Next Gen Ophthalmic Imaging for Neurodegenerative Diseases and Oculomics

Petteri Teikari, PhD
https://www.linkedin.com/in/petteriteikari/
Version “Fri, April 5, 2024“
Next Gen
Ophthalmic
Imaging for
neurodegenerative
diseases and
oculomics:
#1/2: Imaging
Hardware

About the slides
What are these?
Shallow literature analysis on recent trends in (multimodal) ophthalmic imaging
with focus on neurodegenerative disease imaging / oculomics. Open-ended
literature review on what you could be building next.
Format?
Even though these are slides, these are not meant as presentation aids. More like a
“visual literature review” without being as factually detailed as a “real review”.
Who are these for?
For data scientists, engineers, clinicians, entrepreneurs, investors, accelerators, etc.
People who might not be so familiar with the retinal imaging technology, but are
involved in imaging projects. Retinal imaging scientists probably won’t learn too
much from this?

Executive
Summary
Next-generation retinal
imaging device for
neurodegenerative disease
screening and prognostics.
Let established companies do
their incremental product
updates

1st
Gen: Unimodal with vintage imaging tech
Fundus images classified with the state-of-the-art nets of the time.
Note! Generation divides now by the author
A deep convolutional neural network was trained using a retrospective development
data set of 128,175 retinal images, which were graded 3 to 7 times for diabetic
retinopathy, diabetic macular edema, and image gradability by a panel of 54 US
licensed ophthalmologists and ophthalmology senior residents between May
and December 2015.
A variety of cameras were used, including Centervue DRS, Optovue iCam, Canon
CR1/DGi/CR2, and Topcon NW using 45° fields of view.

2nd
Gen: Multiple with retrospective data
Include OCT in addition to CFP, with or without multimodal fusion

Next-Gen Multimodal Imaging Systems
MERLIN EU Project
Handheld AOSLO (confocal +
non-confocal split-detection
for phase contrast) & AOCT
Hagan et al. (2020) Duke
Moon 2020 project, in vivo retinal Raman
imaging + OCT- Sentosa et al. (2023)
Spectroscopic OCT - Drexler (2004)
‘Neurodegenerative disease imaging’ converging with cutting-edge next-gen ophthalmic imaging designs? As in you won’t be having
“separate gadgets” just for Alzheimer’s screening, but the advanced capabilities are part of the high-end ophthalmic imaging equipment?
Structure
AO-OCT
OCT-A
ORG
Structure
Off-axis SLO
FLIM
Polarimetry
Spectroscopic OCT
Molecular imaging
Molecular Imaging
in vivo Raman
Novel Biomarkers
from Phase Contrast?
’glial imaging’
‘inflammation imaging’
Portable OCT, Prof Chao Zhou
Portable &
Computational
innovations

Next-Gen Portable Fundus
Previous generation d-Eyes and Peek Retinas had suboptimal image quality,
could a combination of metalenses and computatinal technique enable low-cost fundus?
Huang et al. (Feb 2022): “Full-Color Metaoptical
Imaging in Visible Light”
Praneeth Chakravarthula (2023):
Thin On-Sensor Nanophotonic
Array Cameras
In this work, we investigate flat
nanophotonic computational
cameras as an alternative that
employs a metalens array of
skewed lenslets and a learned
reconstruction approach.
Ji et al. (2023): “mHealth hyperspectral
learning for instantaneous spatiospectral
imaging of hemodynamics”
Li (2023, PhD thesis): “Robust deep learning
for computational imaging through random
optics”
Diffuser-based computational lensless imaging
system.

Note on foundation models
How well in practice the existing foundation models trained on large-scale retrospective data
perform for unseen future technology coming in small-scale from universities, spin-offs and startups?
SLO images can be classified by a model
trained on old-school CFP images
Sarah Matta et al. (2023): Impact of training data diversity
on the generality of automated diabetic retinopathy
screening in fundus photographs
Despite the visual difference existing between conventional and
confocal-based CFPs, the algorithms trained on conventional CFPs
showed good performance for detecting moderate DR or worse in
confocal-based DRSplus CFPs.
Abraham Olvera-Barrios et al. (2023): Diagnostic accuracy
of diabetic retinopathy grading by an artificial intelligence-
enabled algorithm compared with a human standard for
wide-field true-colour confocal scanning and standard
digital retinal images
EyeArt identified diabetic retinopathy in EIDON images with similar
sensitivity to standard images in a large-scale screening programme,
How about hyperspectral / molecular data
further away from CFP/Fundus?
Open question

Refresher: The Eye Anatomy #1
Modified from Heidelberg’s OCT image,
https://www.linkedin.com/feed/update/urn:li:
activity:6788811651245215745/
See e.g. “Quantification of Retinal Ganglion
Cell Morphology in Human Glaucomatous
Eyes” by Zhuolin Liu et al. (2021) for glaucoma
analysis

Posterior vs Anterior Segment
“Optics”
“Sensory
Processing”
anterior segment examinations
posterior segment examinations

Refresher: The Eye Anatomy #2 Anterior Segment
1 – True dense 3D volume. Image 2 – Anterior segment B-scan (horizontal solid yellow line in Image 4 shows the volume
reference). Image 3 – Z-axis or enface axis B-scan (horizontal yellow line in Image 5 shows the reference), shows an iris
cyst at 9 o’clock. Image 6 shows a vertical B-scan along the vertical dotted line in Image 4.
https://www.cyliteoptics.com/resource/volume-capture-how-it-works/
‘Everything layered’
including the
crystalline lens
- Gupta et al. (2023)

Refresher: The Eye Anatomy #3 Retina (Posterior Segment)
Ferrara et al. (2021)
https://www.heidelbergengineering.com/int/news/know-your-retinal-layers-33401465/

Posterior+Anterior imaged together #1
M. Kendrisic et al. (2023): “Low-cost long-range SS-OCT
for imaging the human eye in-vivo from anterior to
posterior segment”
The trend towards homecare and point-of-
care devices in medical care triggers the
need for more compact and economic
medical diagnostic technology. With this
goal, we developed a flexible low-cost long-
range SS-OCT capable of imaging the
human eye from anterior to posterior
segment, based on a single-mode tunable
VCSEL at 850nm. The system runs at A-scan
rates of 2-25kHz with a measured maximum
sensitivity of 97dB. In this work, we present in
vivo results of full eye imaging with an
imaging range of 5cm including both
anterior and posterior segment B-scans.
Ireneusz Grulkowski et al. (2012): “Retinal, anterior segment and full eye imaging using ultrahigh
speed swept source OCT with vertical-cavity surface emitting lasers” Cited by 389
Becoming a thing instead of having two separate devices?

Intalight
DREAM OCT™

https://www.cyliteoptics.com/

Retinal Imaging Segments
Off-the-shelf technology for clinical use.
’Experimental/emerging’ imaging from research labs reviewed later

Opthalmoscope no imaging, for GPs
Arclight A pocket all-in-one ophthalmoscope-loupe-
otoscope for the 21st century – an innovative and easy
to use tool for examining the front and back of the eye
and the ear canal.
Ideal for doctors (including GPs and Pediatricians),
Nurses, Midwives and trainees as well as specialists
(ophthalmologists, optometrists).

Smartphone/Flat optics low-cost imaging
D-EYE, The Modern
Day Digital Ophthalmoscope
Form factor would be nice for GPs, low-income countries, etc., but the image quality in practice have been
rather unsatisfactory. Waiting for the metaphotonic/computational revolution (Metalenz)?
Peek Retina https://peekvision.org/

Desktop and ‘high-end portable’ fundus
Optomed Aurora IQ
https://www.optomed.com/optomed-aurora-iq/
Relatively pricey still while not OCT expensive. Losing the clinical utility battle to OCT
Topcon NW500

(Confocal) Scanning Laser Ophthalmoscope ((c)SLO)
Scanning the image with a single-pixel detector. Confocal gives rejection of out-of-focus
signal leading to better image quality
https://www.optos.com/ OPTOS Ultra-Wide Field (UWF)
200deg pseudocolor SLO The SLO simultaneously scans the
retina using two low-power lasers (red – 633 nm and green – 532
nm) that enable high-resolution, color imaging of retinal
substructures.
Revenio iCare Eidon SLO
White LED used instead of a
laser
3-laser multicolor SLO
Heidelberg Spectralis

Optical Coherence Tomography (OCT)
Volumetric 3D images captures allowing layer-level analysis, starting to become the standard
in ophthalmology. Multimodal OCT+fundus cameras exist facilitating the transition from
fundus imaging to OCT
OCT + SLO on Heidelberg

2D vs 3D Imaging
Fundus vs OCT

CFP Standard Coaxial Design
Optical Designs for Fundus Cameras
https://www.slideshare.net/PetteriTeikariPhD/optical-designs-for-fundus-cameras
Color Fundus Photography

CFP Robotic Extensions
Reducing the effect of the operator skill on the fundus image quality
Nexy

CFP Illumination does not have to be done through pupil
Trans-pars-planar illumination
https://doi.org/10.1038/s41598-018-27112-x
https://doi.org/10.1364/TRANSLATIONAL.2018.CF3B.8
http://dx.doi.org/10.1364/BOE.9.003867
Through-the-noise-illumination
Vielight Neuro (Toronto, ON) - NIR light energy
penetration through a human cadaver.
https://www.vielight.com/de/brain-photobiomodulation/
Transscleral optical phase imaging
of the human retina
Timothé Laforest et al.
Nature Photonics (Lausanne, 2020)
https://doi.org/10.1038/s41566-020-0608-y

2D vs 3D Imaging
cSLO (“upgraded fundus”)
vs OCT

Multicolor SLO vs Conventional Fundus (CFP)
Terasaki et al. (2021) Commercial clinical confocal SLOs SPECTRALIS MultiColor SLO by Heidelberg Engineering., Mirante (Nidek,
Gamagori, Japan) and CLARUS (Carl Zeiss Meditec Inc., Dublin, CA, USA; and the polychromatic white LED-based Revenio iCare line,
and non-confocal SLO from Optos.
Heidelberg Spectralis, with the
Flex module (mount), allowing
imaging of bedbound patients
in supine positions

Revenio iCare Eidon (ex Centervue)
iCare EIDON widefield
TrueColor Confocal fundus
imaging system
- Widefield, ultra-high-resolution
imaging
- Capability to image through
cataract and media opacities
- Dilation-free operation
(minimum pupil 2.5 mm)
- Flexibility of fully automated and
fully manual mode
460nm
pumped
white LED
Commercial SLO-like imaging with white LED (with suboptimal CRI?) instead of laser(s)
Less of orange “choroid cast” saturating the image
compared to conventional fundus

Retinal Imaging of Protein Biomarkers of Neurodegenerative
Diseases in the Brain
Melanie CW Campbell at I2Eye 2023, Paris
LumeNeuro spinout from University of Waterloo
SLO designs for more ‘advanced retinal imaging’
e.g. for polarimetric imaging (Mueller imaging)
Gramatikov (2014): “Modern technologies for retinal scanning and
imaging: an introduction for the biomedical engineer”

Image Scanning Microscopy
e.g. for fluorescence lifetime imaging (FLIM)
Paul Bernstein et al. (2019): “Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO)”
Healthy eyes with different characteristic macular pigment (MP)
distribution patterns.

