Possible future avenues for ophthalmic imaging combining advanced techniques and deep learning. "Bubbling under the surface, and inspiration from ‘bioimaging’ in general"

1. Petteri Teikari, PhD
Singapore Eye Research Institute (SERI)
Visual Neurosciences group
http://petteri-teikari.com/
Version “Mon 7 May 2018“
Advanced
Retinal Imaging
Bubbling under the
surface, and inspiration
from ‘bioimaging’ in
general
Collage by Rodrigo de Filippis

2. About the
Presentation
Who is this for: Biophotonics enthusiasts, i.e. biomedical optics
engineers and scientists interested in bringing something new to
ophhtalmic imagingfrom other bio-optics fields.
Presentation type: Snippets of the literature to give a quick
overview of the existing techniques without going too in-depth
analysis.
Adaptive opticsOCT– Cellular resolution retinal imaging
https://zmpbmt.meduniwien.ac.at/wissenschaft-forschung/optical-imaging/advanced-imaging-technologies/adaptive-optics-oct/

3. That was just
plain old
fundus
photography
and OCT
https://www.slideshare.net/PetteriTeikariPhD/portable-retinal-imaging-and-medical-diagnostics

4. Retinal
Imaging
Multimodal
imagingforbetter
clinicaldiagnostic
power
E
ColorFundus Red-free Fluorescence
Angiography
Fundus
Autofluorescence
MultiFunction RetinalCameraTopcon TRC-NW8Fplus
http://a20121031152928.sub.redwall.com.tw/index.php/web/pro_i/480
Addedvalueofinfrared,red-freeand
autofluorescencefundusimagingin
pseudoxanthomaelasticum
JulieDeZaeytijdetal.British Journal of Ophthalmology2010; 94
http://dx.doi.org/10.1136/bjo.2009.162644
Aspect of feathered type of angioid streaks in the right eye of
young female patient 8 on white light (A), infrared (B),
autofluorescence (C) and red-free imaging (D); note the better
visibility of the streaks as whitish areas on near-infrared
reflectance imaging and red-free imaging than on white light
fundus imaging; streaks appear as dark areas on the
autofluorescenceimage.
A B C D
In AREDS2, the best digital images
matched the best film. Overall, however,
digital provided lower contrast of retinal
detail. Digital images taken with higher
G-to-R ratio showed better brightness
and contrastmanagement.
- Hubbardetal. (2008)

5. MultimodalOCT
Imaging
3D OCT becoming the defacto
standard forretinal imaging as it
offers better diagnostic
capability (e.g. for glaucoma,
Blumberg et al. 2016)
Multimodal imaging of the right eye of a 54-year-old female from the control group.
The white line in the CF image (A) designates the scan line of the PS-OCTB-scan image
(E–I). Neither the NIR image (B), NIR-AF image (C), SW-AF image (D), norstandard OCTB-
scan image (E) provides information about the melanin density in the choroid. The DOPU
B-scan image (F) and composite DOPU B-scan OCT image (G) show the melanin
distribution in the RPE and choroid. Binarization was applied for the standard OCT B-scan
image to detect the choroidal interstitial area (H) and red lines indicate manuallysegmented
lines of the choroid (H). The red lines in the B-scan image for the calculation of percentage
area of low DOPU (I) show the boundary of the choroidal interstitial stroma. The scale bar in
B-scan images represents 500 μm × 500μm.
Polarization-SensitiveOptical CoherenceTomographic
DocumentationofChoroidal MelaninLossin Chronic Vogt–
Koyanagi–HaradaDisease Miura etal. (Sept2017) doi:10.1167/iovs.17-22117
Optical coherencetomography angiography: an overview ofthe
technology andan assessmentofapplicationsforclinical research
AndrewKoustenisJr etal. (2016) doi: 10.1136/bjophthalmol-2016-309389
The resulting outputs of OCT-
A offer improvements over
that of alternative imaging
modalities. Compared with
Heidelberg retinal flowmetry
(HRF), OCT-A offers insight to
vessel density, whereas HRF
only provides insight into
blood flow velocity and the
amount of avascular zone
presentina field of view
Process of bandwidth segmentation, used by the
split-spectrum amplitude decorrelation angiography
algorithm to increase the signal-to-noise ratio. The
bandwidth was segmented into four sections to
maintain appropriate image resolution(Jia et al. 2012)
While OCT-A has advantages over other imaging modalities, it is important to
recognise limitations. For example, it must be acknowledged that OCT-A
specifically analyses the posterior retinal and choroidal microvasculature. While a
specialised adaptor to image capillaries in the anterior segment has been
developed (Anget al.2015)
, not all of the relevant ocular vasculature is imaged, such as
the retrobulbar vessels. Also, if one is interested in deeper vessels of the retina,
one must be aware that the super- ficial vessels may obscure the layer of interest
(Jia et al.2012)
By providing high-resolution images and vessel density quantification
of the retina and the optic nerve extending into the lamina cribrosa,
understanding of the retinal vascular component in
neurodegenerative diseases may be furthered with OCT-A.
Advancing the understanding of the pathophysiology of diseases
with retinal vascular components allows for more effective
treatmentof diseasesand management of vision and health.
Yarmohammadi et al. (2016)

6. Retinal
spectral
properties

7. Spectral
Sensitivity
Photoreceptors
Teikari thesis (2012)
Enezi et al. 2011.
Stockmann And Sharpe (2000), CVRL
Govardovskii et al. 2000
Note! Melanopsin and S-cones do not seem to
contributetocentral vision luminance perception
Quantally defined daytime sensitivity (2º central
vision, Sharpe etal., 2005): V*(l) = [1.891·l(l) +
m(l)]/2.80361
Where l islong-wavelength ('red') cone sensitivity,
and m medium-wavelength (green) cone sensitivity
Stockman, A., & Sharpe, L. T. (2008). Spectral sensitivity InThe
Senses:A Comprehensive Reference, Volume 2:Vision II (pp. 87-
100)

8. Spectral
Sensitivity
“Non-idealities in
photoreception” Self-screening effect changes the width/peak of the
absorption spectrum. (A) Percentage absorption spectra of various
concentrations of photopigment (OD - optical density in log units). (B) An
illustration of self-screening in at various photoreceptor lengths. Human rod
photoreceptor is ~25 mm, (Pugh and Lamb 2000) and the cone
photoreceptor 13 mm (Baylor et al. 1984). The longest known
photoreceptor has been found in dragonfly, the length being 1,100 mm (
Labhart and Nilsson 1995).
The sidelobe on the short-wave
side come from the beta band
(see template from
Govardovskii et al. 2000)
The absorbance spectrum of an exemplary vertebrate rhodopsin
(lmax ~ 500 nm), considered as a sum of absorbance bands,
indicated by alpha (a), beta (b), gamma (g), sigma (s) and epsilon
(e) normalized to the peak absorbance of the alpha-band (after
Stavenga and van Barneveld 1975, from Stavenga 2010).
Without the
crystalline lens
(aphakic eye), visual
sensitivity would
extend to
ultraviolet
Goodeve et al., 1942

9. Ocular spectral
properties

10. Ocular
Media
Crystalline lens
and cornea
vandeKraats and van Norren (2007): “Optical densityof the aging human ocularmedia in
the visible and theUV”. J. Opt. Soc. Am. A/ Vol. 24, No. 7/ July2007
https://doi.org/10.1364/JOSAA.24.001842 | Cited by124 articles
PetteriTeikari, Raymond P. Najjar, Kenneth Knoblauch, Dominique Dumortier, Pierre-
Loïc Cornut, Philippe Denis, Howard M. Cooper and ClaudeGronfier "Refined flicker
photometrytechniqueto measureocular lens density" J. Opt. Soc. Am. A Vol. 29, Issue
11, pp. 2469-2478 (2012) https://doi.org/10.1364/JOSAA.29.002469
Human crystalline
lensgetsmore
yellowwith age

11. Macular
Pigment
Across the macula, macular protective pigment (MPP) distribution
takes the form of a mountain, peaking centrally at the foveola and
declining to nil at an eccentricity of 7°. L – lutein, Z – zeaxanthin.
optometricmanagement.com
Recovered spectra for one normal subject. The four spectra recovered by NMF. The
fourth spectrum denotes the MP spectrum (solid line). The two peaks between 450
and 500 nm are the classic bifid spectrum previously reported (Hammond etal.2005) in
vitro.Thesecondarypeaksat 425nmhavealso beenreported invitro. - Fawzi etal. (2011)
Clinical imagingof macularpigment optical densityand
spatialdistribution
Christopher M Putnam | College ofOptometry,University of Missouri-St Louis
Clin Exp Optom, 100: 333–340. doi: 10.1111/cxo.12500
Spectralis optical coherence tomography (OCT)
provides a cross-section of the central retina of a
healthy human subject. The layers of the retina have
been identified with arrows. The vertical
distribution pattern of macular pigment (MP) is
identified primarily within the photoreceptor
axons that comprise the outer plexiform layer (OPL),
the inner plexiform layer (IPL) and Henle fibre layer
within the macula. Lesser concentrations of macular
pigment have also been identified at the level of the
retinal pigment epithelium (RPE) and photoreceptor
outer segments.
Macular pigment is deposited preferentially in the fovea in the Henle fiber layer which
consists of the foveal cones’ axons, and in the parafovea, macular pigment is also located in
the inner plexiform layers of the retina (Snodderly, Auran &Delori,1984;
Trieschmann,etal., 2008).
Macular pigment optical densitymap of oneeye included in
the study. - VerônicaCastroLimaet al. (2013)

