophthalmoscopy (cSLO) (Fig.  6).  It  is capable of producing high-contrast retinal images by raster scanning a laser spot on fundus and detecting fluorescence emission(signal) through a confocal pinhole.

Fig. 6 General concept of confocal scanning laser ophthalmoscopy (cSLO). BS: beam-splitter; DM: Dichroic mirror; FOV: field-of-view. Caution: the   beam profile shown in the image is only for illustration, should NOT be treated as rigorous reference for calculation in the questions.

A  simplified  design  schematic  of  a  cSLO  system  is  shown  in  Fig.   7  -  designed  for Fluorescence angiography (FA) as well as Fundus autofluorescence (FAF) imaging. The generalized design includes separate optical pathways for illumination and collection through a common telescope (formed by 2 lenses L1 and L2). The two paths are separated by a beam-splitter. The telescope is configured in a way such that the subject’s pupil plane L3 and the scanning mirror plane M1 form the pair of conjugate planes. For the sake of simplicity, only one mirror is shown in Fig. 7. (In practice, two mirrors are needed for 2D scanning).

Fig. 7 Simplified system configuration of cSLO. Note that the drawing is not to scale.

The mirror scanner M1 has a maximum steering angular range of θmirror=10o  bounded by the blue and green lines, as shown in Fig. 7. Based on the system configuration shown in Fig. 7 and the ray-tracing technique, sketch how the two scanning beam paths (along the blue and green lines) are projected (focused) onto the retina.

2. (6%) Figure 8 shows the excitation and emission spectra of lipofuscin, fluorescein, and ICG. Hence, what are the emission colors of FAF images, fluorescein angiograms and ICG angiograms?

Fig. 8 Fluorescence excitation (dashed curves) and emission (solid curves) of lipofuscin, fluorescein, and ICG.

3. (6%) In order to perform. multimodal retinal imaging, i.e. FAF imaging, fluorescein angiography and ICG angiography are  performed in the same platform, multiple lasers are required to excite different fluorophores.  In view  of cost effectiveness, the  number of lasers used in the system should be kept at minimum. Based on this criterion and Fig. 8, choose the proper lasers (from Table 1) to be used in this multimodal retinal imaging system.

4. (10%) Draw a system schematic explaining how such a multimodal retinal cSLO system works (Hints: How many laser sources/photodetectors do we need? What are the specifications of the spectral filters used in the system so that it can perform. multicolour imaging of FAF, fluorescein angiography and ICG?)

5. (12%) It has been argued that ICG angiography is more appropriate than fluorescein angiography for imaging of the choroidal circulation below RPE. Based on what you have learnt from Tissue Optics, explain if you support this argument.   (Hint:   RPE consists of high density of  pigment melanin;  what  are excitation/emission wavelengths of ICG/fluorescein?).

6.  (8%) Let’s consider a spectral-domain OCT system, which is to be integrated with the cSLO system we studied in Questions 1-4. If it is required to achieve an axial resolution no worse than 7 μm, choose the best light source from the list shown in Table 2 for this OCT system. Explain your choice. (*Assuming all four sources have the Gaussian spectral shape)

7. (8%) If it is required to achieve real-time 3D imaging (512(x) × 512(y) × 1024(z) voxels) at a speed of 1 frame per second  (fps), i.e.  512 x 512 A-scans in  1 second, choose the best line-can camera  from the list shown in Table 3 and Fig. 9. Note that you should also make your choice based  on  the  source  you  choose  in  question  6.  Explain  your  choice. (**x  and y  are  along the transverse  directions whereas z is along the axial direction.)

Figure 9: (left) Typical configuration of a line-scan camera. (right) Spectral responses of four different line-scan cameras for OCT.

8. (10%) To visualize blood flow in retinal and choroidal vasculatures, OCTA is needed. According to Fig. 5, OCTA detects the changes in the OCT intensity signal between repeated B-scans (up to 4 B-scans) acquired at the same location (pixel). One simple approach of evaluating such changes is to compute the variance of the intensity of an image pixel over N repeated B-scans:

Where Ii is the intensity of the pixel in the ith B-scan. is the average intensity of the pixel over N repeated B-scans.

Let’s take the following simple example which displays the intensity values of the 6 pixels along an A-scan direction (Pixel 1 to 6) captured in 4 repeated consecutive B-scans. Calculate the variances of all 6 pixels over N=4 B-scans (*this array of 6 pixels is essentially the OCTA signal along the A-scan direction).

9. (8%) Explain why the approach of detecting the OCT intensity change (e.g., by calculating the variance of intensity in Question 8) can yield a map of blood flow in vasculatures? (hints: what do we expect, on the other hand, the variance value from the static tissue region?)

10. (10%) Choroidal neovascularization (CNV) is a hallmark of neovascular age-related macular degeneration (AMD) and is classified into three main subtypes based on the anatomical location. Understanding these subtypes is essential for selecting the appropriate imaging modality and guiding treatment decisions.

• Type 1 CNV arises from the choroid and grows beneath the retinal pigment epithelium (RPE), often presenting as pigment epithelial detachments (PEDs).

• Type 2 CNV extends above the RPE into the subretinal space and is usually associated with prominent leakage on FA, corresponding to "classic" CNV.

• Type 3 CNV, also known as retinal angiomatous proliferation (RAP), originates within the retina and can progress to form. retinal-choroidal anastomoses, often requiring multimodal imaging for accurate detection.

A 75-year-old patient presents with blurred central vision and metamorphopsia in one eye. Fundus examination reveals drusen and subtle pigmentary changes. An ophthalmologist suspects choroidal neovascularization (CNV) secondary to AMD. She has access to the following imaging modalities:

•     Fluorescein angiography (FA)

•     Indocyanine green angiography (ICGA)

•     Spectral-domain optical coherence tomography (OCT)

•     Optical coherence tomography angiography (OCTA)

Which of the following statements correctly match the CNV subtype with the most appropriate or helpful imaging modality for its detection and characterization? Select all that apply.

A. Type 1 CNV (occult, beneath the RPE) is best detected using indocyanine green angiography (ICGA) or OCTA due to its choroidal origin and lack of dye leakage on FA.

B. Type 2 CNV (classic, above the RPE) is readily identified using fluorescein angiography (FA), which shows early leakage and well-defined lesion boundaries.

C.  Type  3  CNV  (retinal  angiomatous  proliferation)  is  best  visualized   using   OCT  alone,  as  other angiographic modalities provide limited additional information.

D. OCTA is capable of detecting both Type 1 and Type 3 CNV by identifying flow patterns in the outer retina and choriocapillaris layers, even in the absence of leakage.

E. Fluorescein angiography is the most reliable modality for detecting Type 1 CNV due to its ability to show occult leakage from sub-RPE vessels.

(Hints: think about the tissue properties of RPE; Light-tissue interaction in these layers of retina, choroidal layers)

**A challenge to think (not counted as the Homework 2,s grade): How to integrate OCT, OCTA, FAF, fluorescein and ICG angiography in a unified system. (hint: You could think of a schematic  diagram design of the entire system to explain the basic operation principles)