Fundus cameras
becoming like fax
machines? Obsolete?
What next?
OCT taking over (but
things take time)

Probably no special issues on “New Frontiers in Fundus Imaging”?
https://tvst.arvojournals.org/ss/forauthors.aspx#oct
Excluding maybe the “metaphotonics fundus papers” enabling current robotic desktop quality at a
significantly lower price? Think of Peek Retina with better image quality, and thus better clinical utility?

Ophthalmic Imaging Market
Esquenazi et al. (2022)
OCT News
fluorescein angiography
Fundus
OCT
https://www.specsavers.co.uk/eye-test/oct-scan
2023 Optical Coherence Tomography
Market Report

What next? ORG, FLIO and better OCT
The future of retinal imaging (2020)
https://doi.org/10.1097/ICU.0000000000000653
FLIO, OCT, AO SLO-FA
The Development and Clinical Application of Innovative Optical
Ophthalmic Imaging Techniques (2022)
https://doi.org/10.3389/fmed.2022.891369
Phase-sensitive OCT, ORG, OCT/OCT-A, AO-OCT, SLO, AO-SLO

OCT
Ian Rubinoff et al. (2023): ”Optical coherence
tomography (OCT) enabled noninvasive retinal
imaging at a spatial resolution of a few
micrometers and has been considered as the
“gold standard” for examining structural
damages or therapeutic efficacy in nearly all
vision-threatening diseases.”
Leitgeb et al. (2023) “30 Years of Optical
Coherence Tomography: introduction to the
feature issue” Issue 10
Huang et al. (1991): ”Optical Coherence
Tomography” Cited by 18260

OCT Variants
Time-domain (TD-OCT) -> Spectral-domain (SD-OCT) -> Swept-source (SS-OCT)
OCT B scan of the same eye imaged using SD-OCT, and SS-OCT. Note the increased OCT signal
penetration (with SS-OCT) and resulting improvement in visualization of the choroid and choroido-
scleral junction (yellow arrows) - Alibhai et al. (2018)
Fourier-domain (FD-OCT)

OCT innovation for increased depth
The team from the University of Adelaide, Australia,
Technical University of Denmark (DTU), the University of
St Andrews, Scotland and Aerospace Corp., USA, has
discovered an alternative viewpoint – that selective
collection of multiple scattered light can lead to
improved image contrast at depth, particularly in
highly scattering samples. The team believe their
breakthrough is poised to defy convention and lead to
a step change in recovering images at depth. The
team are further bolstered by having both granted
and filed intellectual property (IP) in this area and are
keen to see translation. In 2021 the OCT market was
valued at US$1.3 billion and is set to triple by the end
of the decade.
Untracht et al. (2023): “Spatially offset optical coherence
tomography: Leveraging multiple scattering for high-contrast
imaging at depth in turbid media” Cited by 1
The pursuit of imaging at depth has been
largely approached by extinguishing
multiple scattering. However, in OCT,
multiple scattering substantially contributes
to image formation at depth. Here, we
investigate the role of multiple scattering in
OCT image contrast and postulate that, in
OCT, multiple scattering can enhance
image contrast at depth. We introduce an
original geometry that completely
decouples the incident and collection fields
by introducing a spatial offset between
them, leading to preferential collection of
multiply scattered light. A wave optics–
based theoretical framework supports our
experimentally demonstrated improvement
in contrast. The effective signal attenuation
can be reduced by more than 24 decibels.
Notably, a ninefold enhancement in image
contrast at depth is observed in scattering
biological samples. This geometry enables a
powerful capacity to dynamically tune for
contrast at depth.

Fully Robotic OCT from Duke
Development started already before COVID see ARVO 2022
Get rid of operator effect on image quality
https://pratt.duke.edu/about/news/robotic-scanner-automates-diagnostic-imaging-eye
https://people.duke.edu/~mtd13/research/robotic-oct/
Allows for example
1) automated image montaging (higher FOV) with the
pupil tracking
2) Henle’s fiber layer imaging with the controllable pupil
entry position. (Amit Narawane et al. 2022)

Fundus vs OCT Angiography
Retinal vasculature has a layered structure (plexus), and again you would to have plexus-specific
analysis of vascular structure, blood flow, etc.
Kur et al. (2012)
Physiological Anatomy of the Retinal Vasculature S.S. Hayreh, in
Encyclopedia of the Eye, 2010
This 2D
projection is
what you see
in fundus
photographs
and in fundus
angiography

Fundus vs OCT Angiography (OCTA)
Retinal vasculature has a layered structure (plexus), and again you would to have plexus-specific
analysis of vascular structure, blood flow, etc.
This is what 3D volumetric OCT
Angiography more or less looks like
Taylor et al. (2024):
The role of the retinal
vasculature in age-
related macular
degeneration: a spotlight
on OCTA
Retinal imaging of a
patient with intermediate
age-related macular
degeneration: red-free
(top left), structural OCT
(top right), OCT
angiography of superficial
vascular complex (bottom
left), OCT angiography of
deep vascular complex
(bottom right).

OCT-A with SS-OCT -> Volumetric Retinal Vasculature #1
Hormel et al. (2021): “Plexus-specific retinal vascular anatomy and pathologies as
seen by projection-resolved optical coherence tomographic angiography”
Campbell et al. (2017): “Detailed Vascular Anatomy of the Human Retina by
Projection-Resolved Optical Coherence Tomography Angiography”
Chen (ARVO 2022, EMA): Plexus-specific retinal erythrocyte velocity:
“...We have shown that blood flow in the SVP, ICP, and DCP can be precisely
quantified and can differ between plexuses.”

OCT-A with SS-OCT -> Volumetric Retinal Vasculature #2
Kiyoko Gocho (2022): Multimodal and multiscale clinical high-resolution retinal imaging

OCT getting wider too (UWF OCT) #1
As in Optos UWF happened to fundus/SLO

75deg OCT (23mm x 23 mm, in 6 sec) ->
105deg UWOCT (Jia, Casey Eye Inst)
“Widefield optical coherence tomography
angiography imaging with distortion corre
ction” 220deg UWOCT Intalight

J. Peter Campbell
LinkedIn Post (7 March 2024)
Associate Professor, Casey Eye Institute, Oregon Health
& Science University
Here is an example of what is going
to be possible when we have ultra-
widefield OCT available for neonatal
retinal imaging. Note the variations
in extra-retinal neovascularization
that are visible in 3D.
Thankful for super smart colleagues
Yifan Jian and Ben Young, and
National Eye Institute (NEI) and RPB
for their support. (Scan taken in
awake baby, using contact-lens
approach, 140 degree visual angle,
<1.5 seconds).
Hanif et al. (2024): “Implementation of optical coherence tomography in
retinopathy of prematurity screening”

Full-Field OCT (FFOCT) #1
Full field OCT is the parallel version of OCT, instead of acquiring axial lines in the
sample and then scan spatially to reconstruct 2D images (BScan) or 3D
volumes (CScan), FFOCT uses a camera to acquire directly a 2D plane,
perpendicular to the illumination. FFOCT works quite the same way as a
conventional full field microscope, hence its name. One of the advantage of
FFOCT compared to traditional OCT is its simplicity, due to the absence of
scanning. Also, acquiring a full frame in one shot is often better, especially for
moving samples such as the eye - Jules Scholler (2019).
Hari Nandakumar and Shailesh Srivastava (2019): “Low Cost Open-Source OCT Using
Undergraduate Lab Components” Time-domain full-field (TD-FF-) OCT device has been
assembled with Arduino control, which yields sub-4- m axial and lateral resolutions.
μ
Kate Grieve et al.

Full-Field OCT (FFOCT) #2
Paul Balondrade et al. (2022): Multi-spectral matrix microscope

Dynamic Full-Field OCT (FFOCT)
Monfort et al. (2023): “Dynamic full-field optical coherence
tomography module adapted to commercial microscopes allows
longitudinal in vitro cell culture study”
Dynamic full-field optical coherence tomography (D-FFOCT) has
recently emerged as a label-free imaging tool, capable of resolving
cell types and organelles within 3D live samples, whilst monitoring their
activity at tens of milliseconds resolution. Here, a D-FFOCT module
design is presented which can be coupled to a commercial
microscope with a stage top incubator, allowing non-invasive label-
free longitudinal imaging over periods of minutes to weeks on the
same sample. Long term volumetric imaging on human induced
pluripotent stem cell-derived retinal organoids is demonstrated,
highlighting tissue and cell organization processes such as rosette
formation and mitosis as well as cell shape and motility. Imaging on
retinal explants highlights single 3D cone and rod structures. An
optimal workflow for data acquisition, postprocessing and saving is
demonstrated, resulting in a time gain factor of 10 compared to prior
state of the art. Finally, a method to increase D-FFOCT signal-to-noise
ratio is demonstrated, allowing rapid organoid screening.
D-FFOCT in the photoreceptor layer of a porcine retinal
explant, imaged under culture conditions. The cone and rod
photoreceptor mosaic were revealed in en face (a, b) and axial (c)
slices. The depth positions of a and b are indicated by the black
arrows next to (c). White arrows highlight three cones which can be
visualized in all three views. Scale bar, 10 µm.

‘Smartphone Visible LF-OCT’ (SmartOCT)
Joseph D. Malone et al. (2023): “SmartOCT: smartphone-integrated
optical coherence tomography”
Here, we demonstrate smartOCT, a smartphone-integrated OCT system that
leverages built-in components of a smartphone for detection, processing and
display of OCT data. SmartOCT uses a broadband visible-light source and
line-field OCT design that enables snapshot 2D cross-sectional imaging.
The smartOCT system provides several advantages compared to
traditional OCT systems. Mainly, the use of a smartphone integrates several
components (camera, PC, display) that are normally separate entities into a
single compact device. As such, the cost is lower (<$6,000) than other
comparable visible-light OCT systems, including the phone (market value
<$300) and excluding the light source.
The current design is a proof-of-concept benchtop system that we believe
can be improved to provide a portable all-in-one smartOCT system. For
example, a major limitation of this work is the use of a supercontinuum laser
source, which is a common source for visible-light OCT and was helpful to
ensure sufficient power for imaging. Recently, there has been progress on
using broadband LED sources for visible-light OCT Wang and Liu 2021
. With
additional improvements to the technology in this space, LED light sources
may be viable for future smartOCT designs.