12. Irisand
Ocular
Wall
“In special cases such as with intraocular straylight measuruments (
Ijspeertetal. 1990; vanden Berg etal.2009; Michael etal.2009), the
transmural (ocular wall) and iris transmittance need to be explicitly
addressed in contrast of assuming them to be light-tight structures (
van den Berg et al. 1990). The translucency of iris and the ocular wall are
exploited by ophthalmologists when performing diaphanoscopy (cf
Greenwood 1913), in which a light guide is positioned against the sclera
andthepupil isseentoglowfromwithin (La Heyetal.1993).
Vanden Berg etal.1991 estimated the irises of blue-eyed individuals to
attenuate the red light only 0.72 log units and the green for 1.48 log units,
whereas the corresponding attenuation values were 2.27 for red and 2.64
log unitsforgreen lightin brown-eyed individuals.
In addition to the translucency of the iris and the surrounding ocular wall,
fundal reflections (Vos1963; van deKraatsand vanNorren2008) might
contribute to the pigmentation-related differences. Furthermore, the iris
pigmentation have been shown to correlate directly with choroidal
pigmentation (Weiteretal. 1985) and to be reduced with aging (
Schmidtand Peisch 1986).”
SPEED:SPectraleyevidEo database
AnaGebejes,PauliFält, RomanBednarik, MarkkuHauta-Kasari
University ofEastern Finland,Joensuu,Finland
UbiComp '16 https://doi.org/10.1145/2968219.2968335
Ahyperspectralimagingsystemforthe
evaluationofthehuman irisspectralreflectance
Luca Di Cecilia;FrancescoMarazzi;Luigi Rovati
Univ. ofModenaandReggioEmilia, Italy
SPIE BiOS,2017,doi: 10.1117/12.2252184
Hyperspectral opticalimagingofhuman
irisin vivo: characteristicsof
reflectancespectra
JoseM.Medina, LuísM. Pereira,Helder T.Correia,andSérgio M.C.
Nascimento,UniversityofMinho, Portugal
Journal of Biomedical Optics16(7),076001 (July 2011)
Reflectance factor as a function of the wavelength
measured with the hyperspectral system. Reflectance
data correspond to (a) dark (orange), (b) light
pigmented iris (cyan)
Our study provides evidence for hyperspectral imaging
being suitable for the characterization of melanin and the
noninvasive diagnosis ofoculardiseases and iris color.
Iris reflectances in thevisible/near-infrared spectral region.
Light blue iris (blue line), hazelnut-green iris (green
line)and darkbrown iris (brown line).
(right) RGB image generated form the liquid crystal
tunable filter (LCTF) spectral image; Bottom – spectral
signatures of the points selected from the spectral image.
These are mean spectra from a 10x10 pixel areas sampled
fromthefeatures ofinterest marked on theRGB image.

13. Sclera
Vogel et al. (1991):Optical Properties ofHuman Sclera, and TheirConsequences for
Transscleral Laser Applications. LasersinSurgeryand Medicine11:331340(1991)

14. Eyelid
M. J. Moseley, S. C. Bayliss and A. R. Fielder (1988) Light transmission
through the eyelid:doi:10.1111/j.1475-1313.1988.tb01043.x
Spectral transmittance of arbitrary unit amounts of hemoglobin,
melanin, and bilirubin used for predicting the spectral transmittance of
eyelidskin.
Bierman etal. (2011): Measuring and predicting eyelid spectral
transmittance.J.of BiomedicalOptics,16(6),
MillisecondFlashesofLightPhase Delay theHuman Circadian
Clockduring Sleep JamieM.Zeitzer,RyanA. Fisicaro, Norman F. Ruby,H.
Craig Heller.StanfordUniversity.Journal of Biological Rhythms201429(5): 370-
376. doi: 10.1177/0748730414546532
“Confirmation that the flashes penetrated the
eyelids is presented by the occurrence of an
evokedresponseintheEEG.“

15. Multispectral
Imaging

16. Fundus
Multi/Hyperspectral
Imaging
“Hyperspectralimaging
measurescontiguousspectral
bands,asopposedto
multispectralimagingwhich
measuresspacedspectralbands.”
AnnidisRHA Multispectralfundusimagingsystem
http://www.annidis.com/page/technology
Multi-spectral imaging forin vivo imaging
ofoxygentensionand -amyloidβ
Dr. TosTJM Berendschot,Prof.dr. Carroll AB
WebersUniversityEyeClinicMaastricht – PhD
Project
Li, Shanshan, etal."In VivoStudy ofRetinal
TransmissionFunction in Different
SectionsoftheChoroidal Structure Using
Multispectral Imaging" Investigative
Ophthalmology&Visual Science56.6 (2015): 3731-
3742.doi: 10.1167/iovs.14-15783
Lian, Jian, et al. "Measuring Spectral
Inconsistency of Multispectral
Images for Detection and
Segmentation of Retinal
Degenerative Changes." Scientific
Reports 7 (2017). doi:
10.1038/s41598-017-11730-y
A collection of MSI images captured by Annidis RHA from a patient aged 60 and diagnosed with diabetic retinopathy.
Ordered from left to right and from top to bottom, the first 11 sequential images are captured with short wavelengths of
MSI-550, MSI-580, MSI-590, MSI-620, MSI-660, MSI-690, MSI-740, MSI-760, MSI-780, MSI-810 and MSI-850,
respectively.

17. Multi/Hyperspectral
Imaging
Oximetryin general, a
goodexample ofthe
utilityofspectral in
biomedical
engineering
Photoplethysmgraphy (PPG)-based technique for pulse oximetry (left) Oxygen
saturation is defined as the measurement of the amount of oxygen dissolved in
blood, based on the detection of Hemoglobin (HbO2) and
Deoxyhemoglobin (HbR). (right) A photodetector in thesensor perceives the
non-absorbed light from the LEDs. The DC component represents the light
absorption of the tissue, venous blood, and non-pulsatile arterial blood. The AC
component represents the pulsatile arterial blood. - Ahmed etal. (2014);
Abayand Kyriacou (2017)
Photoplethysmgraphy with an IR distance sensing IC (e.g. Vishay
VCNL3020, ~£1.30/100pcs) (left) Structural features of digital
optical proximity sensors, and they can be used for PPG (
Chandrasekaretal.2012), (right) Basic photoplethysmographic
(PPG) approach to measuring heart rate
http://embedded-lab.com/blog/?p=5508.
Hyperspectral images showing mouse foot pad and comparing the blood
oxygenation of a ischemic foot and a control foot
https://www.quora.com/Which-method-theoretical-or-not-analyses-blood-without-extracting-it-from-the-vein
Absorption spectra of major endogenous contrast agents in biological tissue at
normal concentrations. Oxy-hemoglobin (HbO2) and deoxy-hemoglobin
(HbR), 150g/L in blood; lipid, 20% by volume in tissue; water, 80% by volume in
tissue; DNA and RNA, 1g/L in cell nuclei; melanin, 14.3g/L in medium human skin;
reduced myoglobin (MbR) and oxy-myoglobin (MbO2), 0.5% by mass in skeletal
muscle;bilirubin, 12mg/Lin blood. - Yao and Wang (2014)
Optical heart ratesensor
on Samsung phones

18. Multi/Hyperspectral
Imaging
Especially useful for retinal
vasculature measurements
Going beyond structural
measurementswith “standard
OCT angiography
Age-Related AlterationsintheRetinal
Microvasculature,Microcirculation,and
Microstructure
Yantao Wei etal. IOVS 2017, Vol.58, 3804-3817.
doi:10.1167/iovs.17-21460
RetinalMicrovascular Networkand
MicrocirculationAssessmentsinHighMyopia
Li etal AmJ Ophthalmol.2017Feb;174:56-67.doi: 10.1016/j.ajo.2016.10.018
Department of Ophthalmology Bascom PalmerEye Institute
OCT-angiography:Aqualitativeandquantitative
comparisonof 4OCT-Adevices
MarionR. Munk | Department ofOphthalmology,Inselspital,Bern University Hospital
PLoS ONE12(5): e0177059. https://doi.org/10.1371/journal.pone.0177059
Theage-related
changesand interlink
among retinal
microstructure,
microvasculature, and
microcirculation. Overall,
aging played a roleinthe
thinning of theretinal
innersegment,lossof
retinalmicrovasculature,
and decreased venular
blood flowvelocity
(BFV). Thechangesin
microvasculaturewere
relatedto thinning of the
innerretinaand venular
blood flow.
Deep capillary
plexus Each ofthefour
evaluated OCT-A modules had
particularstrengths, which
differentiated it from their
competitors.
Superficial capillary
plexus TopconDRI-OCTTriton
Swept-sourceOCT, Optovue
RTVue-XR, a prototype Spectralis
OCT2, Heidelberg-Engineering and
Zeiss Cirrus 5000-HD-OCT

19. Multi/Hyperspectral
Imaging
In some cases the clinically
relevantinformation iseasier
toacquire with multiple
spectral bandsbeyond typical
RGB color imaging.
Oxygensaturationmeasurementsof the
retinalvasculatureintreatedasymmetrical
primary open-angleglaucomausing
hyperspectralimaging
Mordant etal.(2014) Eye 28, 1190–1200;
doi: 10.1038/eye.2014.169; July2014
Pseudo-color images of the oxygen saturation values along the
retinal arterioles (a, left) and venules (b, left) in a normal eye.
Mean optical density spectra (red filled circles and whiskers),
non-linear fitting oximetry algorithm (red line), and oxygen
saturation calculations of respective selected vessel segments
areshown in the plotstothe right.
Spatialand SpectralCharacterizationof Human
RetinalPigmentEpitheliumFluorophoreFamiliesby
Ex VivoHyperspectral AutofluorescenceImaging
Tal Ben Ami etal. (2016) Invest.Ophthalmol.Vis.Sci.. 2016; 57(12):54.
doi: 10.1167/tvst.5.3.5
Nonnegative matrix factorization (NMF) spectral output (36-year-old
male, near-periphery). S0 is the usual BrM spectrum; SX is a second BrM
spectrum. S1, S2, and S3 are the basic lipofuscin/melanolipofuscin
(LF/ML) spectra.
Hyperspectral AutofluorescenceImagingOf Drusen
And RetinalPigment Epithelium InDonorEyesWithAge-
relatedMacularDegeneration
Tong etal. (2016) Invest. Ophthalmol. Vis. Sci..2016; 57(12):54.
doi: 10.1097/IAE.0000000000001325
large
drusen
small/interm.
drusen

