AI‑generated visualization of retinal cones.
Adaptive optics is reshaping retinal imaging. Engineered by Marcel Bernucci & Yan Liu, this new, compact, integrated fixation–stimulus channel introduces precise eye control and spectrally pure light into one streamlined system. Powered by NKT Photonics’ broadband laser and fast spectral tuning, the system makes high‑resolution functional imaging accessible and reproducible.
Pioneering a Standard for AO Imaging
The team of researchers at Miller Lab, Indiana University School of Optometry, Bloomington, develops advanced adaptive optics (AO) retinal imaging systems to study the living human eye at the cellular level.
AO ophthalmoscopy gives users unprecedented access to photoreceptors, ganglion cells, and other microscopic retinal structures and their function. AO can be implemented using either Flood Illumination, Scanning Laser Ophthalmoscopy (SLO), or Optical Coherence Tomography (OCT).
Flood illumination is analogous to snapping a photo with a smartphone: a brief flash of light illuminates the retina, and the reflected light is captured as a single image by a 2D detector. In contrast, SLO sweeps a laser spot across the retina to generate a sharp, surface‑level image, while OCT uses light interference to map the retina in depth, producing cross‑sectional and 3D views.
Why Fixation and Stimulus Matter in AO
To examine specific cells reliably, users must precisely control both where the eye is looking and what light reaches the retina – called fixation and stimulus.
These challenges become especially obvious in AO ophthalmoscopy. The tiny field‑of‑view and high magnification make precise fixation control and stimulus delivery essential for generating reliable, reproducible measurements.
The Challenge: No Standard AO Solution
AO laboratories have tried to address these challenges with custom‑built fixation displays and limited‑bandwidth stimulus sources that vary widely from lab to lab. However, system performance is still insufficient for many applications, and the lack of standardization makes it difficult to reproduce experiments and compare results across sites.
The Solution: An Integrated Fixation and Stimulus Channel
To overcome this lack of capability and standardization, the research team designed a fully integrated fixation and stimulus channel (Figure 1) that is:
- compact
- low-cost
- high-performance
- based on stock components
- open-source
Short, Powerful Flashes for Functional Imaging by Optoretinography
L, M, and S cones are the retina’s three color‑sensitive photoreceptors: L‑cones detect long (red) wavelengths, M‑cones detect medium (green) wavelengths, and S‑cones detect short (blue) wavelengths. Together, they enable human color vision.
The optoretinogram (ORG) measures tiny light‑evoked changes of these retinal photoreceptors, enabling identification of individual S, M, and L cone cells. ORG cone classification is a non‑invasive way to “tell apart the different types of cone cells” based on how each one reacts to tiny, precisely controlled flashes of light.
This is achieved through the stimulus subsystem, powered by NKT Photonics’ broadband white‑light laser, tunable filter, and modulation hardware. Together, these elements deliver short, powerful, spectrally pure flashes of light across the visible spectrum with the precise timing needed for reliable ORG cone classification.
The research team is also using the versatility of the stimulus subsystem to deliver high‑intensity, near‑monochromatic light. This helps them measure how different cones respond and map their spectral sensitivity in the living human eye.
Making Functional AO Imaging Accessible
The system makes advanced fixation control and spectrally precise stimulation available to AO laboratories and uses stock components and a fully characterized design. This makes it easy to reproduce, lowers the barrier to functional AO imaging, and opens up more retinal regions for study.
Fixation Subsystem Enabling Broader AO Reach
A stable, flexible fixation subsystem is essential for accurate beam placement, even though much of the system design centers on the stimulus subsystem.
The fixation subsystem provides a wide steering range (39.0° × 36.6°), an extended working distance (up to 42 mm), a 25‑diopter Badal optometer for correcting refractive error, and low distortion (<0.4°) across the entire field.
While this subsystem supports AO‑OCT imaging even at far peripheral retinal locations, functional imaging requires both fixation control and simultaneous stimulus delivery. For that, the stimulus subsystem – and the performance of its light source and supporting hardware – is critical.
Spectrally Pure, High‑Power Visible Stimuli
For the stimulus subsystem (Figure 1), the researchers chose the SuperK FIANIUM FIU‑15 supercontinuum laser for its high power across the full visible range (3901)–850 nm) and its especially strong output at shorter wavelengths (400–500 nm).