Systems that combine simultaneous color imaging with OCT
OPTOS Monaco - Silverstone
Topcon Maestro 2
Heidelberg Spectralis

Visible-light OCT #1
Opticent spin-off
Aurora X2
(510-610 nm)
Xiao Shu et al. (2017):
“Visible-light optical
coherence tomography: a
review” Cited by 200
“The development of vis-OCT is
primarily motivated by two
considerations:
(1) with comparable bandwidth,
shorter illumination wavelengths
improve imaging resolution
(2) vis-OCT can retrieve unique
tissue scattering and absorption
contrasts within the visible
spectral range.” The second generation dual-channel VIS-OCT alleviates the
trade-off between micron-level axial resolution and
imaging depth, improving the practical use in clinical
setting. The linear-in-K spectrometer, reference pathlength
modulation and active noise cancellation dramatically
improved the viability of VIS-OCT in clinical settings.
Yi et al. (2023): “Second generation dual-channel visible light
optical coherence tomography (VIS-OCT) for retinal imaging in
clinics”

Visible-light OCT #2
Naoto Ujiie et al. (2023) (Tsutomu Kume lab): “Differential roles of FOXC2 in the
trabecular meshwork and Schlemm’s canal in glaucomatous pathology”
Impaired development and maintenance of Schlemm’s canal (SC) are associated
with perturbed aqueous humor outflow and intraocular pressure (IOP). The
angiopoietin (ANGPT)/TIE2 signaling pathway regulates SC development and
maintenance, whereas the molecular mechanisms of crosstalk between SC and the
neural crest (NC)-derived neighboring tissue, the trabecular meshwork (TM), are
poorly understood. Here, we show NC-specific forkhead box (Fox)c2 deletion in mice
results in impaired SC morphogenesis, loss of SC identity, and elevated intraocular
pressure. Visible-light optical coherence tomography analysis further
demonstrated functional impairment of the SC in response to changes in
intraocular pressure in NC-Foxc2-/- mice, suggesting altered TM biomechanics.
Single-cell RNA-sequencing analysis identified that this phenotype is predominately
characterized by transcriptional changes associated with extracellular matrix
organization and stiffness in TM cell clusters, including increased matrix
metalloproteinase expression, which can cleave the TIE2 ectodomain to produce
soluble TIE2. Moreover, endothelial-specific Foxc2 deletion impaired SC
morphogenesis because of reduced TIE2 expression, which was rescued by deleting
the TIE2 phosphatase VE-PTP. Thus, Foxc2 is critical in maintaining SC identity and
morphogenesis via TM–SC crosstalk.

Hyeong Soo Nam and Hongki Yoo (2017): “Spectroscopic optical
coherence tomography: A review of concepts and biomedical
applications”
Optical coherence tomography (OCT) is a 3-dimensional high-
resolution imaging modality based on an interferometry and is widely
used in a large variety of medical fields. Spectroscopic OCT (S-OCT) is
a signal-processing method (15, 16) that uses the raw interferograms
generated by OCT to investigate depth-resolved spectroscopic
profiles of a sample. The spectroscopic information provided by S-OCT
can be used to enhance the contrast of OCT images and overcome the
limitations of gray-scale OCT images that describe only morphology.
In this review, we present the concepts behind S-OCT as well as
acquisition methods and description of obtainable spectroscopic
properties. Furthermore, this review covers the biomedical applications
of the spectroscopic information that can be obtained with S-OCT,
including measurements of hemoglobin concentrations, blood oxygen
saturation levels, atherosclerotic plaque detection, evaluation of burn
injuries, contrast enhancement using exogenous contrast agents, and
detection of precancerous lesions.
Spectroscopic OCTa
“Depth-resolved hyperspectral imaging”

Hyeong Soo Nam and Hongki Yoo (2017): “Spectroscopic
optical coherence tomography: A review of concepts and
biomedical applications”
Broadly speaking, S-OCT can be performed in one of two
ways: hardware-based and software-based.
Hardware-based S-OCT employs two or more light
sources with different bands of wavelength and collects
the light separately. Thus, hardware-based S-OCT
enables spectroscopic analysis based on multiple light
sources comprising distinct wavelength bands.
In this review, we only discuss software-based S-OCT,
which does not require hardware modification of the
typical OCT system. Therefore, software-based S-OCT
can provide not only a depth-resolved structure of a
sample, but also spectroscopic information for a
specific depth within the band of the light source, both
of which can be obtained simultaneously using a post-
processing technique and any FD-OCT system. In
other words, with S-OCT, backscattering spectra
containing spectroscopic properties of the sample, such
as wavelength-dependent absorption and scattering,
are provided for every pixel in an OCT image of a
sample without any additional hardware
requirements
Spectroscopic OCTb
Spectroscopic analysis for software-based S-OCT can be accomplished by
time–frequency transformation (TFT) of OCT interferograms. The method
used for TFT and the determination of window type and size have a
significant effect on S-OCT performance. Thus, careful consideration of the
method and the parameters is necessary and depends on the intended
application

Hyeong Soo Nam and Hongki Yoo (2017): “Spectroscopic
optical coherence tomography: A review of concepts and
biomedical applications”
Depth-resolved spectra are generally
fitted to the above exponential equation
(Eq. (4)) along the depth direction, from
which the total attenuation coefficient
can be calculated. However, the total
attenuation coefficient is a summation of
absorption and scattering coefficients,
which are independent of each other.
Separation of absorption and scattering
contribution can thus be useful for
characterizing the spectroscopic features
of a sample tissue. Indeed, several studies
have described extracting absorption and
scattering coefficients from spectra by S-
OCT Hermann et al. (2004);Bosschaart et al. (2009);Xu et al. (2004);
Robles and Wax (2010)
Spectroscopic OCTc
Metric analysis demonstrated with phantoms containing
different sizes of microspheres. The first two rows
represent the gray-scale OCT images of phantoms. The
following two rows represent the colorcoded OCT images
of the phantom by the metrics SOM-RGB, PCA-RGB, SUB-
RGB, and COM, respectively. The last two rows represent
the color-coded OCT images by the classification results
according to K-means clustering based on the PCA
metric. Clear characterization of the areas with different
microspheres is visible in the overlaid images

Spectroscopic OCT (Hyperspectral OCT): NIR #1
This method allows the spectrum of backscattered light to
be measured over the entire available optical bandwidth
(650-1,000 nm) simultaneously in a single measurement.
Specific spectral features can be extracted by use of digital
signal processing without changing the measurement
apparatus. - Morgner et al. (2000)
Image above from Drexler (2004)
It is important to note that OCT image contrast results
from a combination of absorption and scattering.
Incident light is attenuated by scattering and absorption
as it propagates through the tissue, then is
backscattered from the internal structure that is being
imaged and is again attenuated as it propagates out of
the tissue. Thus the optical properties absorption and
scattering of deep structures are convolved with the
properties of the intervening structures, making it
challenging to determine the exact optical properties of
a given internal structure. However, OCT provides more
information than other spectroscopic imaging
techniques that integrate continuous wave
backscattered light from multiple depths within tissue.
http://dx.doi.org/10.1007/978-3-540-77550-8_8
First proof-of-concept published by Kulkarni and Izatt (1996) Cited by 41

Oldenburg et al. (2007): “Spectroscopic Optical
Coherence Tomography and Microscopy”
Imaging biological tissues using optical
coherence tomography (OCT) is enhanced with
spectroscopic analysis, providing new metrics
for functional imaging. Recent advances in
spectroscopic optical coherence tomography
(SOCT) include techniques for the discrimination
of endogenous tissue types and for the
detection of exogenous contrast agents. In this
paper, we review these techniques and their
associated signal processing algorithms, while
highlighting their potential for biomedical
applications. We unify the theoretical framework
for time- and frequency-domain SOCT and
introduce a noise correction method. Differences
between spectroscopic Mie scatterers are
demonstrated with SOCT, and spectroscopic
imaging of macrophage and fibroblast cells in a
3-D scaffold is shown.
Comparison between
transmission spectra of optical
filters measured with a
spectrometer and SOCT, using
a method which subtracts the
lowest order shot noise
contribution (corrected) and a
simple normalization method
(uncorrected). (a) 830-nm 10-
nm bandpass filter. (b) 780-nm
low-pass filter. (c) 800-nm low-
pass filter. (d) 825-nm low-pass
filter. (e) 840-nm low-pass filter.
(f) Reference beam spectrum.
The bandpass interference filter
was tilted for SOCT to avoid
strong backreflection, which
blue-shifted its frequency
response.

Volker Jaedicke et al. (2014): “Performance comparison of different
metrics for spectroscopic optical coherence tomography”
When light interacts with a scattering medium, the spectrum of the incident light
undergoes changes that are dependent on the size of the scatterers in the
medium (Xu et al. 2004; Robles and Wax 2010)
. Spectroscopic Optical Coherence Tomography (S-
OCT) is a method that can be used to ascertain the resulting spatially-dependent
spectral information. In fact, S-OCT is sensitive to structures that are below the
spatial resolution of the system, making S-OCT a promising tool for diagnosing
many diseases and biological processes that change tissue structure, like cancer.
The most important signal processing steps for S-OCT are the depth-resolved
spectral analysis and the calculation of a spectroscopic metric. While the former
calculates the spectra from the raw OCT data, the latter analyzes the information
content of the processed depth-resolved spectra.
We combine the Dual Window spectral analysis with different spectroscopic
metrics, which are used as an input to colorize intensity based images. These
metrics include the spectral center of mass method, principal component (PCA) and
phasor analysis. To compare the performance of the metrics in a quantitative
manner, we use a cluster algorithm to calculate efficiencies for all methods. For this
purpose we use phantom samples which contain areas of microspheres of different
sizes. Our results demonstrate that PCA and phasor analysis have the highest
efficiencies, and can clearly separate these areas. Finally we will present data
from cartilage tissue under static load in vitro. These preliminary results show that S-
OCT can generate additional contrast in biological tissue in comparison to the
pure intensity based images.

Visible light range OCT https:/
/www.photonics.com/Articles/VIS-OCT_Opens_Eyes_to_New_Approaches/a65021
Yet, current clinical ophthalmic imaging diagnostics such
as Optical Coherence Tomography often use near-
infrared (NIR) light. While near-infrared light can delineate
layers in the retina based on NIR light scattering, visible
light offers new diagnostic approaches, such as
measuring concentrations of important retinal
chromophores or assessing light scattering from smaller
structures, such as microtubules, which may be sensitive
to early axonal changes in glaucoma. Preliminary human
images are shown on the right. The innovation over
current clinical OCT systems is improved axial resolution
and new avenues to assess retinal function, as described
here.
Spectroscopic OCT (Hyperspectral OCT): Visible #1

Robles et al. (2011): “Molecular imaging true-
colour spectroscopic optical coherence
tomography” Cited by 239
Molecular imaging holds a pivotal role in medicine due to its
ability to provide invaluable insight into disease mechanisms
at molecular and cellular levels. To this end, various
techniques have been developed for molecular imaging,
each with its own advantages and disadvantages. For
example, fluorescence imaging achieves micrometre-scale
resolution, but has low penetration depths and is mostly
limited to exogenous agents. Here, we demonstrate
molecular imaging of endogenous and exogenous
chromophores using a novel form of spectroscopic optical
coherence tomography (OCT). Our approach consists of
using a wide spectral bandwidth laser source centred in the
visible spectrum (centre wavelength of 575 nm and a
bandwidth of 240 nm), thereby allowing facile assessment of
haemoglobin oxygen levels, providing contrast from
readily available absorbers, and enabling true-colour
representation of samples. This approach provides high
spectral fidelity while imaging at the micrometre scale in
three dimensions. Molecular imaging true-colour
spectroscopic optical coherence tomography (METRiCS
OCT) has significant implications for many biomedical
applications including ophthalmology, early cancer
detection, and understanding fundamental disease
mechanisms such as hypoxia and angiogenesis.