20. Multi/Hyperspectral
Imaging
Amyloid- astheβ
biomarkerfor
Alzheimer’sDisease?
Structural imaging of
retinal amyloid plaques ,for
Alzheimer disease diagnosis.
With or withoutlabeling
Label-freeimagingofamyloidsusing their
intrinsic linearand nonlinearopticalproperties
Patrik K. Johansson and Patrick Koelsch
Biomedical OpticsExpressVol.8, Issue2, pp. 743-756 (2017)
https://doi.org/10.1364/BOE.8.000743
The optical properties of amyloid fibers are often distinct from those of the source
protein in its non-fibrillar form. These differences can be utilized for label-free imaging
or characterization of such structures, which is particularly important for understanding
amyloid fiber related diseases such as Alzheimer’s and Parkinson’s disease.
We demonstrate that two amyloid forming proteins, insulin and -lactoglobulin ( -LG),β β
show intrinsic fluorescence with emission spectra that are dependent on the excitation
wavelength. Additionally, a new fluorescence peak at about 430 nm emerges for -LGβ
in its amyloid state. The shift in emission wavelength is related to the red edge
excitation shift (REES), whereas the additional fluorescence peak is likely
associated with charge delocalization along the fiber backbone.
Thepresented results suggest that amyloiddepositscanbeidentified and
structurally characterized basedontheirintrinsicopticalproperties, whichis
important for probe-less and label-freeidentification and characterization ofamyloid
fibers invitro and in complex biological samples.
EarlyDetectionof AmyloidopathyinAlzheimer's
MicebyHyperspectralEndoscopy
More etal. (2016) Invest. Ophthalmol. Vis. Sci..June2016, Vol.57, 3231-3238.
doi: 10.1167/iovs.15-17406 | Cited by7
Optical spectra recorded from
humanand mouseretina
samples. (A) Comparisonof
spectral shapes from
Alzheimer's and normal retina.
(B) Micespectra from(A)
shown withstandard
deviations at eachwavelength.
At wavelengths lower than 570
nm, error bars fromeach mean
spectrumdo not overlap.
Amyloid-betaintherodentretina exhibitsa
characteristichyperspectralprofile
ChristineTramOanhNguyen etal.(2016)
ARVO Annual Meeting Abstract | September2016
http://iovs.arvojournals.org/article.aspx?articleid=2560704
“In vitro andin vivo exogenous preparations ofA exhibitsimilarhyperspectral reflectanceβ
profiles to 5xFAD mice, all producing consistent differences at 459 to 481nm when
compared to their respective controls. This opens the possibility of using hyperspectral
imaging as a non-invasive tool to detect A in retina in theabsence ofcontrastmedium. “β
Hyperspectralimagingsignaturesdetect
amyloidopathyinAlzheimer's mouseretinawell
beforeonset of cognitivedecline
More etal. (2014) ACS Chem.Neurosci., 2015,6 (2), pp 306–315
DOI: 10.1021/cn500242z |Citedby14
Amyloidosis inRetinalNeurodegenerativeDiseases
Masuzzoetal. (2016)FrontNeurol. 2016; 7: 127.
doi: 10.3389/fneur.2016.00127| Cited by3
bovinemilk (a–f) and bovine insulin (g–l)
Preparationand characterizationofamyloidfibersandspherulites. (a)Insulin
and -lactoglobulin formfibers or spherulites, depending on theexperimentalβ
conditions.

21. Multi/Hyperspectral
Imaging
Amyloid‘labelengineering’
leadingtobetterdrug
targetingaswell?
Structural imaging for
retinal amyloid plaques ,for
Alzheimer disease diagnosis
PhotochemicalIdentificationofMolecular
BindingSitesontheSurfaceofAmyloid-β
FibrillarAggregates
Amir AliyanThomasJ. Paul, BoJiang, Christopher Pennington,
Gaurav Sharma, Rajeev Prabhakar, Angel A. Martí
Chem (2017)https://doi.org/10.1016/j.chempr.2017.09.011
FromPhys.org: A probe invented at Rice University that lights up when it
binds to a misfolded amyloid beta peptide—the kind suspected of causing
Alzheimer's disease—has identified a specific binding site on the protein
that could facilitate better drugs to treat the disease. Even better, the lab has
discovered that when the metallic probe is illuminated, it catalyzes oxidation
of the protein in a way they believe might keep it from aggregating in the
brainsof patients.
Finding the site was relatively simple once the lab of Rice chemist Angel
Martí used its rhenium-based complexes to target fibrils. The light-switching
complex glows when hit with ultraviolet light, but when it binds to the fibril it
becomes more than 100 times brighter and causes oxidation of the amyloid
peptide.
"There's an interest in finding medications that will quench the deleterious
effects of amyloid beta aggregates," he said. "But to create drugs for these,
we first need to know how drugs or molecules in general can bind and
interact with these fibrils, and this was not well-known. Now we have a better
idea of whatthemoleculeneedsto interactwiththesefibrils."
"It's a very attractive system because it useslight, which is a cheap resource,"
Martí said. "If we can modify complexes so they absorb red light, which is
transparent to tissue, we might be able to perform these photochemical
modificationsin living animals, and maybesomedayinhumans."
RiceUniversity|DiscoverylightspathforAlzheimer'sresearch
https://youtu.be/fmqKs1rEliE
AsystemicviewofAlzheimerdisease—
insightsfromamyloid- metabolismbeyondβ
thebrain
Amir AliyanThomasJ. Paul, BoJiang, Christopher Pennington,
Gaurav Sharma, Rajeev Prabhakar, Angel A. Martí
Chem (2017) https://doi.org/10.1016/j.chempr.2017.09.011

22. Multi/Hyperspectral
Imaging
Labeling retinal amyloid
beta invivo with
curcumin
1 g curcumin (4g of Longvida®,
8capsa day) wastaken once
aday, either for 2 daysor for 10
daysprior of imaging
Retinal amyloid pathologyandproof-of-concept
imagingtrialinAlzheimer'sdisease
Koronyo etal. (2017) JCI Insight. 2017 Aug17;2(16).pii: 93621.
Departmentof Neurosurgery, MaxineDunitzNeurosurgicalResearchInstitute,Cedars-SinaiMedicalCenter,Los
Angeles; NeuroVisionImagingLLC; DohenyEye Institute
doi:10.1172/jci.insight.93621
Noninvasive detection of retinal amyloid deposits
in live AD patients (A) Multistep manual postacquisition
image processing and analysis to detect and quantify spots
with increased curcumin fluorescence signal in the retina (B)
Curcumin fluorescence fundography (C) Higher-
magnification image (D and E) Representative optical
coherence tomography (OCT) of a selected curcumin-
positive regioninan AD patientwith nomaculopathy
Increased retinal amyloid index in ADpatients—a proof-of-concept
human trial.
The geometric distribution and increased burden of retinal amyloid pathology in
AD, together with the feasibility to noninvasively detect discrete retinal amyloid
deposits in living patients, may lead to a practical approach for large-scale AD
diagnosisand monitoring.
“In a proof-of-concept retinal imaging trial (n = 16), amyloid
probe curcumin formulation was determined and
protocol was established for retinal amyloid imaging in live
patients”

23. Multi/Hyperspectral
Imaging:
DeviceDesigns
Portable fastand compact
cameradesigns
Fastandcompact internalscanning
CMOS-basedhyperspectralcamera:
the Snapscan
JulienPichette; WouterCharle; AndyLambrechts
IMEC(Belgium); SPIEOPTO2017
doi: 10.1117/12.2253614
BayerFilterSnapshot Hyperspectral
FundusCameraforHumanRetinal
Imaging
JoelKaluznyetal.,CurrEyeRes42,2017 – Issue4
http://dx.doi.org/10.1080/02713683.2016.1221976
Normalized macular pigment optical densities (yellow) overlaid with
contrast-enhanced retinal vessel maps (red) from (a) a 24-year-old
volunteer and (b) an 80-year-old volunteer. Field of view, 35°.
Clinical imaging
system.
(a)ThecompactImec
snapshothyperspectral
detector(inset)is
integrated witha
commercial fundus
camera. (b)Optical
schematic of detector
and relaylenssystem.
(c)Diagramof the
detectorwith thefilter
mosaics. Each filter
mosaic isa4× 4array of
filters printedon a4× 4
area of pixels.
Miniaturemultispectralcameraandits
applicationinmachinevision
Cui Ma ; Hui Lin ; GuodongZhang ; RuxuDu
Information and Automation (ICIA),2016IEEE
https://doi.org/10.1109/ICInfA.2016.7831890

24. Multi/Hyperspectral
Imaging:
DeviceDesigns
Smartphone-based
hyperspectral sensing
VTTcreateshyperspectraliPhonecamera
30Nov 2016
The adjustable tiny MEMS filter
is integrated with the camera
lens and its adjustment is
synchronised with the camera's
imagecapturesystem.
Rissanenetal.2016, 2017
Video demo:
https://youtu.be/OcAstSnbVX4
Koronyo-Hamaoui etal. (2011) used
curcumin to label amyloid plaques
(a biomarker or not in Alzheimer
disease) from the retina, and AOTF-
based multispectral imager from
ChromoDynamics
Could youget OptosUltraWide-
Field typeof opticstoa
smartphone?
Wide field scanninglaser
opthalmoscope
DCAnderson,RA Lucas,R
Henderson -OptosPlc
US Patent 5,815,242,1998
Retinalimagingapparatus
andmethod
DGray,SPemberton,DSwan,M
Thomson -OptosPlc
USPatent9,743,831,2017
Ultrawide-fieldoptical
coherencetomography
JMMHorn,MAndre,CJRVBaker,
OWIRTH -CarlZeiss
USPatent9,427,151,2016
Exampleapplication:
Amobilephone-based retinal
camera forportablewidefield
imaging
Robi NMaamari JeremyDKeenan,Daniel A
Fletcher, Todd P Margolis
http://dx.doi.org/10.1136/bjophthalmol-2013-303797
Cited by 66
British Journal ofOphthalmology2014;98:438-441.
“Retinalfield-of-viewof
approximately55°”
Trans-palpebralillumination:
anapproachforwide-angle
fundusphotographywithout
theneed forpupildilation
DevrimToslak, DamberThapa, Yanjun Chen,
MuhammetKazimErol,R.V. PaulChan,and
Xincheng Yao
https://doi.org/10.1364/OL.41.002688
Optics Letters Vol. 41, Issue 12, pp. 2688-2691 (2016)
“Retinalfield-of-view(interiorangle
of152°,and exteriorangle105°”