Figure 1: Left: 3D CAD rendering of the fixation-stimulus channel. Right: Light stimulation to the eye is delivered by the NKT Photonics FIU-15 supercontinuum laser to the NKT Photonics SELECT AOTF, which is triggered by signals relayed from the OCT Control Computer via an Arduino UNO, a BNC 555 series Delay Generator, and a Fast Wavelength Switching Interface (NKT Photonics COMMAND).
They then used the NKT Photonics SELECT acousto‑optic tunable filter (AOTF) with the VIS 1× crystal – covering 430–670 nm, providing a 0.5–1.8 nm full-width at half-maximum (FWHM) spectral bandwidth, and offering >10 dB sidelobe suppression – to shape and control the stimulus light. This setup allowed precise adjustment of wavelength, power, and pulse duration (typically 5 ms, Figure 2A).
Fast wavelength switching through NKT Photonics’ COMMAND interface enabled modulation rates up to 2 kHz. To maintain high throughput and an even beam across the 2° stimulus patch, the filtered light was delivered by multimode fiber, achieving <5% nonuniformity at the retinal plane.
Stimulus power – up to 1.2 mW at the cornea (Figure 2C) – was controlled through NKT Photonics CONTROL software as the light passed through the AOTF. The stimulus wavelengths produced by the NKT Photonics SELECT were then further spectrally tuned to generate twelve near‑monochromatic stimuli by sending them through dual cascaded ultra‑narrow bandpass filters (Figure 1, Figure 2C, D).
This multistage filtering approach preserved the SELECT’s performance characteristics while suppressing spectral sidelobes by more than 60 dB, producing <2 nm FWHM near‑monochromatic flashes with minimal leakage from adjacent wavelengths. These highly pure stimuli improve the robustness of ORG‑based cone classification and spectral sensitivity measures by isolating cone responses to each specific test wavelength.
Figure 2: (A) Double-flash ORG imaging protocol uses two high-intensity 5-ms flashes (red) of 434.1 nm and 606.8 nm light spaced 1 s apart for cone classification. (B) Image of a 606.8 nm stimulus beam (<5% beam nonuniformity). (C) Twelve test wavelengths sampled across the visible spectrum and (D) overlaid on the human cone fundamentals. (E) ORG path length change (ΔOPL) responses following a double-flash ORG protocol from 255 cones are used to classify cones into S (blue), M (green), or L (red) spectral types. (F) Cone classification reveals the trichromatic cone mosaic.
Precise ORG Imaging at Extreme Angles
Paired with the fixation subsystem, the stimulus subsystem enabled successful double‑flash ORG at 19.5° temporal retina, a region where both gaze steering and signal quality are pushed to their limits.
Using NKT Photonics‑powered 434.1 nm and 606.8 nm flashes (Figure 2E), the team classified 255 cones and measured L:M:S ratios of 52%:38%:10% (Figure 2F), consistent with known distributions in the peripheral retina.
The strength of these ORG signals highlights how critical the stimulus subsystem is – particularly the brightness, spectral selectivity, and beam uniformity delivered by NKT Photonics’ FIU‑15 supercontinuum source, SELECT AOTF filtering optics, and COMMAND modulation hardware2).
Conclusion: A Reproducible Foundation for Functional AO
This integrated fixation and stimulus channel marks a significant step forward for functional AO ophthalmoscopy.
The fixation subsystem broadens the retinal regions accessible at high resolution, while the stimulus subsystem – powered by NKT Photonics’ high‑brightness, full‑visible‑spectrum supercontinuum laser – delivers the precision needed for reliable ORG measurements.
Because the design uses stock components and is fully characterized, it provides AO laboratories with a reproducible, ready‑to‑integrate foundation they can incorporate directly into their own systems to achieve robust fixation control and spectrally precise stimulation.
Get All the Technical Details in the White Paper
Read the published manuscript describing the integrated fixation-stimulus channel in detail.
1) Since this work was completed, NKT Photonics has updated the FIU-15 to cover 390 nm – 850 nm in the visible range.
2) Since this work was completed, NKT Photonics has released a new version of the SuperK SELECT tunable filter with fast wavelength switching functionality built in. This eliminates the need to interface with the external COMMAND unit to achieve fast switching. The built-in feature allows users to switch between wavelengths in under 50 microseconds.