Song et al. (2020): “Visible light optical
coherence tomography angiography (vis-
OCTA) facilitates local microvascular
oximetry in the human retina”
We report herein the first visible light optical coherence
tomography angiography (vis-OCTA) for human retinal
imaging. Compared to the existing vis-OCT systems, we
devised a spectrometer with a narrower bandwidth to
increase the spectral power density for OCTA imaging,
while retaining the major spectral contrast in the blood.
We achieved a 100 kHz A-line rate, the fastest acquisition
speed reported so far for human retinal vis-OCT. We
rigorously optimized the imaging protocol such that a
single acquisition took <6 seconds with a field of view (FOV)
of 3×7.8 mm2
. The angiography enables accurate
localization of microvasculature down to the capillary level
and thus enables oximetry at vessels <100 µm in
diameter.
“One advantage of vis-OCT is its spatio-spectral analysis within
the microvasculature for label-free oximetry (i.e. measuring
hemoglobin oxygen saturation, sO2). Compared to 2D
hyperspectral fundus imaging modalities [Palsson et al. 2012],
vis-OCT’s precise 3D localization of blood vessels excludes a
myriad of confounding factors from other tissue depths,
enabling accurate and reliable oximetry measurements.

Rasmus Eilkær Hansen (2023, PhD Thesis): “Mid-Infrared
Supercontinuum based Spectroscopic OCT” Technical University of Denmark
This thesis provides a thorough review of mid-infrared (mid-IR)
supercontinuum (SC) laser sources (NKT Photonics) and their application
within optical coherence tomography (OCT). Based on cascaded mid-IR SC
sources, OCT is performed with a centre wavelength of 4 µm. A centre
wavelength in the mid-IR typically allows increased penetration in samples
compared to the more conventional near-IR systems.
The 4 µm centre wavelength allowed penetrating through paper with a
thickness of 90 µm, such that the thickness of the sample could be
measured simultaneously with the refractive index. It was further shown
that the OCT system can show the roughness of the paper surface, and
that it detect defects in the cases of tears, voids and contamination by a
droplet of oil.
By spectrally subdividing the OCT images in the post processing it is
possible to obtain spectral information such as spectrally dependent
scattering or absorption of the sample. This data analysis technique was
applied to show a proof-of-concept of spatially and temporally resolved
imaging of CO2 gas in channels inside a 3D printed epoxy resin cube.
Spectroscopic OCT (Hyperspectral OCT): Mid-IR

Hope et al. (2021): “Inverse spectroscopic optical coherence
tomography (IS-OCT) for characterization of particle size and
concentration”
Inverse spectroscopic optical coherence tomography (IS-OCT) methods
apply inverse problem formulations to acquired spectra to estimate
depth-resolved sample properties. In the current study, we modelled the
time-frequency-distributions using Lambert-Beer’s law and implemented
IS-OCT using backscattering spectra calculated from Mie theory, then
demonstrated the algorithm on polystyrene microspheres under idealized
conditions. The results are significant because the method generates depth
dependent estimates of both the concentration and diameter of
scattering particles.
Inverse Spectroscopic OCT #1
Estimate retinal scatterers?
Rayleigh fitting parameter for
Aβ-aggregates
More et al. (2019): “In Vivo
Assessment of Retinal Biomarkers by
Hyperspectral Imaging: Early
Detection of Alzheimer’s Disease”
Backscattering spectra
for polystyrene
microspheres diameters
0.1 to 10 µm analyzed
across a wavelength
range of 0.4 to 1.4 µm

Leopold Veselka et al. (2023, Vienna): “Quantitative
Parameter Reconstruction from Optical Coherence
Tomographic Data”
Quantitative tissue information, like the light scattering
properties, is considered as a key player in the detection
of cancerous cells in medical diagnosis. A promising
method to obtain these data is optical coherence
tomography (OCT). In this article, we will therefore discuss
the refractive index reconstruction from OCT data,
employing a Gaussian beam based forward model. We
consider in particular samples with a layered structure,
meaning that the refractive index as a function of depth is
well approximated by a piece-wise constant function. For
the reconstruction, we present a layer-by-layer method
where in every step the refractive index is obtained via a
discretized least squares minimization. For an
approximated form of the minimization problem, we
present an existence and uniqueness result. The
applicability of the proposed method is then verified by
reconstructing refractive indices of layered media
from both simulated and experimental OCT data.
Inverse Spectroscopic OCT #2

Optical Coherence Elastography (OCE) Retinal Biomechanics #1
Morgan J. Ringel et al. (2021): “Advances in multimodal
imaging in ophthalmology”
OCE uses OCT imaging to detect micron-scale displacements caused by an external
mechanical stimulus to extract biomechanical properties of tissue (Kennedy et al. 2014
). Phase-decorrelation OCT (PhD-OCT) is an alternate method for measuring tissue
biomechanics that uses the decorrelation of scattered light from Brownian motion as a
surrogate measure of tissue viscosity (Blackburn et al. 2019). Initial OCE demonstrations
used OCT speckle tracking of axial displacements from a static loading force to quantify
tissue strain and derived Young’s modulus from the linear stress–strain relationship (
Schmitt 1998). OCE can also be used to measure Young’s and shear moduli by
combining dynamic loading forces, such as steady-state harmonic loading and
transient excitation sources, with advanced wave propagation models (Liang et al. 2010
). These dynamic OCE methods have been used to non-invasively measure
biomechanical properties of the human cornea in vivo, showing the potential for
clinical translation and utility (Ramier et al. 2020).
Cellular changes in AMD can alter the elasticity of retinal tissue, making OCE a potential
technology for early disease diagnosis (Krishnan et al. 2007). ARF-OCE studies have
shown distinct elasticity differences in retinal layers in in vivo rabbit and ex vivo porcine
models (Qu et al. 2018a). Decreased retinal stiffness observed in in vivo rabbit AMD eyes
was hypothesized to result from lymphocyte infiltration, but initial results did not show
statistical significance (Qu et al. 2018b). OCE studies have also shown that increased
optic nerve head Young’s modulus and posterior scleral stiffness are correlated with
increasing IOP, which suggests that OCE can also be used to monitor progression of
glaucoma (Du et al. 2019).
OCE imaging of the (a), (b), cornea and (c), (d) retina. (a) Structural OCT and (b) OCE elastogram
cross sections of in vivo rabbit cornea pre-, post-, and 1 week after CXL treatment (top to bottom,
respectively). (c) Structural OCT and (d) OCE elastogram cross sections of ex vivo porcine retina
showing differences in retinal layer stiffness. CXL, corneal collagen crosslinking; OCE, optical
coherence elastography; OCT, optical coherence tomography.

Retinal Biomechanics without OCE?
Braeu et al. (2024): “AI-based clinical assessment
of optic nerve head robustness superseding
biomechanical testing”
To use artificial intelligence (AI) to: (1) exploit
biomechanical knowledge of the optic nerve head
(ONH) from a relatively large population; (2) assess
ONH robustness (ie, sensitivity of the ONH to changes in
intraocular pressure (IOP)) from a single optical
coherence tomography (OCT) volume scan of the ONH
without the need for biomechanical testing and (3)
identify what critical three-dimensional (3D) structural
features dictate ONH robustness.
We propose an AI-driven approach that can assess
the robustness of a given ONH solely from a single
OCT volume scan of the ONH, and without the need to
perform biomechanical testing. Longitudinal studies
should establish whether ONH robustness could help us
identify fast visual field loss progressors.
Hannay et al. (2024): “A noninvasive clinical
method to measure in vivo mechanical
strains of the lamina cribrosa (LC) by optical
coherence tomography”
To measure mechanical strain of the lamina
cribrosa (LC) after intraocular pressure (IOP)
change produced one week after a change in
glaucoma medication. LC mechanical strains
can be effectively measured by changes in eye
drop medication using OCT and are related to
degree of visual function loss in glaucoma.

“OCT Thermometry”? Monitor laser treatments
See MR thermometry (e.g. Rieke and Pauly 2008 Cited by 1405
) for further non-ocular inspiration for thermal dosimetry
Heike H. Müller et al. (2012): “Imaging thermal expansion and retinal tissue
changes during photocoagulation by high speed OCT”
Visualizing retinal photocoagulation by real-time OCT measurements may considerably
improve the understanding of thermally induced tissue changes and might enable a better
reproducibility of the ocular laser treatment. High speed Doppler OCT with 860 frames per
second imaged tissue changes in the fundus of enucleated porcine eyes during laser irradiation.
Tissue motion, measured by Doppler OCT with nanometer resolution, was correlated with the
temperature increase, which was measured non-invasively by optoacoustics. In enucleated
eyes, the increase of the OCT signal near the retinal pigment epithelium (RPE) corresponded well
to the macroscopically visible whitening of the tissue. At low irradiance, Doppler OCT revealed
additionally a reversible thermal expansion of the retina. At higher irradiance additional
movement due to irreversible tissue changes was observed. Measurements of the tissue
expansion were also possible in vivo in a rabbit with submicrometer resolution when global tissue
motion was compensated. Doppler OCT may be used for spatially resolved measurements of
retinal temperature increases and thermally induced tissue changes. It can play an
important role in understanding the mechanisms of photocoagulation and, eventually, lead to
new strategies for retinal laser treatments.
Veysset et al. (2022): “Interferometric imaging of thermal expansion for temperature control in
retinal laser therapy”
Burri et al. (2023): “Real-time OCT feedback-controlled RPE photodisruption in ex vivo porcine eyes
using 8 microsecond laser pulses”

“OCT Thermometry”? with ORG or/and ERG?
Ari Koskelainen (2016): A device and method for non-invasive monitoring of retinal tissue
temperature
Marja Pitkänen (2019): In vivo monitoring of mouse retinal temperature by ERG
photoresponses
Ossi Kaikkonen et al. (2021): “Retinal Temperature
Determination Based on Photopic Porcine
Electroretinogram”
Subthreshold retinal laser therapy (SLT) is a treatment modality where the
temperature of the retinal pigment epithelium (RPE) is briefly elevated to trigger
the therapeutic benefits of sublethal heat shock. However, the temperature
elevation induced by a laser exposure varies between patients due to individual
differences in RPE pigmentation and choroidal perfusion. This study describes
an electroretinography (ERG)-based method for controlling the
temperature elevation during SLT.
The described ERG-based temperature estimation model could be used to
control SLT treatments such as transpupillary thermotherapy. Significance: The
introduced ERG-based method for controlling SLT could improve the
repeatability, safety, and efficacy of the treatment of various retinal disorders.
Mooud Amirkavei et al. (2022): “Novel subthreshold retinal laser treatment with ERG-based
thermal dosimetry activates hormetic heat response in pig RPE in vivo”
Jukka-Pekka Alanko et al. (2023): (Modulight, Maculaser) “Novel ophthalmic laser system
paired with focal electroretinography for subthreshold laser therapy of DME”

OCT+SLO for “Precision Photodamage” in animal models
Rico-Jimenez et al. (2023): “MURIN: Multimodal Retinal
Imaging and Navigated-laser-delivery for dynamic
and longitudinal tracking of photodamage in murine
models”
Laser-induced photodamage is a robust method for investigating retinal
pathologies in small animals. However, aiming of the photocoagulation laser
is often limited by manual alignment and lacks real-time feedback on lesion
location and severity. Here, we demonstrate MURIN: MUltimodal Retinal Imaging
and Navigated-laser-delivery, a multimodality OCT and SLO ophthalmic
imaging system with an image-guided scanning laser lesioning module
optimized for the murine retina. The proposed system enables targeting of focal
and extended area lesions under OCT guidance to benefit visualization of
photodamage response and the precision and repeatability of laser lesion
models of retinal injury.
Real-time MURIN imaging concurrent with laser lesioning allowed us to
visualize lesion formation dynamics and any corresponding changes in
retinal morphology. We observe increasing fluorescence photoconversion on
SLO and scattering contrast on OCT. Significant morphological changes are
visible on MURIN after high-severity photodamage. OCT cross-sections show the
spatial extent of the lesions contract over time from diffusion areas of increased
scattering to granular scatterers and corresponding SLO images show a radial
pattern surrounding severe focal lesions, which may be a result of a change in
Müller cell shape or orientation in response to injury. The inner plexiform layer is
distorted and increased RPE thickness and scattering are observed, all of which
are confirmed on corresponding hematoxylin and eosin (H&E) histology and
differential interference contrast (DIC) microscopy.