25. NeurovascularUnit
(NVU)functioninthe
retina

26. Multi/Hyperspectral
Imaging
Vasculature analysis isahot
topicwith fractal dimension
(“structure”),blood flow and
functional imagingstudying
neurovascular coupling
(“function”)
First peakfractal analysisof opticalcoherence
tomographyangiography in glaucomatouseyes
Bing Q Chiu;Edmund Tsui; SarwarZahid;Nicole K Scripsema; Emma Young;
PatriciaM. Garcia;Joseph Panarelli;Paul A Sidoti;Richard B Rosen;JoshuaA
Young. ARVO AnnualMeetingAbstract | June2017
http://iovs.arvojournals.org/article.aspx?articleid=2638810
Retinal hemodynamicoxygen reactivity
assessed byperfusion velocity,blood oximetry
and vesseldiametermeasurements
OliverNiels Klefter, Anne Øberg Lauritsen, Michael Larsen Acta
Ophthalmologica2014 http://doi.org/10.1111/aos.12553
To test the oxygen reactivity of a fundus photographic method of
measuring macular perfusion velocity and to integrate macular
perfusion velocities with measurements of retinal vessel diameters
and blood oxygen saturation.
Breakdown of self-similarity for POAG occurred at significantly higher
resolution than that of controls or NTG, and more so for moderate to
severe POAG subgroups, suggesting their increased loss of
microvasculature and highlighting the potential of fractal analysis in
establishing quantitative parameters for evaluation of glaucoma.
Spectroscopicoximetry in the eye: areview
Lewis. E. MacKenzie, Andy. R. Harvey, Andy. I. McNaught Expert Review of
Ophthalmology Volume 12, 2017 - Issue 4doi: 10.1080/17469899.2017.1318067
Retinal oximetry has provided insights into the development of
diabetic retinopathy and glaucoma, and may enhance the evaluation
and treatment of retinal vessel occlusion. The development of more
sophisticated phantoms that resemble in vivo environments has
helped validate oximetry applications. New insights into ocular
physiology and disease are likely to be gleaned from future studies.
Neurovascular coupling is a process through which neuronal activity
leads to local increases in blood flow in the central nervous system.
Our findings demonstrate that hyperoxia does not alter neurovascular
coupling in vivo, ensuring that active neurons receive an adequate
supply of nutrients.
Oxygen modulation of neurovascularcoupling in
the retina
Anusha Mishra, ArifHamid, and Eric A. Newman PNAS vol.108 no. 43 doi:
10.1073/pnas.1110533108
The evidence supports a conceptual shift in the mechanisms of neurovascular
coupling, from a unidimensional process involving neuronal-astrocytic signaling
to local blood vessels to a multidimensional one in which mediators released
from multiple cells engage distinct signaling pathways and effector systems across
the entire cerebrovascular network in a highly orchestrated manner. The recently
appreciated NVU dysfunction in neurodegenerative diseases, although still
poorly understood, supports emerging concepts that maintaining neurovascular
health promotes brain health.
The NeurovascularUnit Coming of Age:A
Journeythrough NeurovascularCoupling in
Health and Disease
Costantino Iadecola Neuron vol. 96issue 1, p17-42
doi: 10.1016/j.neuron.2017.07.030

27. Multi/Hyperspectral
Imaging
Visualization ofwhatcould be
measured from neurovascular
unitdysfunction and structural
changesvia fractal analysis.
Thepipelinefor calculatingthefractaldimensionfromacolor
fundusimage.Fan Huang etal.(2016): “Reliability of Using Retinal Vascular
Fractal Dimension as a Biomarker in the Diabetic Retinopathy Detection”
Each line represents the mean of 12 retinal vessel diameters of all 12
participants average flicker profiles for arteries (top panel) and veins
(bottom panel) for each flicker duration (5, 7, 10, and 20
seconds). Vertical gray line denotes flicker onset; horizontal gray
line denotes baseline diameter normalized to 100%.
Heitmar and Summers (2015): “Reliability of Using Retinal Vascular Fractal
Dimension as a Biomarker in the Diabetic Retinopathy Detection”
A schematic representation of the neurovascular unit (NVU) showing cellular
elements regulating cerebral blood flow along the vascular tree. - Kisler etal. (2017):
Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease
Network model for the retinal vasculature. The vasculature is divided into
five main compartments: the CRA, arterioles, capillaries, venules, and the CRV. Each
compartment includes resistances (R) and capacitances (C). Diameters of venules and
intraocular and translaminar segments of the CRA and CRV are assumed to vary passively with
IOP, whereasarteriolesare assumed to be vasoactive. - Guidoboni et al. 2014

28. NeurovascularUnit
imagingfromanimal
brain
’Traditional techniques’

29. NeurovascularUnit
imagingfromanimal
brain
Multimodal multi-photon and
functional ultrasound imaging
Understandingthe neurovascularunit
atmultiple scales:advantagesand
limitationsofmulti-photonand
functionalultrasoundimaging
Alan Urbanetal. (2017)| AdvancedDrugDeliveryReviews
https://doi.org/10.1016/j.addr.2017.07.018
This review focuses on the advances in two complementary techniques: multi-photon laser scanning microscopy (MPLSM) and functional
ultrasound imaging (fUSi). MPLSM has become the gold standard for in vivo imaging of cellular dynamics and morphology, together with cerebral
blood flow. fUSi is an innovative imaging modality based on Doppler ultrasound, capable of recording vascular brain activity over large scales (i.e.,
tens of cubic millimeters) at unprecedented spatial and temporal resolution for such volumes (up to 10 µm pixel size at 10 kHz). By merging these two
technologies, researchersmay have accesstoa more detailed view of the variousprocessestaking place at the neurovascular interface. TPLSM and
fUSiare also good candidatesfor addressingthemajor challenge of real-time delivery, monitoring, and in vivo evaluation ofdrugsin neuronal tissue.

30. Molecular
Imaging
Fluorescentprobesfor
invivohumanimaging

31. “Molecular”Imaging
Emerging technology with
fluorescentprobesfor clinical
diagnosis
DiabeticRetinopathy
MolecularImagingof Subclinical
DiabeticRetinopathy
ChristophRussmann andMansoorM.Amiji
J Ophthalmic VisRes.2017 Apr-Jun; 12(2): 129–131.
doi: 10.4103/jovr.jovr_54_17
Dr. Hafezi-Moghadam's (Frimmel etal. 2017) laboratory developed
the first generation of fluorescent nano-probes with a variety of
surface moieties that mimic leukocyte rolling and adhesion to the
vascular endothelium. The nano-probes injected into the blood
stream of live animals circulate throughout the animal's vasculature,
including the retinal vessels. The probes' interactions with the
inner vessel wall are tracked by epifluorescence microscopy,
and provide an unprecedented temporal and spatial
resolution that gives precise knowledge about the presence of
target molecules in the retinal microvessels. Using their custom-
designed probes in combination with scanning laser
ophthalmoscopy (SLO), Frimmel etal.2017 in their current work
elegantly visualize early molecular signs of diabetic retinopathy
DR in vivo. These results could ominously foretell the course of the
disease before it becomes too late to intervene. . The hope
exists that one day evasive conditions such as Alzheimer's and
atherosclerosis could be diagnosed and staged through
molecular imaging in theretina
MolecularImagingofRetinal
EndothelialInjuryinDiabeticAnimals
SonjaFrimmeletal.(2017)
J Ophthalmic VisRes.2017Apr-Jun; 12(2): 175–182.
doi: 10.4103/jovr.jovr_243_16
Leukocyte accumulation and ICAM-1 expression in the diabetic retina. (a)
Leukocyte adhesion in retinal vessels of normal and diabetic rats. (b) Quantification of
leukocytes in retinal arteries and veins (n = 6). (c) Immunohistochemistry of firmly adhering
leukocytes (red) for CD18, aligand for theendothelial ICAM-1.
Early detection by molecular imaging precedes structural damage. Retinas of
normal and diabetic animals were trypsin-digested to visualize vascular changes in the
diabetic retina. PAS and hematoxylin-stained flatmounts of trypsin-digested normal retinas
show patent retinal capillaries, which are comprised of endothelial cells and are surrounded
by pericytes (a). At three weeks of diabetes, retinas of diabetic animals show no signs of
structural damage (b). In contrast, long-term diabetic animals (six months) display obliterated
acellular capillaries (arrow) (c). Bar, 50 μm.

32. “Molecular”Imaging
Emerging technology with
fluorescentprobesfor clinical
diagnosis
Glaucoma and
Alzheimer’sdisease
Invivo imaging of retinal neurodegeneration at the
single cell level inhumans
Jochen Herms Christian Schön
Brain, Volume 140, Issue 6, 1 June2017, Pages1542–1543,
https://doi.org/10.1093/brain/awx100
In this issue of Brain, Cordeiro andcolleagues describe the results of a phase I clinical trial testing a
novel diagnostic approach for glaucoma (Cordeiro etal.,2017). The authors make use of
fluorescently-labelled annexin 5 (ANX776) to visualize apoptotic cells in the retina, the
presenceof which isanearlysign of glaucoma disease(Quigley, 2011).
Due to the clear optical media of the eye, the retina can be repeatedly and longitudinally studied non-
invasively with microscopic resolution by confocal laser scanning ophthalmoscopy (SLO). Cordeiro
etal. namethenewtechnique‘Detection of Apoptosing Retinal Cells’ (DARC).
A further challenge for fluorophore-based diagnostic techniques is finding a feasible route of
application. . In the current study, Cordeiro et al. show that a systemic (intravenous) application
of ANX776 also results in a positive labelling of apoptotic cells in the retina. This finding is not self-
evidentbecauseANX776 hasto crosstheblood–retinal barrierto reach theapoptoticRGCs.
So far, a definitive diagnosis of Alzheimer’s disease is only possible post-mortem and the search for
useful biomarkers for early diagnosis is an active and contentious field (Limetal.,2016;
van Wijngaardenetal.,2017). It was reported back in 1989 that RGCs degenerate in patients with
Alzheimer’sdisease(Blanksetal., 1989 Citedby198
).
As discussed by Cordeiro et. al, the kinetics of RGC loss in glaucoma patients are quite fast (77 to
90 lostcells per day) and thereforeDARCisa valid tool to visualizethesecellswithin theshort time
period theglaucomapatientisexamined.
However, it seems improbable that DARC will be useful for patients with other neurodegenerative
diseases such as Alzheimer’s disease that have a slower progression. Importantly, ANX776 is a
specific fluorescent marker for the detection of apoptotic RGC loss and is therefore distinct from
non-specific fluorescent compounds like curcumin. It has been claimed that the latter can be used
to detect fibrillary amyloid-b accumulations in the retina in vivo; however, we (Schönetal.,2012) and
others (Ho etal.,2014) have never observed such accumulations in the retina of patients with
Alzheimer’sdiseaseon histological sections.
To circumvent the problem of non-specific signals due the autofluorescent accumulations, it will be
essential to examine the retina of the patient at baseline before a fluorophore is applied. This concept
is not only relevant for clinical applications but also for the development of fluorophore-based
diagnostic approaches in preclinical models, as we described in a recent protocol (
Schön and Herms,2017).
Real-timeimagingof single
neuronalcellapoptosisin
patientswithglaucoma
Maria F. Cordeiro et al. (2017)UCL, London
Brain, Volume 140, Issue 6, 1 June2017, Pages 1757–1767,
doi:10.1093/brain/awx088
InVivoImagingof Tau
AggregatesintheMouseRetina
ChristophRussmannand Mansoor M. Amiji
J Ophthalmic Vis Res. 2017Apr-Jun;12(2):129–131.
doi:10.1007/978-1-4939-6598-4_24

33. Photoacoustic
Imaging (PAI)
Photoacousticimagingis a
promisingimagingtechnique thatis
particularly usefulforimagingdeep
retinalandchoroidalstructures
withoutcompromisingthespatial
resolution
PAI isbased onoptical excitation and ultrasonic detection.
Ashort pulse laser (nanosecond pulseduration)
illuminatesand excitesthe eye. The retinaoftheeye
absorbssome of thedelivered laser energy, generates
heat, undergoestransient thermoelastic expansion, and
producesultrasonic signal. An ultrasound transducer is
focused on the retinal surface and detects ultrasonic
signals, which arethen imaged for both anatomic PAI and
functional analysis[Liu et al. 2016].