OCT Thermometry ‘easily integrated’ to iOCT systems?
Marc B. Muijzer et al. (2022): “Clinical applications for intraoperative
optical coherence tomography: a systematic review”
Sommersperger et al. (2021): “Real-time tool to layer distance estimation
for robotic subretinal injection using intraoperative 4D OCT”
Maierhofer et al. (2023): “iOCT-guided simulated subretinal injections: a
comparison between manual and robot-assisted techniques in an ex-
vivo porcine model”
Peiyao Zhang et al. (2024): “Autonomous Needle Navigation in Subretinal
Injections via iOCT”
Robert M. Trout et al. (2023): “Methods for real-time feature-guided image
fusion of intrasurgical volumetric optical coherence tomography with
digital microscopy”
4D-microscope-integrated optical coherence tomography (4D-MIOCT) is an
emergent multimodal imaging technology in which live volumetric OCT (4D-
OCT) is implemented in tandem with standard stereo color microscopy. 4D-OCT
provides ophthalmic surgeons with many useful visual cues not available in
standard microscopy; however it is challenging for the surgeon to effectively
integrate cues from simultaneous-but-separate imaging in real-time. In this
work, we demonstrate progress towards solving this challenge via the fusion of
data from each modality guided by segmented 3D features. In this way, a
more readily interpretable visualization that combines and registers important
cues from both modalities is presented to the surgeon.
Surface shading pipeline, sample was a soft-tip tool
contacting the retina.
Time series intrasurgical 4D-MIOCT image data for the
approach, contact, grasp and withdrawal (columns 1-4
respectively) of forceps at the retina
Dr. Fanny Nerinckx, Leica Microsystems (2024): RPE65 Gene Therapy Subretinal Injection:
Benefits of Intraoperative OCT
iOCT – intraoperative OCT for visualizing ophthalmic surgical operations

OCT-guided ERG
Michael Carlson et al. (2022): “OCT guided micro-focal ERG system with
multiple stimulation wavelengths for characterization of ocular health”
Inherited retinal disorders and dry age-related macular degeneration
(AMD) are characterized by the degeneration and death of different
types of photoreceptors at different rate and locations. Advancement
of new therapeutic interventions such as gene replacement10
,
optogenetic gene therapy11
, and regenerative cell12
(transplant) therapies
are dependent on electrophysiological measurements at cellular
resolution.
Here, we report the development of an optical coherence tomography
(OCT) guided micro-focal multi-color laser stimulation and
electroretinogram (ERG) platform (Nanoscope NS-Neel) for highly
localized monitoring of retina function. Functional evaluation of wild
type and transgenic pigs affected by retinal degeneration was carried
out using OCT guided micro-focal ERG ( fERG) with selected
μ
stimulation wavelengths for S, M and L cones as well as rod
photoreceptors. In wild type pigs, fERG allowed functional recording
μ
from rods and each type of cone photoreceptor cells separately.
Furthermore, functional deficits in P23H transgenic pigs consistent with
their retinal degeneration phenotype were observed, including
decrease in the S and M cone function and lack of rod photoreceptor
function.
OCT guided fERG based monitoring of physiological function will
μ
enable characterization of animal models of retinal degenerative
diseases and evaluation of therapeutic interventions at the cellular
level.
WhitePaper: New Perspectives on OCT Guided Visualization and Manipulation

‘Desktop OCT‘
Innovators
New emerged
companies outside the
established players

Cylite (Melbourne, Australia)
https://www.cyliteoptics.com/

Intalight (San Francisco, USA)
Vitreous Body Imaging and coalesce cistern visualization with SS OCT
Dream OCT from Intalight - Adil EL Maftouhi
Centre ophtalmologique de Rive , Genève, Institut Parisien d'Opthalmologie, Medical DevEyes
Feb 2024
https://intalight.com/

Portable OCT for
home/bedside/
pediatric Imaging

Automatic/Home OCT for disease management
From A review of low-cost and portable optical coherence tomography, e.g. the Notal device
Efficacy of Notal Vision OCT device demonstrated by a series of scientific and clinical work, and ARVO 2022
presentation of paradigm change of patient interaction in OCT (with their robotic platform)

Portable OCT (finally) getting somewhere? #1
Song et al. (2021): “A review of low-cost and
portable optical coherence tomography””
Recently, the commercialization of OCT
engines and components has accelerated
[50], and several groups have aimed to
implement small and portable OCT
systems with handheld scanners. These
efforts include the use of a
microelectromechanical (MEMS) mirror for
scanning [51], rather than the traditional
use of galvanometers, and minimizing
components within the handheld scanner [
52–54]. In spite of these efforts,
commercially available OCT systems
remain fairly bulky in size, limiting their
portability. However, the most significant
limitation of using OCT at the point-of-care
is its high cost. In the field of
ophthalmology, the price of a commercial
OCT system can range between $40 000
and $150 000 [55], and thus availability is
usually restricted to large eye centers or
hospitals [56]. Several efforts have been
made to reduce the cost of various OCT
components using off-the-shelf optics and
custom electronics [57, 58].
Chao Zhou, a professor of biomedical engineering in
the McKelvey School of Engineering at Washington
University in St. Louis, will lead work to develop a
portable OCT scanner.
With the ARPA-H funding, Zhou and collaborators will
assemble the components in a photonic chip using
advancements in CMOS processes that have
benefitted the semiconductor industry. This will
streamline manufacturing and lower costs. Once
functioning, the collaborators will conduct studies
using the device on adult and pediatric patients. The
proposed system is >50× faster than existing state-
of-the-art commercial OCT systems at a fraction of
the cost, the researchers said.

Hagan et al. (2020, Duke): “Wavefront sensorless multimodal handheld adaptive
optics scanning laser ophthalmoscope for in vivo imaging of human retinal
cones”
Hagan et al. (2023): “Dual modality handheld adaptive optics optical coherence
tomography probe for in vivo 3-D photoreceptor imaging”
Adaptive optics optical coherence tomography (AO-OCT)
has allowed for the reliable 3-D imaging of individual retinal
cells. The current AO-OCT systems are limited to tabletop
implementation due to their size and complexity. This work
describes the design and implementation of the first dual
modality handheld AO-OCT (HAOOCT) and scanning
laser ophthalmoscope (SLO) probe to extend AO-OCT
imaging to previously excluded patients.
Simultaneous SLO imaging allows for tracking of imaging
features for HAOOCT localization. Pilot experiments on
stabilized and recumbent adults using HAOOCT, weighing
only 665 grams, revealed the 3-D photoreceptor structure
for the first time using a handheld AO-OCT/SLO device.
ARVO 2022 1x1deg AO-OCT, 3.9x3.9deg AOSLO

Kaveri A. Thakoor (2022): “Enhancing Portable OCT
Image Quality via GANs for AI-Based Eye Disease
Detection”

Milana Kendrisic et al. (2023): “Thermally-tuned
VCSEL at 850 nm as a low-cost source alternative
for full eye SS-OCT” ophthalmologytimes.com
Swept-source optical coherence tomography (SS-OCT)
demonstrates superior performance in comparison to
spectral domain OCT with regard to depth ranging. The
main driver of cost for SS-OCT systems is, however, the
price of the source. In the following, we demonstrate first in
vivo results for full-eye biometry and anterior segment SS-
OCT with a thermally tuned VCSEL at 850 nm (TRUMPF ULM-
850-B2) used as a low-cost swept source.
Based on our results, we believe that this technology can be used
as a cost-effective alternative OCT for point-of-care diagnostics..
Furthermore, there is a current trend for developing homecare
and point-of-care OCT devices. These might trade-off image
quality for system costs, provided that the image still allows the
recognition of signs of pathologies. e.g., open-angle glaucoma or
retinal pathologies [von der Burchard et al. 2021].
Dierck Hillmann (2021): “OCT on a chip aims at
high-quality retinal imaging”
OCT systems are still not available for <10,000 USD. Apart from using
nonstandard techniques such as multireference OCT Neuhaus et al. 2017
or
full-field time-domain OCT Sudkamp et al. 2016, Vabre et al. 2002
, changing
components for cheaper alternatives or using more cost-effective
production processes for standard spectrometer-based OCT
(spectral-domain, SD-OCT) appears to be the most promising way to
maintain high image quality while still producing systems in large
numbers at reduced production costs.
Probably, the most promising approach to cut costs and size is to replace
optical components of SD-OCT systems with photonic integrated
circuits (PICs). This technique is comparable to the microchip production
process but uses optical waveguide-based components instead of
electrical circuits and transistors. One of the most critical of these SD-
OCT components is the spectrometer. It is the heart of any SD-OCT
system and determines its performance. When implementing an OCT
spectrometer with PICs, it is commonly realized as an arrayed waveguide
(AWG) Smit 1988, Li and Fainman 2021, Xu et al. 2023
. A recent paper by
Elisabet Rank et al. (2021) demonstrated a considerable step towards
high-quality low-cost OCT on a chip.

Ni et al. (Nov 2023): “Panretinal Optical
Coherence Tomography”
We introduce a new concept of panoramic retinal
(panretinal) optical coherence tomography (OCT)
imaging system with a 140° field of view (FOV). To
achieve this unprecedented FOV, a contact
imaging approach was used which enabled faster,
more efficient, and quantitative retinal imaging with
measurement of axial eye length. The utilization of
the handheld panretinal OCT imaging system
could allow earlier recognition of peripheral retinal
disease and prevent permanent vision loss. In
addition, adequate visualization of the peripheral
retina has a great potential for better
understanding disease mechanisms regarding the
periphery. To the best of our knowledge, the
panretinal OCT imaging system presented in this
manuscript has the widest FOV among all the retina
OCT imaging systems and offers significant values
in both clinical ophthalmology and basic vision
science.
En face OCT image from an infant with ROP
stage 1

OCT
Post-Processing
Radiomics-style Meyer et al. (2019)
possible post-processing
issues?

OCT speckle itself can carry pathological signal
VB Silva et al. (2021): “Signal-carrying speckle in
Optical Coherence Tomography: a methodological
review on biomedical applications” Cited by 10
The studies have been clustered according
to the nature of their analysis, namely static
or dynamic, and all features were described
and analysed. The results show that
features retrieved from speckle can be
used successfully in different applications,
such as classification and segmentation.
However, the results also show that speckle
analysis is highly application-dependant,
and the best approach varies between
applications. Conclusions: Several of the
reviewed analysis were only performed in a
theoretical context or using phantoms,
showing that signal-carrying speckle
analysis in OCT imaging is still in its early
stage, and further work is needed to
validate its applicability and
reproducibility in a clinical context.
Machine Learning in OCT Imaging (May 7, 2021) hosted by
Center for Biomedical OCT Research & Translation:
Lei Tian: “Adaptive Deep Learning for Imaging in Scattering Media”
Nestor Uribe-Patarroyo: “Quirks and Twists of OCT Imaging for the Computational Scientist”

Denoising gives nicer looks for sure
Probabilistic tissue polarimetry dramatically enhances spatial
resolution in intravascular OCT. IV-PS-OCT imaging of intimal
thickening in a patient. Birefringence ( n) given in º/ m, in-plane
Δ μ
optic-axis angle ( ) visualization “ n– ” is a luminance( n)–hue( )
φ Δ φ Δ φ
overlay. The media layer birefringence and optic axis appear more
uniform, even in the region (blue arrow) where they are lost in
conventional processing. The green inset box shows a region (at 4
o’clock, outside of main image) where a thin strip of high
birefringence is seen in probabilistic processing, matching the
boundary between adventitia and peri-adventitial region.
Is some pathological signal lost here?
Carlos Cuartas-Vélez et al. (2018): “Volumetric non-
local-means based speckle reduction for optical
coherence tomography” octresearch.org
Chintada et al. (2023): “Probabilistic volumetric speckle suppression in OCT using deep learning”
We present a deep learning framework for volumetric speckle reduction in optical coherence
tomography (OCT) based on a conditional generative adversarial network (cGAN) that leverages
the volumetric nature of OCT data. We demonstrate fast, effective, and high-quality despeckling
of the proposed network in different tissue types acquired with three different OCT systems
compared to existing deep learning methods. The open-source nature of our work facilitates re-
training and deployment in any OCT system with an all-software implementation, working
around the challenge of generating high-quality, speckle-free training data.
Contrast-enhanced boxes show superior speckle
suppression ability of DL-TNode-3D compared to cGAN-2D,
which exhibits high-frequency artifacts along the slow-
scan axis

Adaptive Optics
for future OCT and SLO
systems

Adaptive optics in retinal imaging
Correct for individual (static and dynamic) distortions -> sharper image
Idea from ground-based astronomy, and
correction for the effect of turbulence to
image quality

Adaptive optics: Retinal Imaging motivation
Small FOVs though, expensive optics, have not become clinically popular yet
“The promise of AO”: Cellular-level in vivo imaging
Jonnal et al. (2016): “A Review of Adaptive Optics
Optical Coherence Tomography: Technical
Advances, Scientific Applications, and the Future” (
Cited by 105)

Volume electron microscopy (EM) for nanometer scale #1
Could you train “deep super-resolution”/ image translation with the EM ground truths to further improve AO-OCT/SLO
resolution? Paired ex vivo samples imaged both by AO-OCT and EM?
Christine Curcio: Check this new publication, in IOVS. From the
Human Foveal Connectome group #AndreasPollreisz; #DennisDacey;
#DeepayanKar; using Dragonfly #MikeMarsh
Maximilian Lindell et al. (2023): “Volumetric
Reconstruction of a Human Retinal Pigment Epithelial
Cell Reveals Specialized Membranes and Polarized
Distribution of Organelles”
Specialized membranes at the apical and basal side of the
retinal pigment epithelium (RPE) cell body involved in
intercellular uptake and transport represent over 90% of the
total surface area. Together with the polarized distribution of
organelles within the cell body, these findings are relevant
for retinal clinical imaging, therapeutic approaches, and
disease pathomechanisms.
Our new data can launch studies of how membrane
specializations contribute to OCT imaging. … Basolateral
infoldings are relevant to a hyporeflective band visualized
in visible light OCT (Zhang et al. 2013, Chong et al. 2017)
prototype ultra-high-resolution OCT (Chen et al. 2023), and
adaptive optics OCT (AO-OCT, Liu et al. 2019) in healthy eyes.
A hyporeflective band separating hyper-reflective RPE cell
body and Bruch's membrane in young adults, attributed to
basolateral infoldings, was invisible in persons at mid-life.
Intriguingly this band appeared in aged persons and
appeared thick in AMD, and thought to represent basal
laminar deposits (Chen et al. 2023). The prospects of
observing basolateral infoldings over the lifespan and in
relation to fluid balance through in vivo imaging are thus
good.

Volume electron microscopy (EM) for nanometer scale #2
Kar et al. (2023): “Volume electron microscopy reveals
human retinal mitochondria that align with reflective
bands in optical coherence tomography”
Mitochondria are candidate reflectivity signal
sources in optical coherence tomography (OCT)
retinal imaging. Here, we use deep-learning-
assisted volume electron microscopy of human
retina and in vivo imaging to map mitochondria
networks in the outer plexiform layer (OPL),
where photoreceptors synapse with second-
order interneurons. We observed alternating
layers of high and low mitochondrial abundance
in the anatomical OPL and adjacent inner nuclear
layer (INL). Subcellular resolution OCT imaging of
human eyes revealed multiple reflective bands
that matched the corresponding INL and
combined OPL sublayers.
Data linking specific mitochondria to defined
bands in OCT may help improve clinical
diagnosis and the evaluation of mitochondria-
targeting therapies.
Optical coherence tomography reveals bands correlating with
ultrastructurally defined mitochondria layers.

Flood vs. Scanning AO Systems #1
Burns et al. (2018)
High resolution pattern projection in the retina for phase contrast imaging
Pierre Senée, Léa Krafft, Pedro Mecê, Serge Meimon at I2Eye 2023, Paris

Flood vs. Scanning AO Systems #2
https://www.pariseyeimaging.com/Research-Themes/fb9bec8b9a-Theme-Flood-and-Scanning-Technology.en.htm
PARIS Eye Imaging
Hampson et al. (2021): “Adaptive optics for high-resolution imaging”

Clinical AO Imaging Imagine Eyes rtx1
https://www.imagine-eyes.com/

MERLIN EU Project driven by Imagine Eyes

MERLIN EU Project driven by Imagine Eyes
Quinze-Vingts hospital

Multimodal AO-OCT
Liu et al. (2022, FDA): “Ultrahigh-speed multimodal adaptive optics system for
microscopic structural and functional imaging of the human retina”
We describe the design and performance of a multimodal and
multifunctional adaptive optics (AO) system that combines scanning
laser ophthalmoscopy (SLO) and optical coherence tomography
(OCT) for simultaneous retinal imaging at 13.4 Hz. The high-speed AO-
OCT channel uses a 3.4 MHz Fourier-domain mode-locked (FDML) swept
source. The system achieves exquisite resolution and sensitivity for pan-
macular and transretinal visualization of retinal cells and structures while
providing a functional assessment of the cone photoreceptors. The
ultra-high speed also enables wide-field scans for clinical usability
(3°×3° AO-SLO scans) and angiography for vascular visualization. With a
few exceptions, most previously reported AO systems limited the
imaging FOV to 2° or less, preventing wide adoption of AO into clinical
practice. System complexity, imaging speed, and small FOV have thus
limited the full development of multimodal AO approaches.
The FDA FDML-AO system is a powerful platform for studying various
retinal and neurological diseases for vision science research, retina
physiology investigation, and biomarker development.

Multifunctional AO-OCT
Kazuhiro Kurokawa and Morgan Nemeth (2024):
“Multifunctional adaptive optics optical coherence
tomography allows cellular scale reflectometry, polarimetry,
and angiography in the living human eye”
Clinicians are unable to detect glaucoma until substantial loss
or dysfunction of retinal ganglion cells occurs. To this end, novel
measures are needed. We have developed an optical imaging
solution based on adaptive optics optical coherence
tomography (AO-OCT) to discern key clinical features of
glaucoma and other neurodegenerative diseases at the
cellular scale in the living eye. Here, we test the feasibility of
measuring AO-OCT-based reflectance, retardance, optic axis
orientation, and angiogram at specifically targeted locations in
the living human retina and optic nerve head.
Multifunctional imaging, combined with focus stacking and
global image registration algorithms, allows us to visualize
cellular details of retinal nerve fiber bundles, ganglion cell layer
somas, glial septa, superior vascular complex capillaries, and
connective tissues. These are key histologic features of
neurodegenerative diseases, including glaucoma, that are now
measurable in vivo with excellent repeatability and
reproducibility. Incorporating this noninvasive cellular-scale
imaging with objective measurements will significantly
enhance existing clinical assessments, which is pivotal in
facilitating the early detection of eye disease and
understanding the mechanisms of neurodegeneration.

Handheld AOSLO+ OCT
(Hagan, Duke): Handheld OCT. Initial
sensorless design upgraded with a
wavefront sensor. 1x1deg AO-OCT,
3.9x3.9deg AOSLO. Zemax OpticStudio
Tolerance Stack Analysis. Solidworks for
custom optomechanics design. Custom
model (phantom) eye with 5 micron
spacing 5 micron diameter hexagonal
cones. 3.75 uW for OCT, 50 uW for
wavefront sensing, rest 100 uW for SLO.
Custom deformable mirrors ordered
with custom optomechanics for
reproducible mechanical mounting. See
e.g. Hagan et al. (2020)

Tomography for AO systems without intrinsic sectioning
Pedro Mecê et al. (2020) (Institut Langevin): “Optical
Incoherence Tomography: a method to generate tomographic
retinal cross-sections with non-interferometric adaptive optics
ophthalmoscopes”
We present Optical Incoherence Tomography (OIT): a
completely digital method to generate tomographic
retinal cross-sections from en-face through-focus
image stacks acquired by non-interferometric imaging
systems, such as en-face adaptive optics (AO)-
ophthalmoscopes. We demonstrate that OIT can be
applied to different imaging modalities using back-
scattered light, including systems without inherent
optical sectioning and, for the first time, multiply-
scattered light, revealing a distinctive cross-sectional
view of the retina. The axial dimension of OIT cross-
sections is given in terms of focus position rather than
optical path, as in OCT. We explore this property to
guide focus position in cases where the user is “blind”
focusing, allowing precise plane selection for en-face
imaging of retinal pigment epithelium, the vascular
plexuses and translucent retinal neurons, such as
photoreceptor inner segments and retinal ganglion
cells, using respectively autofluorescence, motion
contrast and split detection techniques.
Flood-Illumination Ophthalmoscopes (FIO)
Scanning Laser Ophthalmoscopes (SLO)

AO OCT Still in research labs, not in clinical practice
Hammer et al. (2023, FDA): Adaptive Optics
Imaging of Outer Retinal Diseases
Sabesan et al. (2023): “Introduction
to the Feature Issue on Adaptive
Optics for Biomedical Applications”

AOSLO Phase Imaging
for novel biomarkers
To reveal transparent
retinal structures and
cells

Off-Axis (Phase Contrast) Imaging | AOSLO #1
New biomarkers coming up, for translucent structures
Multi-aperture AO-SLO
retinal imaging
Mircea Mujat, Ankit Patel, Nicusor Iftimia

Multi-aperture AO-
SLO retinal imaging
Mircea Mujat, Ankit
Patel, Nicusor Iftimia
i2eye 2022, Paris

Label-free in vivo imaging of inflammation
at the level of single cells in the living huma
n eye
- Ethan Rossi

High-tech phase imaging (non-confocal split detection)
For rare eye disorders
"The NEI's long-term investment in imaging technology is changing our
understanding of eye diseases," said NEI Director Michael F. Chiang, M.D.
"This study is just one example of how improved imaging can reveal
subtle details about pathology in a rare eye disease that can inform the
development of therapeutics.”
Tao Liu et al. (2022): “Photoreceptor and Retinal Pigment Epithelium
Relationships in Eyes With Vitelliform Macular Dystrophy Revealed by
Multimodal Adaptive Optics Imaging” Cited by 7
Multimodal adaptive optics (AO) imaging was performed
in 11 patients with vitelliform macular dystrophy (VMD)
using a custom-assembled instrument. Non-confocal
split detection and AO-enhanced indocyanine green
were used to visualize the cone photoreceptor and RPE
mosaics, respectively.
Assessment of cones and RPE in retinal locations outside
of the macular lesions reveals a pattern of cone and RPE
disruption that appears to be gene dependent in VMD.
These findings provide insight into the cellular
pathogenesis of disease in VMD.