34. Photoacoustic
Imaging(PAI)
PhotoacousticTomography: InVivo
ImagingfromOrganellestoOrgans
LihongV. Wang,Song Hu
Science 23Mar2012: Vol.335, Issue6075,pp.1458-1462
DOI: 10.1126/science.1216210
Photoacoustic tomography (PAT) can create multiscale
multicontrast images of living biological structures ranging from
organelles to organs. This emerging technology overcomes the high
degree of scattering of optical photons in biological tissue by making
use of the photoacoustic effect. As a rule of thumb, the achievable
spatial resolution is on the order of 1/200 of the desired imaging
depth, which can reach up to7 centimeters. PATprovidesanatomical,
functional, metabolic, molecular, and genetic contrasts of vasculature,
hemodynamics, oxygen metabolism, biomarkers, and gene
expression. We review the state of the art of PAT for both biological
and clinicalstudies and discussfuture prospects.
Multiscale PAT of organelles, cells, tissues, and organs in vivo. (A) Subwavelength (SW) PAM
of melanosomes in the ear of a black mouse. (B) OR-PAM of individual red blood cells
traveling along a capillary in a mouse ear. (C) AR-PAM of a nevus on a human forearm. (D)
PACT of a human breast. (E) Imaging depth versus spatial resolution in PAT. SM,
submicrometer;LA, linear array.
ResearchersBreakFundamental
Barrierof PhotoacousticImaging: Can
ViewCapillariesatSuperResolution
Nov9TH, 2017 www.medgadget.com/2017/11
osa.org/en-us/about_osa/newsroom/news_releases/2017
Super-resolutionphotoacoustic fluctuation
imagingwithmultiplespeckleillumination
ThomasChaigneetal.(2016) |Optica Vol. 3,Issue1, pp. 54-57 (2016)
https://doi.org/10.1364/OPTICA.3.000054

35. Photoacoustic
Imaging(PAI)
PhotoacousticImagingin
Ophthalmology
HuZ, WangX,LiuQ,Paulus YM | UniversityofMichigan
IntJ OphthalmolEyeRes03(8),126-132.
doi: 10.19070/2332-290X-1500027
Photoacousticimagingfeaturesof
intraoculartumors:Retinoblastoma
and uvealmelanoma
Guan Xu et al.(Feb 2017)|PLOSOne
https://doi.org/10.1371/journal.pone.0170752
A real-time photoacoustic imaging (PAU) and ultrasonography (US) parallel
imaging system based on a research US platform was developed to examine
retinoblastoma in mice in vivo and human retinoblastoma and uveal melanoma
ex vivo. PA signals were generated by optical illumination at 720, 750, 800, 850,
900 and 950 nm delivered through a fiber optical bundle. PAI could be a
potential tool complementary to other diagnostic modalities for characterizing
intraocular tumors.
Relative optical absorption spectra complied by combining data
acquired animal in vivo and human tissues ex vivo. (A) retinoblastoma
in mice in vivo. (B) Retinoblastoma in human ex vivo. (C) Uveal melanoma in
human exvivo.
(A)mouseexperiment invivo.(B)human eyeglobeimaging ex vivo.
Simultaneously acquired SD-OCT (A and C) and PAOM (B and D)
fundus images. A) and B) are acquired from an albino rat, and C) and
D) are acquired from a pigmented rat. RV: retinal vessel; CV: choroidal
vessel;RPE: retinal pigmentepithelium. Bar: 500μm. [Song etal.2013]
Based on previous work,Song etal.(2012) further developed the
multimodal imaging platform integrating Photoacoustic
Ophthalmoscopy (PAOM) with spectral-domain optical coherence
tomography (SD-OCT), scanning laser ophthalmoscopy (SLO), and
fluorescein angiography (FA) to provide optical absorption, optical
backscattering, and fluorescence properties of the albino and
pigmented ratretina.

36. Multi-view
and multi-
illumination
Retinal
Imaging

37. Retinal
Imaging
Detection
scheme
Think of automatic
“compressed sensing” here to
get the structure-of-interest as
fast and sharp as possible
Multioffset configuration allows better
imaging of various retinal structure
Blood vessel contrast can be enhanced or nearly completely minimized depending on multioffset
configuration. The contrast of the blood vessel running diagonally in A from lower left to upper right is
enhanced with this multioffset configuration (A, Inset) but minimized in Bwith the orthogonal
configuration (B, Inset). Imagesobtained fromanormalhumanretina. (Scalebar, 50µm.)
Multioffset images of ganglion cell layer neurons in monkeys with high light levels ( 7 mW) required for∼
TPEF (A)arestill visibleatthelowerlightlevels( 270 µW)typically used for human imaging with thesame∼
sizeaperture(B)orwith anaperturewith twicethediameter (C).(Scalebar,25 µm.)
Ethan A. Rossiet al. (2017)
doi: 10.1073/pnas.1613445114
On-axis diagram of the various detection schemes
used in AOSLO. … In a triangular multioffset detection
pattern (F), the aperture is positioned at several points
arranged in a triangular grid. Black outlines enclose
detectionareas.

38. Retinal
Imaging
Illumination
optimization
combined withthe
detection scheme
optimization
AOSLO confocal (left) and dark-field (right) retinal images in
four different subjects, all collected at the foveal center (center of
fixation). The confocal images show the cone photoreceptor
mosaic, while the dark-field images show the characteristic
hexagonal RPE cell mosaic. The scale bar is 100 μm across.
-DrewScolesetal. (2013)
By collecting the scattered light through the pupil, the partially
coherent illumination produces dark field images, which are combined
to reconstruct a quantitative phase image with twice the numerical
aperture given by the eye's pupil. We then report, to our knowledge, the very
firsthuman in vivophaseimagesofinnerretinalcells withhighcontrast.
a. Trans-epidermal illumination by means of flexible PCB containing LEDs placed in contact with the
skin of the eyelid. Light is then transmitted inside the eyeball. After scattering off the eye fundus, the light
passing through the retinal’s cell layers is collected by the eye lens. b. Flexible PCB holding 4 red LEDs. c.
Recording and reconstruction procedure for in-vivo measurement. d. Experimental setup. The light
scattered from theretina is collected by lens L1. The4f system composed of thelensesL1 and L2isadjusted
for defocus thanks to a badal system. The lens L2 forms an image of the pupil plane at its focal distance,
while the lens L3 forms an image of the retina on the EMCCD camera. Dic: dichroic mirror. Synchronization
between the LEDs and the camera is performed thanks to a programmable board. -
TimothéLaforestetal. (2017)

39. Multiphoton
Imaging

40. Two-photon
Imaging for
phototransduction
analysis
Thefluorescence response
increased followinglight
stimulation, whichcould
provideafunctionalmeasure
oftheeffectsoflighton
photoreceptors.
Imagesof photoreceptorsin living primate eyesusing adaptive opticstwo-photon
ophthalmoscopy
JenniferJ.Hunter,Benjamin Masella,AlfredoDubra, Robin Sharma,LuYin, WilliamH.Merigan, GrazynaPalczewska,Krzysztof
Palczewski,andDavid R. WilliamsFlaumEye Institute /Institute of Optics/CenterforVisual Science,Universityof Rochester.FlaumEye Institute,PolgenixInc.
BiomedOptExpress.2011Jan 1; 2(1):139–148.
Publishedonline2010Dec 17.doi: 10.1364/BOE.2.000139
Two-photon fluorescence imaging has a number of advantages for retinal imaging. Our group was the first to
demonstrate two-photon imaging in the living primate eye (Hunter etal,2011) we are using those results as a stepping
stoneforthefollowing projects:

41. Two-photon
fluorescence adaptive
optics scanning light
ophthalmoscope
(TPF-AOSLO)
This invivo survey of two-photon
autofluorescencethroughout
the primate retinademonstrates
awider variety of structural detail
in theliving eyethan is available
through conventionalimaging
methods, and broadens theuse
of two-photon imaging of normal
and diseased eyes.
Two-PhotonAutofluorescence Imaging RevealsCellularStructuresThroughout the
Retina of the Living Primate Eye
Robin Sharma; DavidR.Williams; GrazynaPalczewska; KrzysztofPalczewski; JenniferJ. Hunter
FlaumEye Institute /Institute of Optics/CenterforVisual Science,Universityof Rochester.FlaumEye Institute,PolgenixInc.
IOVS| MultidisciplinaryOphthalmic Imaging | February2016 |Vol.57,632-646.doi: 10.1167/iovs.15-17961
Relative two-photon autofluorescence levels in the primate retina. (a)
Optical coherence tomography image of a retinal location 5 mm temporal to
the fovea. Labels refer to the various retinal layers that can be distinguished [
Staurenghiet al. 2014]. (b) Two-photon autofluorescence signals plotted
for 730 and 920 nm excitation.
These data were collected at the same retinal location on two different imaging sessions,
almost 4 weeks apart. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform
layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; EZ,
ellipsoid zone, outer segment junction; OS, outer segment; RPE CC, retinal pigment
epitheliumand choriocapillaris. Scalebars: 50 μm.
Through-focus image stack
of a blood vessel in the
retina is shown here.
Reflectance images are
shown in the left in (a), (c),
(e), and (g), with (a) being
the most vitreal and (g) the
most scleral. The
corresponding two-
photon images at 730
nm excitation are shown on
the right, (b), (d), (f), and (h).
In (b), edges of the vessel
walls were almost
indistinguishable and the
two-photon fluorescence
signal from the en face
vessel wallswasdominant.
Comparison of in vivo and ex vivo two-photon
autofluorescence, λex
= 730 nm from the same retinal location,
8° nasal to the fovea. (a) Two-photon image of the living eye.
(b) Two-photon imageof fixed tissuefromthe sameeyeat the
same location. (c) Radial average of the Fourier transform for
both images plotted on the same scale. The peak
correspondsto acellspacing ranging from10 to 18 μm.