Polarimetric
retinal imaging
or Polarization-
sensitive OCT (PS-OCT)

Polarimetric fundus imaging
‘Full’ (Müller Imaging) 2D polarimetric imaging not so common in retinal context
Twietmeyer et al. (2008): “Mueller matrix retinal imager
with optimized polarization conditions”
Polarization parameter images for ONH (optic nerve head, optic disc) of right eye, for
one subject. These images derive from the Mueller matrix image with corneal
compensation. (a): normalized average intensity; (b): linear retardance; (c): retardance
orientation; (d): depolarization index; (e) diattenuation magnitude; (f) diattenuation
orientation.
Melanie C. W. Campbell et al. (2005): “Enhanced confocal
microscopy and ophthalmoscopy with polarization imaging”
Juan M. Bueno et al. (2007): “Improved scanning laser fundus
imaging using polarimetry”

What is Mueller Matrix Imaging?
4 polarization angles (0,45,90,-45) for both PSG and PSA. In the vanilla design you need to
acquire 16 (4x4) images, sequentially typically to get the Mueller matrix of the sample
(e.g. retina) that will give you the polarization parameters.
Filter illumination
Filter Reflectance
Bu et al. (2022)
Chang and Gao (2019): “Method of interpreting
Mueller matrix of anisotropic medium”

Polarization sensitive optical coherence tomography (PS-OCT)
Plenty of structures altering the polarization of the light, including Amyloid plaques for example
Pircher et al. (2011): “Polarization sensitive optical
coherence tomography in the human eye” Cited by 309
Optical coherence tomography (OCT) has
become a well established imaging tool in
ophthalmology. The unprecedented depth
resolution that is provided by this technique yields
valuable information on different ocular tissues
ranging from the anterior to the posterior eye
segment. Polarization sensitive OCT (PS-OCT)
extends the concept of OCT and utilizes the
information that is carried by polarized light to
obtain additional information on the tissue.
Several structures in the eye (e.g. cornea, retinal
nerve fiber layer, retinal pigment epithelium) alter
the polarization state of the light and show
therefore a tissue specific contrast in PS-OCT
images.
Example of tissue discrimination based on PS-OCT. (A) intensity image, (B) pseudo color coded
structural images. The light brown corresponds to conjunctiva, green indicates sclera, dark yellow
indicates trabecular meshwork, blue indicates cornea, and red indicates uvea.
PS-OCT B-scan images of healthy eyes. Analysis of the origin of atypical scanning laser
polarimetry retardation patterns. (A) Intensity images and (D) retardation images from eye with
normal retardation pattern. (G) Intensity and (J) retardation images from eye with atypical
retardation pattern.

Triple-input PS-OCT (TRIPS-OCT)
Liu et al. (2023): “Posterior scleral birefringence measured
by triple-input polarization-sensitive imaging as a
biomarker of myopia progression”
In myopic eyes, pathological remodelling of collagen in
the posterior sclera has mostly been observed ex vivo.
Here we report the development of triple-input
polarization-sensitive optical coherence tomography (PS-
OCT) for measuring posterior scleral birefringence. In
guinea pigs and humans, the technique offers superior
imaging sensitivities and accuracies than dual-input
polarization-sensitive OCT. In 8-week-long studies with
young guinea pigs, scleral birefringence was positively
correlated with spherical equivalent refractive errors
and predicted the onset of myopia. In a cross-sectional
study involving adult individuals, scleral birefringence was
associated with myopia status and negatively correlated
with refractive errors. Triple-input polarization-sensitive
OCT may help establish posterior scleral birefringence as
a non-invasive biomarker for assessing the
progression of myopia
TRIPS and dual-input reconstruction methods on
guinea pig retina in vivo.

Emerging techniques
from the research labs

Moon 2020 Project
https://moon2020.meduniwien.ac.at/fileadmin/moon2020/Flyer-Projet-Moon-8_RVB_.pdf
How to improve the early diagnostics of ophthalmic diseases?
1,135 views Nov 19, 2018 -
https://www.youtube.com/watch?v=c2Ud8K7eEsw&ab_channel=euronews
Alzheimer mentioned
in vivo retinal Raman imaging #1

Sentosa et al. (MOON project) (2023): “Towards in vivo
molecular imaging of the retina: OCT-guided Raman
spectroscopy”
Here we investigated the potential diagnostic capability of in vivo Raman spectroscopy
(RS) of the retina in a clinical setting. With our multimodal ophthalmic imaging device,
we can acquire interpretable Raman spectra from AMD subjects and healthy controls. Our
results confirmed the potential of RS to differentiate healthy and diseased retina tissue
in vivo. In future clinical studies, we will investigate that the molecular information
obtained by RS could enable an earlier diagnosis of retina diseases.
Rainer Leitgeb (MOON project) (2023): “Multimodal
retinal tissue assessment combining wide field
OCT/OCTA and Raman Spectroscopy”
Multiple sclerosis, Parkinson’s and Alzheimer’s

Rainer Leitgeb (MOON project) (2023): “Multimodal
retinal tissue assessment combining wide field
OCT/OCTA and Raman Spectroscopy”

in vivo FTIR?
If you would have some trick to get 6 micron signal through water
Sean D. Moran and Martin T. Zanni (2014): “How to Get Insight into Amyloid Structure
and Formation from Infrared Spectroscopy” Cited by 161 (1615-1630cm-1 6192-6135 nm)
→
T. Sylvestre et al. (2021): “Recent advances in supercontinuum
generation in specialty optical fibers [Invited]”

Mid-infrared hyperspectral imaging
Water absorption in the human eye still high for this range.
Upconversion trick to help finding optical components for the visible range instead of the mid-infrared
Fang et al. (Feb 2024): “Wide-field mid-infrared
hyperspectral imaging beyond video rate”
Mid-infrared hyperspectral imaging has become an indispensable tool to
spatially resolve chemical information in a wide variety of samples. However,
acquiring three-dimensional data cubes is typically time-consuming due to
the limited speed of raster scanning or wavelength tuning, whichimpedes real-
time visualization with high spatial definition across broad spectral bands.
Here, we devise and implement a high-speed, wide-field mid-infrared
hyperspectral imaging system relying on broadband parametric
upconversion (replica in the visible region) of high-brightness
supercontinuum illumination at the Fourier plane. The upconverted replica is
spectrally decomposed by a rapid acousto-optic tunable filter (AOTF), which
records high-definition monochromatic images at a frame rate of 10 kHz based
on a megapixel silicon camera.
Consequently, the hyperspectral imager allows us to acquire 100
spectral bands over 2600-4085 cm−1
(3,846-2448 nm) in 10 ms,
corresponding to a refreshing rate of 100 Hz. Moreover, the angular
dependence of phase matching in the image upconversion is
leveraged to realize snapshot operation with spatial multiplexing for
multiple spectral channels, which may further boost the spectral
imaging rate. The high acquisition rate, wide-field operation, and
broadband spectral coverage could open new possibilities for high-
throughput characterization of transient processes in material and
life sciences.

Zhongya Qin et al. (2020): “Adaptive optics two-photon microscopy enables near-
diffraction-limited and functional retinal imaging in vivo” Cited by 22
https://twitter.com/VPOptics/status/1564175352708141056?t=MRCqLHpxVN-Tl-L7Mm_Hzg&s=19
https://www.eye-tuebingen.de/schwarzlab/
AO-Two-Photon Imaging (AO-2PM) #1

AO-Two-Photon Imaging (AO-2PM) #2
Zhang et al. (2023): “Retinal microvascular and neuronal
pathologies probed in vivo by adaptive optical two-photon
fluorescence microscopy”
The retina, behind the transparent optics of the eye, is the only neural
tissue whose physiology and pathology can be non-invasively
probed by optical microscopy. The aberrations intrinsic to the mouse
eye, however, prevent high-resolution investigation of retinal
structure and function in vivo. Optimizing the design of a two-photon
fluorescence microscope (2PFM) and sample preparation
procedure, we found that adaptive optics (AO), by measuring and
correcting ocular aberrations, is essential for resolving putative
synaptic structures and achieving three-dimensional cellular
resolution in the mouse retina in vivo. Applying AO-2PFM to
longitudinal retinal imaging in transgenic models of retinal
pathology, we characterized microvascular lesions with sub-
capillary details in a proliferative vascular retinopathy model, and
found Lidocaine to effectively suppress retinal ganglion cell
hyperactivity in a retinal degeneration model. Tracking structural
and functional changes at high-resolution longitudinally, AO-
2PFM enables microscopic investigations of retinal pathology and
pharmacology for disease diagnosis and treatment in vivo.

Two-Photon Imaging (2PM)
Zhang et al. (2023): “In vivo two-photon microscopy of the human eye”
Cited by 40
Two-photon (2P) microscopy is a powerful tool for imaging and exploring label-free
biological tissues at high resolution. Although this type of microscopy has been
demonstrated in ex vivo ocular tissues of both humans and animal models, imaging the
human eye in vivo has always been challenging. This work presents a novel compact
2P microscope for non-contact imaging of the anterior part of the living human eye.
The performance of the instrument was tested and the maximum permissible exposure to
protect ocular tissues established. To the best of our knowledge, 2P images of the in vivo
human cornea, the sclera and the trabecular meshwork are shown for the very first time.
Acquired images are of enough quality to visualize collagen arrangement and
morphological features of clinical interest. Future implementations of this technique
may constitute a potential tool for early diagnosis of ocular diseases at submicron scale.
Kaushik et al. (2023): “Two-photon excitation fluorescence in ophthalmology:
safety and improved imaging for functional diagnostics”

Two-Photon Excited Fluorescence Lifetime
Reveals Differences in Biochemical
Composition Between Retinal Cells in the
Living Monkey and Mouse
Huynh, Khang T.
University of Rochester
https://www.proquest.com/openview/3a1659ca83957f24c42372ea4c4014
0d/1.pdf?pq-origsite=gscholar&cbl=18750&diss=y
AOFLIO images at 730 nm excitation of TPEF intensity (a) and mean TPEF lifetime (b) of the
photoreceptors at 20° eccentricity.
∼ Cones can be distinguished from rods by their longer lifetime.
in vivo 2PM+FLIO+AO

Ophthalmic
Imaging
Standards
Not like the ‘clinical imaging’
devices are that well
standardized compared to
experimental lab devices

Call for Standards #1
Michael F. Chiang (2021): “NEI joins call for standardization of ophthalmic
imaging devices”
DICOM compliance is low for ophthalmic imaging technologies. Even so-called DICOM-
compliant devices fail to meet DICOM standards with significant limitations, such as
the embedding of patient identifiers on the image. In the past, the Academy has used its
resources extensively to encourage standard-setting activities and to develop
standards collaboratively with device manufacturers.
Recommendations The Academy strongly encourages imaging device manufacturers
and PACS manufacturers to implement existing DICOM standards. These are 2 specific
examples of implementation that would benefit ophthalmologists:
●
Provide machine-readable, discrete data for user-selected reports of ophthalmic
imaging or functional testing.
●
Use lossless compression for pixel or voxel data to encode the same raw data as
used by manufacturers.
The Academy’s efforts are focused on making sure that medical technology is more
relevant to the needs of the end user, the ophthalmologist, by ensuring that there is
interoperability, that is, that there can be a seamless interface that allows the
communication and comprehension of image data between 2 parties. Once
ophthalmic imaging device manufacturers implement globally recommended
standards, then the field of ophthalmology can rapidly progress along the path of
efficient electronic workflow, interoperability, and artificial intelligence systems
that will meet an increased demand for ophthalmic services to the public.
Revie et al. (2016): “Current problems and perspectives on
colour in medical imaging”
The importance of accurate calibration and reproducible image
capture and display has become more apparent, but standards
and best practices in this field are still in development. Since
2013, the International Color Consortium has engaged with the
medical imaging community to help understand the particular
problems encountered and to help develop solutions. Currently the
ICC Medical Imaging Working Group is working on a wide range of
topics including digital microscopy, medical displays,
ophthalmology, medical photography, multispectral imaging, petri
dish imaging, dermatology, skin colour measurement, and 3-D
imaging for surgery. In this overview, the problems in each of these
areas are summarised and the current activity is described.”
Fundus cameras produce widely varying images of the same
retina. Although previous work has addressed the problem of
consistency between different systems [Hubbard and Ferris 2009, Cited by 2; Bull 2009
Cited by 4]
, until now there has been no method of calibrating such
cameras in a way that leads to accurate or consistent colour
images.

Call for Standards #2
Michael F. Chiang (NEI) and Kerry Goetz (NEI) (April 2024): “NEI Informatics & Data-Driven
Insights: Seminars & Dialogue Opportunities for Vision Health” hosted by the National Eye
Institute’s Office of Data Science and Health Informatics (ODSHI)
DICOM for ophthalmic data Kerry E. Goetz et al. (2024): “Accelerating Care: A Roadmap to Interoperable Ophthalmic Imaging Standards
in the United States”
All of Us FHIR interoperability

The interoperable standards a bit of a mess in general
Guy Tsafnat et al. (2023): “Converge or collide? Making
sense of a plethora of open data standards in healthcare:
an editorial”
Interoperabile data is hailed as a near-future solution to many of
these challenges and have been so for decades. Paradoxically,
interoperability efforts have themselves been fractured and
inconsistent, resulting in a plethora of incompatible interoperability
standards, despite widespread acknowledgement that fewer
standards would provide better interoperability. This paper presents
a typology of healthcare data requirements and describes the
challenges and opportunities of open data standards in healthcare.
Recognizing that different data standards represent different points
of view and respond to different needs, and that no single standard
would necessarily be able to meet all the requirements of all
healthcare systems, we distinguish three domains (openEHR,
OMOPP, FHIR) of healthcare data with their own unique
characteristics and challenges, and outline high-level design
requirements. We distinguish between requirements that are
common across all domains, and those that are specific to each
domain.

● Functional Optical Coherence Tomography (OCT):
○ Doppler OCT (DOCT), polarization-sensitive OCT (PS-OCT), optical
coherence elastography (OCE), spectroscopic OCT (SOCT), and molecular
imaging OCT
● Electroretinography (ERG)
○ Optoretinography
● Visually evoked potentials (VEP, EEG)
● Visual field examination
○ with eye tracking (e.g. VR)
○ Fundus (SLO) Microperimetry, e.g. iCare MAIA Confocal Microperimeter iCare
MAIA (Macular Integrity Assessment) and S-MAIA offer the best in confocal microperimetry to
combine visual field tests, fixation loss correction by a real-time retinal tracker and non-mydriatic
confocal SLO fundus imaging, all in one exam. iCare MAIA and S-MAIA detect and monitor functional
changes of the retina with great reliability.
Functional tests
- can the patient see still well, or is the patient losing sight (BCVA, visual fields)
- ‘intermediate’ functional tests that can be used to diagnose and monitor progression

C Light’s Tracking SLO (TSLO) for Eye movements
https://www.clighttechnologies.com/technology

Eye Tracking developed for retinal imaging
Berkeley Center for Innovation in Vision and Optics - CIVO Binocular
eye tracking three different ways. Line scanning ophthalmoscope eye tracking
with arcminute accuracy at rates up to 1kHz simultaneously with pupil tracking
and Dual-Purkinje Image eye tracking at 400 Hz. Collaborative research at its
best. With CIVO faculty Austin Roorda, Jorge Otero-Millan and Jacob Yates, CIVO
visiting professor David Merino and postdoctoral researcher Roksana Sadeghi.
But can be used for functional purposes as highlighted by C Light
Wu et al. 2023: High-resolution eye-tracking via digital imaging of Purkinje reflections

Optoretinography (ORG) Basics
‘Imaging Electroretinography (ERG)’
Tae-Hoo Kim et al. (2022): “Functional Optical Coherence
Tomography for Intrinsic Signal Optoretinography: Recent
Developments and Deployment Challenges”
Intrinsic optical signal (IOS) imaging of the retina, also termed as
optoretinogram or optoretinography (ORG), promises a non-invasive
method for the objective assessment of retinal function. By providing the
unparalleled capability to differentiate individual retinal layers, functional
optical coherence tomography (OCT) has been actively investigated for
intrinsic signal ORG measurements.
Retinal diseases are often quite advanced before they draw clinical
attention, by which time the retina may be functionally abnormal.
Structural and functional abnormalities in the retina are often not
correlated in the spatial location and time window. Therefore, an objective
method for functional assessment of the retina promises early detection and
longitudinal therapeutic assessment of retinal degenerative diseases.
Time-lapse light microscopy and fundus camera have been used for two-
dimensional (2D) IOS imaging study of isolated retinal tissues and intact eyes (21, 22,
28). By providing the unparalleled capability to differentiate individual layers of the
retina, OCT has been actively used for IOS imaging of animal and human retinas (25,
29, 31–45).
(B) Optoretinography reveals functional activity in cone outer segments.
Illumination pattern (three bars) drawn to scale over the line-scan
ophthalmoscopic image. (C,D) The spatial map of OPL changes between the
ISOS and COST before (C) and after stimulus (D), measured at 20-Hz volume
rate. Reprinted with permission from Pandiyan et al. (32).

ORG for vision research as we
Jessica Morgan (2022): Optoretinography and retinal imaging in retinal health and disease
using adaptive optics scanning laser ophthalmoscopy (Cooper et al. 2017)

ORG can be done with clinical-grade OCTs (no AO-OCT needed)
Kari V Vienola et al. (2022): “Phase-based optoretinography with
clinical-grade OCT using tissue velocity” (ARVO 2022), see also
the paper)
Here we present a new objective test capable of measuring functional
responses from retinal neurons, while simultaneously acquiring
microscopic structural images of the same tissue. This method has the
potential to transform the field of clinical retinal disease diagnosis and
to accelerate future drug developments.
To our knowledge, the ORG is the only noninvasive, objective test of neural
function in the retina that can simultaneously reveal its structure,
making it ideal for ophthalmic care and clinical research. However, the
advanced imaging systems used to prove the ORG concept pose some
challenges for clinical translation.
OCT system used a 1060 nm swept-source (SS-OCT; Axsun; Billerica, MA, USA),
with a 100 kHz A-scan rate and 100 nm bandwidth. The results are consistent
with previous ORG responses acquired from photoreceptor's outer segments
using adaptive optics OCT (AO-OCT). Including time for dark adaptation,
imaging, and processing, functional responses can be measured and
visualized within ten minutes providing a feasible clinical pipeline for
larger scale ORG studies.

ORG some interest from clinicians
RETINA CONVERSATIONS: A Conversation About Optoretinography
Ravi S. Jonnal, PhD, and Glenn Yiu, MD, PhD, describe new imaging innovations that could more easily
assess retinal function at the photoreceptor level. By: Jennifer Ford, senior managing editor RETINAL
PHYSICIAN APRIL 1, 2023VOL 20, ISSUE APRIL 2023
https://retinalphysician.com/issues/2023/april/retina-conversations-a-conversation-about-optoretinography/

Erythrocyte-mediated angiography (EMA)
Breanna Tracey et al. (2019)
ICG Angio -> Erythrocyte-mediated angiography (EMA).
AO+EMA for visualizing choriocapillaries. DIstribution of stasis
as “a biomarker” for healthy vs pathological eyes (Li, NIH),
see Gu et al. (2018)
Chen: Plexus-specific retinal erythrocyte velocity: “ In
humans and NHPs, erythrocyte decelerate in arterioles then
accelerate in venules as expected. We have shown that
blood flow in the SVP, ICP, and DCP can be precisely
quantified and can differ between plexuses. Furthermore,
elevated IOP results in decreased erythrocyte velocity and
acceleration.”

Retinal Vessel Analyzer Neurovascular Coupling from fundus
Retinal Vessel Analyzer, https://imedos.com/?lang=en
Tomasso et al. (2017): “Retinal vessels functionality in eyes
with central serous chorioretinopathy”

Retinal Neurovascular Coupling (NVU) Imaging
Pierre Senée et al. (2022): “High resolution pattern projection in the retina for phase contrast imaging”
annulus is
the
projected
pattern

Laser speckle contrast imaging (LSCI) for retinal hemodynamics
Jin et al. (2022): “Laser speckle contrast imaging derived retinal hemodynamics
abnormalities in Alzheimer's disease”
Kyoung-A Cho et al. (2020):
“Portable, non-invasive video
imaging of retinal blood flow
dynamics”
DeBuc et al. (2021): “Use of XyCAM RI for Noninvasive Visualization
and Analysis of Retinal Blood Flow Dynamics During Clinical
Investigations”

Next Gen Ophthalmic Imaging for Neurodegenerative Diseases and Oculomics

Next Gen Ophthalmic Imaging for Neurodegenerative Diseases and Oculomics

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