42. Multiphoton
imaging for
vasculature
imaging
Depending on the label
engineeringonecan
highlight desired
structures
Simultaneously imagemultiple
structures on different spectral
channels
Invivo multiphoton imaging ofadiverse array of fluorophores to investigate deep
neurovascularstructure
David R. Miller, Ahmed M. Hassan, JeremyW. Jarrett, Flor A. Medina, Evan P. Perillo, Kristen Hagan, S. M. ShamsKazmi, Taylor A. Clark, Colin T.
Sullender, TheresaA. Jones, BorisV. Zemelman, and Andrew K. Dunn
Department of Biomedical Engineering,The Universityof Texas at Austin; Department of BiomedicalEngineering,The University of Texasat Austin
BiomedicalOpticsExpressVol.8,Issue7, pp.3470-3481(2017)| doi: 10.1364/BOE.8.003470
In vivo two-photon microscopy images of vasculature labeled with Texas Red. (a)
Three-dimensional reconstruction of a 1,535 μm stack. (b) x–y intensity projections
of stack shown in (a). (c) (top) x–y intensity projection at depth of 1200 μm (note:
image from different stack than shown in (a)). A line scan was performed at the
highlighted red line. (bottom) Line scan at depth of 1200 μm. All scale bars are
50 μmunlessotherwiseindicated.
In vivo two-photon microscopy images of vasculature labeled with
Texas Red and neurons labeled with YFP. (a) Laser speckle contrast
image of mouse craniotomy. The red square indicates the two-
photon imaging location. The zoomed view is a 300 × 300 ×
400 μm3
two-photon maximum intensity projection. (b) x–z intensity
projection of a 1,330 μm stack of vasculature (red) and neurons
(green). (c) x–y intensity projections of stack shown in (b)
demonstrating neuron cell bodies in layer V and a large blood vessel
at a depth of 1,330 μm. All scale bars are 50 μm unless otherwise
indicated.
(a) The SBR as a function of depth for a laser repetition rate of 511 kHz (red) and 255 kHz
(blue). The lines serve as guides for the eye. (b) Comparison of the SBR for a vessel at
z=940 μm for laser repetition rates of 511 and 255 kHz. The vessel images are a 10-frame
average at the respective repetition rate; the images are shown at full scale. The line profile is
averaged over a 6 μm line indicated by the yellow lines in the vessel images. (c) Normalized
intensity profile for a line through a blood vessel at z=1430 μm, demonstrating a SBR above 2
(the dotted black line indicates the calculated background). The yellow line on the vessel
image, which is a 16-frame average, indicates where the line profile was taken. (d) Centerlines
of vessels from 1,200 to 1,450 μm depth encoded by color. The centerlines are overlaid on a
maximum intensity projection of the raw data. All scale bars are 50 μm unless otherwise
indicated.

43. Vasculature-
specific dyes and
techniques
Makingimage
segmentationeasier or
evenpossible
AlexaFluor 633and
fluoresceindextran
Shenetal.(2012)
Qtracker800
quantum dots, emission at
800 nm
3-PM601um
Wanget al. (2015)
2-PM429nm
Vasculature Image Quality.
An example of false fractions in
the structure caused by imaging
imperfectionsand an area of more
artifacts in a maximum-intensity
projection (MIP) slice of a 3-D
fluorescent microscopy image of
microvasculature.Almasietal. (2015)
Label-free live brain imaging
and targeted patching with
third-harmonic generation
microscopy– Witteetal.(2011)
Isolation of blood vessel wall and
perivascular space in vivo. (A–C) Two-
photon image stack acquired through mouse
cranial window in vivo, showing cortical
vasculature stained with fluorescein (green,
restricted to vascular lumen) and Alexa633
(red, specifically located within vascular
lumen and strong staining of elastin within
arteriole vessel walls). Notice that Alexa633
fluorescence is greatly diminished in the
perivascular compartment compared to the
vessel wall compartment isolated in G and H.
Fenrich et al. (2013)

44. From Two to
Three photons
Better opticalsectioning
capability and deeper
transmission.
In practice this enables
imagingofdeep brain
structures such as
hippocampus insmall
animalmodels.
2PM, attenuation z2
from focal plane
3PM, attenuation z4
from focal plane
Horton et al. (2013)
osa-opn.org, November 2013

45. From Two to
Three photons
Rayleighscatter reduceswith
increased wavelength(~λ−4
)
Howeverthereareotherabsorbers
in brainandtheeye,thatdetermine
optimalspectralwindows.Withthe
caseofbrainthree-photon scheme:
λexcitation
at ~1,700 nm, and
λemission
at ~700-900 nm
Invivo three-photon microscopy of subcorticalstructureswithin anintact mouse brain
NicholasG.Horton,KeWang,Demirhan Kobat,CatharineG. Clark,FrankW.Wise, ChrisB. Schaffer&ChrisXu
School of AppliedandEngineering Physics,Cornell University
NaturePhotonics7, 205–209(2013)doi: 10.1038/nphoton.2012.336 |Citedby474articles
The spectral response of oxygenated hemoglobin, deoxygenated
hemoglobin, and water as a function of wavelength. The red highlighted
area indicates the biological optical window where adsorption due to
the body is at a minimum. (Doane and Burda, 2012)
Wavelength-dependent attenuation length in brain tissue and measured laser characteristics. Attenuation
spectrum of a tissue model based on Mie scattering and water absorption, showing the absorption length of
water (la
, blue dashed line), the scattering length of mouse brain cortex (ls
, red dashed-dotted line), and the
combined effective attenuation length (le, green solid line). The red stars indicate the attenuation lengths
reported for mouse cortex in vivo from previous work [Kobat et al., 2009]. The figure hows that the optimum
wavelength window (for three-photon microscopy) in terms of tissue penetration is near 1,700 nm when
both tissue scattering and absorption are considered. Figure from Horton et al. (2013)
Predicted photon transmission properties of biological tissue
as a function of scatter, H2
O-to-Hb ratio, and thickness. (A) The
illumination/detection geometry used for predicting the performance of
Quantum Dots (QDs) for reflectance fluorescence imaging. (B) Using the
model geometry shown in A, the relative number of transmitted photons
as a function of wavelength was simulated on tissues of high H2
O-to-Hb
ratio (left panels) or high Hb-to-H2
O ratio (right panels), at tissue
thicknesses of 0.25 cm (thick solid line) or 1 cm (dashed line). Simulated
tissues exhibited either wavelength-independent scatter (upper panels)
or wavelength-dependent scatter (lower panels). The analysis identified
four possible transmission bands (black bars below ordinate) as
described in the text. The arrow above each transmission band identifies
the peak QD emission wavelength used for subsequent analysis. Figure
fromLimetal. (2003)

46. Beyond 2-PM and
3-PM fluorescence
We review multiphoton microscopy
(MPM) includingtwo-photon
autofluorescence (2PAF), second
harmonicgeneration (SHG), third
harmonicgeneration (THG),
fluorescence lifetime (FLIM), and
coherent anti-StokesRaman Scattering
(CARS) withrelevance to clinical
applicationsin ophthalmology.
MultiphotonMicroscopy for Ophthalmic Imaging
Emily A.Gibson,OmidMasihzadeh,TimC.Lei, DavidA.Ammar,andMalikY.Kahook
Department of Bioengineering,Department ofElectrical Engineering,Department of Ophthalmology,University of ColoradoDenver,
JournalofOphthalmology(2011)doi: 10.1155/2011/870879
Schematic of the eye highlighting the regions of
interest for imaging with multiphoton microscopy.
Light path for imaging of the retina through the
anterior chamber and lensisshown.
Vascular bedofahumanretina imaged by
secondharmonicgeneration(SHG). Serial -
section ofahuman retinashown is collected using
the800 nmnear infrared laser excitation witha
collection window of390–410 nm. Thecollagen
structureofa largeblood vessel is clearlyvisible.
Second harmonic generation (SHG) and two-photon
autofluorescence (2PAF) of trabecular meshwork (TM)
region of a humaneye from a 73-year-old donor. A section of
the eye was flat-mounted with the anterior chamber facing
the microscope objective. Merged image of SHG (blue)
andAF (green) emission. Black scale bar = 50 μm.
Conclusion and Future Prospects Current imaging techniques, such as ultrasound and OCT, have greatly influenced the standards of
clinical and surgical ophthalmic care. Physicians can now detect disease using very sensitive imaging modalities and can also follow the
progression of disease, thus shedding light on the efficacy of applied interventions. While availability of fine structural information is
increasingly available in the clinical setting, the actual function of the imaged structures remains unknown. MPM offers the potential for
obtaining both structural and functional data on a wide range of ophthalmic tissues. For example, it may be possible to image the
trabecular meshwork structure (Ammar et al. 2010) while also establishing the metabolism of individual trabecular meshwork cells by
quantifying NAD(P)H concentrations in real time. Such information could lead toearlier and more precise disease detection, while also
allowingfor more insight intotheeffectsof therapeutic interventionsaimed at preservingvision.
Future applicabilityof MPM in practicewill require further advances in theabilityto penetrate past tissues, such as sclera, that have high
scattering properties (Vogel et al. 1991). The safety of using MPM also requires further studies since some ocular tissues have high melanin
content which may lead to greater energy absorption and related tissue damage. Another obstacle that will need to be addressed is the
difficulty in obtaining data across the relatively long axial length distance noted between the surface of the cornea and the posterior pole.
Fortunately, advances in MPM continue to develop at a rapid pace, and obstacles that existed in the past have been overcome with continued
research. With continued advances, the application of MPM in ophthalmic practice promises to yield valuable clinical information that will
ultimately result in improved patient care, which isthe commongoal of researchersand physiciansalike.

47. Multimodal
imaging
for various
structures
In vivo assessmentof
aqueous drainage
structure
Towardin vivo two-photon analysis of mouse aqueous outflow structure and function
JoseM.GonzalezJr., MinheeK. Ko,AndriusMasedunskas,Young-Kwon Hong,RobertoWeigert,JamesC.H. Tan
DohenyEye Institute andDepartment of Ophthalmology,DavidGeffen School of Medicine,UniversityofCalifornia,Los Angeles,CA,USA
ExperimentalEyeResearchVolume158,May 2017,Pages161-170doi: 10.1016/j.exer.2016.05.009
Deep tissue 2-photon microscopy of the mouse aqueous drainage tissues. A: Light
micrograph of a hematoxylin and eosin-stained cryosection from a Balb/c mouse reveals its
anatomy. B: Mouse TPEF of labeled F-actin (Alexa Fluor 568-conjugated phalloidin; red) and
SHG of collagen (cyan) mirrors the organization shown in panel A. . C: Isosurface volume
reconstruction integrating F-actin TPEF (red) and SHG (cyan, flat) signals, and signal voids of
channel lumen (darker cyan, contoured). D: The image volume is rotated obliquely and
surfacestructures cutawayinthe software to reveal ISP justdeep to theocular surface.
2-photon (2P) navigation of the mouse conventional outflow pathway guided by
autofluorescence, collagen second harmonic generation (SHG) and filamentous actin
(Factin) signals. Eyes were enucleated and not fixed (A, D, G, J) or fixed in paraformaldehyde and
labeled with Alexa-568-conjugated phalloidin (B, C, E, F, H, I, K, L) and imaged with TPEF for
autofluroescence (green; A, D, G, J), SHG (cyan;B, E, H, K), or SHG and F-actin(red;C, F, I, L show
merged SHG and F-actin signals). .Bar = 25 μm.
“Multimodality 2-photon imaging permits high-resolution visualization not only of tissue structural
organization but also cells and cellular function. It is possible to dig deeper into understanding the
cellular basis of aqueous outflow regulation as the technique integrates analysis of tissue structure, cell
biology and physiologyin a waythatcouldalso lead to freshinsightsinto human glaucoma”

48. Adaptive
Optics

49. Adaptive
Optics
Measuring and
correcting individual
ocularaberrations inreal-
time. Sharperimage→
Like turbulent
atmosphere, tear film
behavior is dynamic
makingocularPSFnon-
static
http://www.mpia.de/LINC/adaptive-optics.html
https://www.transparencymarketresearch.com/adap
tive-optics-technology-market.html Uranus in twodifferent wavelength, withand
withouttheAO systemon, creditHammel/de
Pater/Keck
Liuetal.(2016): Adaptive optics OCT cross-
sectional and en faceimages extracted fromthe
photoreceptor-RPE complex of a 48–year-old
subject(S5)ateightretinaleccentricities.
ClinicalHeidelberg
SpectralisOCTB-scan
Adaptive opticsOCT[IndianaAO-OCT
imaging system ( c =790nm)]λ averaged B-scans
and corresponding A-scan profiles(yellow
trace)of volumesreveal distinctreflectance
bandswithinthecomplex labeled IS/OS,
COST, ROST,and RPE. R/COST
Rod/ConeOuterSegmentTips
The datastreamfromthe systemwasprocessed and
displayed using customCUDA softwaredevelopedfor
parallelprocessingbyan NVIDIATitan ZGPU

50. Ultrasound
Steering
Similarishrefraction
correction scheme
usedwithfocused
ultrasound stimulation
tocorrect skull-
induced aberrations
Designofpatient-specificfocused
ultrasound arraysfornon-invasivebrain
therapywithincreasedtrans-skull
transmissionandsteeringrange
Alec Hughes, and KullervoHynynen
SunnybrookResearch Institute; Department of Medical Biophysics,University of Toronto,
Toronto,Canada
Physicsin Medicine & Biology, Volume 62, Number 17
doi:10.1088/1361-6560/aa7cd5
NeurosurgicalApplicationsofHigh-Intensity
FocusedUltrasoundwithMagneticResonance
Thermometry
RivkaR. Colen, ImanSahnoune, JeffreyS. Weinberg
Department ofCancer SystemsImaging and DiagnosticRadiology,The Universityof Texas
NeurosurgeryClinicsofNorth AmericaVolume 28, Issue 4, October 2017,
Pages559-567 https://doi.org/10.1016/j.nec.2017.05.008
Medicalultrasoundsystems
JeffPowers, Frederick Kremkau. Inferface Focus 2011
https://doi.org/10.1098/rsfs.2011.0027
Array steering: (a) delays for steering right; (b)
delays for steering left; and (c) wavefronts
producing asteeredwave.
It was soon found that the image could be significantly
improved by focusing the beam as a function of image
depth. Reflected sound arrives first at the centre of the
array and a few microseconds later at the outer edges.
By delaying the sound at the centre until it is in phase
with that coming from the outer edges, a much more
tightly focused beam can be generated. Owing to the
principle of reciprocity, the transmit beam can also be
focused by applying the transmit pulse earlier to the
outer elements. By changing the delay curvature, the
beammaybefocused atdifferentdepths
Another technique commonly used in ultrasound imaging to determine
the spatial shape of the ultrasound pulses is beam steering. Through
this technique, the angle of the beam with respect to the transducer
can be changed. The technique is implemented in transducer arrays
through electronicbeamforming.www.ultrasonix.com/wikisonix/

51. Adaptive
Optics
Technology
NovelShack-
Hartmanndesigns
HighresolutionShack-Hartmannsensorbased onarrayof
nanostructuredGRINlenses
RafalKasztelanic,AdamFilipkowski,DariuszPysz,RyszardStepien,AndrewJ.Waddie,MohammadR.
Taghizadeh,andRyszardBuczynski
OpticsExpressVol. 25, Issue 3, pp. 1680-1691(2017)
https://doi.org/10.1364/OE.25.001680
Scheme of the Shack-Hartmann setup: a) determining the shift of the spot
for a single gradientindex(GRIN) lens [See Thorlabsselection for
example], b)a fullShack-Hartmann setup.
Design of a preformfor a GRIN lenscomposed of 7651 rodsmade
fromtwo glasses.One isan in-house synthesized low-index silicate glass NC21
(55% SiO2, 1% Al2O3, 26% B2O3, 3% Li2O, 9.5% Na2O, 5.5% K2O, 0.8% As2O3)
and the second acommerciallyavailable high-index lead-silicate glass F2.
Currently reported S-H sensors are based on refractive microlens arrays. They offer a sampling density in the range from 1 to 10 lens/mm when the
minimum diameter of the lens is 100 μm. Smaller lenses are not used due to technological limitation. Lenses below 100 μm in diameter
whose curvature is accurate are difficult to obtain. Moreover, the performance of refractive lensed is limited to air. For liquids, where the
refractive indexexceeds1.33, their performanceisdegraded.
When designing a S-H sensor light economy is an issue. Due to a large number of lenslets the input light intensity is divided into small portions
focused on the camera. The detection of such noisy signal is hard, so it is important that the lenslet array should absorb as little light as possible
and that the fill factor should be close to 100%.
In this paper we present a setup for wavefront measurement based on a S-H sensor, whose main element is a thin hexagonal array of flat GRIN
microlenses with 100% fill factor. The array composed of 469 micro-lenses with 20 µm diameter of single lens and high numerical
aperture NA = 0.5. This provides high sampling density, with can be used with modern CCD or CMOS cameras, where the single pixel size is ~1
μm [30]. High-sampling density, similar to the resolution of the GRIN lenslets array, can be used to study wavefront distortion with ahigh resolution, for
example in microfluidics setups or in biology for imaging plankton. Low thickness (below 100 µm), short focus length, 100% fill factor and flatness
make the array highly light-efficient. Since GRIN array has a flat facade, the optical performance of lenses is not degraded, even if the array is
immersed in medium other than air. This opens a unique opportunity to measure the phase front of the light beam propagating in liquids, not
restricted towater but alsoincluding such high-index mediaas organic liquids.

52. Adaptive
Optics
Invisionscienceand
inopthalmology

53. Adaptive
Optics
Havebeen
emergingfor
sometime
already
Modern Retina | Ophthalmology|Adaptive optics
Howadaptiveopticswillchangeretinal
imagingJuly01, 2017 ByLaird Harrison
http://modernretina.modernmedicine.com/modern-retina/news/how-adaptive-optics-will
-change-retinal-imaging
“Adaptive optics could allow clinicians to monitor the progression of retinal diseases cell
by cell, according to Jacque Duncan, MD. “We’re able to identify the source of vision loss
when there is a problem with patients’ cones,” said Dr. Duncan, professor of clinical
ophthalmology, University of California, San Francisco.”
AOimagingoffersnoveldatafor
understandingretinaldisease
October09, 2017 ByCheryl Guttman Krader
http://modernretina.modernmedicine.com/modern-retina/news/ao-imaging-offers-novel-d
ata-understanding-retinal-disease
https://www.youtube.com/watch?v=2lkCp25Dh-o
“AO is a noninvasive imaging modality that uses a deformable mirror to compensate for
and correct aberrations,” said Dr. Chung,associate professor of ophthalmology, FlaumEye
Institute, University of Rochester, Rochester, NY. “It allows visualization of individual
photoreceptors, retinal pigment epithelial (RPE) cells, and retinal vasculature at 2-µm
resolution.”
SHARP-EYE ProjectID: HPRN-CT-2002-00301
Funded under: FP5-HUMAN POTENTIAL
Adaptiveopticsforretinal imaging andimprovedvision
From 2002-10-01 to 2006-09-30
Participants: National University of Ireland (Galway), Imperial
Collegeof Science, ObservatoiredeParis-Meudon, Technion- Israel
Instituteof Technology,Universidad deMurcia, UniversityCollege
London (UCL),Universityof Crete,UniversitédeParisVII Denis
Diderot
http://cordis.europa.eu/project/rcn/67883_en.html
The fundusphoto has metitsmatch: optical
coherence tomography and adaptive optics
ophthalmoscopy are here to stay
JessicaI. W. Morgan | Department ofOphthalmology,ScheieEyeInstitute, Perelman School ofMedicine, University ofPennsylvania,
OphthalmicPhysiol Opt 2016; 36: 218–239. doi: 10.1111/opo.12289
Comparisonof a
conventional fundus
photograph,an SD-OCT
crosssectional imageobtained
along thegreenlineoverlaid on
thefundusphotograph, and a
montageof confocal AOSLO
imagesacquired within the
whiteboxoverlaid on the
fundusimagein therighteyeof
a 31 year old normalmale.
Coloured box outlineson the
AOSLO montageshowareas
of thephotoreceptormosaic in
highermagnificationbelow.
Adaptiveopticsoptical
coherencetomography
imagesfroma normal control
showing the B-scan, en face,
and projection views from
thevolumedata set
corresponding to theouter
retinallayersat6° superior to the
fovea.
Individual cone
photoreceptorsareresolved
in threedimensions,allowing
measurementsof individual
coneouter segmentlengthsin
theB-scan imageaswellas
visualisation of theconemosaic
in theen faceview.Scalebar
25 μm.

54. Adaptive
Optics
Vision Science #1
Real-time correction of
eye’s opticalaberrations
giving yousharper
images andpossibilityto
focus your stimulus on
theretina
Adaptive optics forstudying visualfunction: A comprehensive review
Austin Roorda| UCBerkeleySchool of Optometry
Journal of Vision June 2011, Vol.11, 6. doi:10.1167/11.5.6 | Cited by 84
In the AOSLO, the stimulus is printed
directly onto the retina by
modulating the scanning laser, line-by-
line, as it scans a raster pattern on the
retina.
The experiments of Sincichet al.
(2009) involved the optical
stimulation of targeted cones
in the monkey along with
simultaneous recording of neural
activity in the lateral geniculate
nucleus.
“In the next ten years, we should expect to see different classes of AO systems for vision science emerge. Low cost systems with
specific uses will likely start coming available for routine clinical use, such as the AO phoropter (e.g. Hamamatsupatent, or Adaptica’s
system). At the same time, it should not be too long before any researcher with appropriate funding will be able to purchase a robust,
user-friendlyAOsystemwithadvancedimagingandvisualtesting capabilities.
Plot of the Strehl ratio as a
function of defocus for an AO-
corrected eye and for a typical
eye (corrected for astigmatism).
Strehl ratio is the ratio of the
peak height of the PSF for the
actual eye to the peak height of
the PSF of a diffraction-limited
eye with the same pupil size.
Strehl ratios range from 0 to 1
where the higher number
indicates a sharperimage.

55. Adaptive
Optics
Vision Science
Imagingthe cone
photoreceptor mosaic
With clinicalapplications
as well
The organization of the cone
photoreceptormosaicmeasured in
the living human retina
LucieSawides, AlbertodeCastro, Stephen A. Burns
Vision Research Volume 132, March 2017, Pages34-44
doi:10.1016/j.visres.2016.06.006
Foveal(top) and
parafoveal
(bottom) montages
for subject S7(a28 yo.
Female). Scale bars
represent 50 μmfor
foveal and 200 μm for
parafoveal montages.
Lines of
isoeccentricityare
drawnand average
coneimages are
shown forselected
locations along the
four primary meridians.
Average cone images
are 12.5 x 12.5 μm for
the foveal montage
and 25 x 25 μm for the
parafoveal montage.
Average cone spacing (panel A) and cone density (panel B)
computed using the local cone average technique for the four
primary meridians (Temporal, Nasal, Superior and Inferior). Error
bars represent plus or minus one standard deviation across
subjects.
We first computed the radial intensity profile and then the
circumferential intensity profile. From the circumferential profile, we
used FFT to determine the degree of hexagonality and the rotation
of the local hexagons, and the circumferential profile maxima to
extracttheangleoftheprincipleaxisandlocalspacing anisotropy.
Pumetal.(1990) found that ‘‘The foveal mosaic from a
glaucomatous eye reveals severe lattice degradation
throughout the rod-free zone, presumably due to extensive
receptor loss”. Because of the simplicity of our approach, and the
fact that it is relatively robust to algorithmically missing some of the
cones, it may be a reasonable alternative to Voronoi analysis, and
could easily be adjusted to also provide estimates of the nature of
the mosaic in non-hexagonal regions from the peak of the FFT
analysis as well as to provide other estimates of spatial
coherence by examining the width of the cone averages at
increasingdistancesfromthecenter.

56. Adaptive
Optics
Vision Science
Imagingthe rod
photoreceptor mosaic
Noninvasiveimagingof thehumanrod
photoreceptormosaicusingaconfocal
adaptiveopticsscanningophthalmoscope
AlfredoDubraet al. | Biomedical Optics ExpressVol. 2,
Issue 7, pp. 1864-1876(2011)
https://doi.org/10.1364/BOE.2.001864
Reflectance images of the human photoreceptor mosaic at three retinal
locations along the temporal meridian for subject DLAB_0008, collected
using 680 nm light and 0.4Airy disk pinhole size. The same imagesare shown
with linear (top row) and logarithmic (bottom row) grayscales, to facilitate
visualization ofthe rod mosaic. The scale barsare 10μmacross.
Comparison of in vivo rod and cone metrics with those from Curcio et al. 1990.
Shown on the left is a plot of the ratio of rods to cones as a function of retinal
eccentricity. The solid line is the mean of Curcio’s measurements taken in the
temporal meridian, and filled circles correspond to the data from this study. On
the right is a plot of photoreceptor density as a function of retinal eccentricity.
Density estimates for our subjects for rods and cones are shown as open
squares and open circles, respectively. Also plotted is the mean rod (solid line)
and cone (dashed line) density values reported by Curcio et al. 1990 for the
temporal meridian.
RODS
CONES
Variationin rod andconedensityfrom thefoveato
themid-peripheryinhealthyhumanretinasusing
adaptiveopticsscanninglaserophthalmoscopy
E M Wells-Gray, SSChoi, ABriesand N Doble
Eye(2016) 30, 1135–1143; doi: 10.1038/eye.2016.107
AO-SLO images (0.7 × 0.9°) were acquired at 680 nm from 14
locations from 30° nasal retina (NR) to 30° temporal retina (TR) in 5
subjects
The ability to image in the mid-periphery will be significant for the
study of retinal diseasessuch as retinitispigmentosa.
We show for the first time in vivo, an increase in the rod center-to-
center spacing at the more peripheral retinal locations.
Photoreceptor mosaic images and corresponding Voronoi plots for
the fovea, 10° NR, and 30° NR for subject N5. Each image is the registered
average of ~ 50 frames, displayed with a logarithmic intensity scaling. For the
Voronoi plots, the color coding of the cell domains indicate the number of
neighboring cells (either cone or rod). Hexagonal packing (six nearest neighbors)
is shown in green. In the 10° and 30° NR Voronoi plots, cells identified as cones
aremarked withblack dots. Thescalebaris50μm

57. Adaptive
Optics
Vision Science
Targeting the
photoreceptor mosaic
Trichromatic reconstructionfromthe
interleavedconemosaic:Bayesianmodel
andthecolor appearanceof smallspots
David H. Brainard; David R. Williams;HeidiHofer
Journal of Vision May2008, Vol.8, 15. doi: 10.1167/8.5.15
Basic data from Hofer, Singer, et al. (2005). The adaptive
optics system was used to briefly present small
monochromatic spots ( 0.3-minute full-width at half-∼
maximum retinal size, <4 ms duration) at the retinal region
where the mosaic had been characterized.
Subjects reported a wide variety of color sensations,
even for long-wavelength stimuli, and all subjects
reported blue or purple sensations at wavelengths for
which S cones are insensitive. Subjects with more L cones
reported more red sensations, and those with more M
cones tended to report more green sensations. White
responses increased linearly with the asymmetry in L to M
cone ratio. The diversity in the color response could not be
completely explained by combined L and M cone
excitation, implying that photoreceptors within the
same class can elicit more than one color
sensation.
UnsupervisedLearningof ConeSpectral
Classesfrom NaturalImages
NoahC. Benson, Jeremy R. Manning, David H. Brainard
PLoS ComputBiol10(6):e1003652. doi: 10.1371/journal.pcbi.1003652
http://color.psych.upenn.edu/supplements/receptorlearning/
Here, we simulate cones in a model human retina and show that
by examining thecorrelation of theresponsesof cones to natural
scenes, it is possible to determine both the number cone classes
present in a retinal mosaic and to explicitly determine the class of
each cone. These findings shed light on the computational
mechanisms that may have enabled the evolution of human
color vision, as well as on the more general question of whether
and when itispossiblefor sensorysystemsto self-organize.
Spatiochromatic Interactionsbetween
IndividualConePhotoreceptorsintheHuman
Retina
WilliamS. Tuten, WolfM. Harmening, Ramkumar Sabesan, Austin Roorda and
Lawrence C. Sincich J Neurosci 2017, 37(39)9498-9509;
doi:10.1523/JNEUROSCI.0529-17.2017

58. Adaptive
Optics
Vision Science
Functionaltesting for
eye movementresearch
Adaptiveopticsscanninglaser
ophthalmoscopeusingliquidcrystalon
siliconspatiallight modulator:Performance
studywithinvoluntaryeyemovement
Hongxin Huang, HaruyoshiToyodaand Takashi Inoue
JapaneseJournal of Applied Physics (2017) 5609NB02
https://doi.org/10.7567/JJAP.56.09NB02
Schematic of an adaptive optics scanning laser ophthalmoscope. LCOS-SLM: liquid
crystal on silicon spatial light modulator, WFS: Shack–Hartmann wavefront sensor,
HS: horizontal scanner, VS: vertical scanner.
Examination of microsaccades and drifts of eye
motion: (a) successive frames from an AO-SLO image
recording. We investigate the effects of involuntary eye
movements, which usually include microsaccades, drift, and
tremor, using both the retinal images and the aberration data
recorded simultaneously. The one at time 30.4 s (top row,
middle column) was partially distorted due to a
microsaccadic eye motion. A large interframe shift was
observed between before and after the motion. Although the
magnitude of the movement wasmore than the size of the view
field, high-contrast images were still observed as shown in the
left and right columns of the top row. Following the motion, a
small interframe displacement could be observed as shown in
the snapshots of the times from 30.5 to 31.0 s. This drift motion
can be confirmed by measuring the positions of bright
spots in the images, as well as the aberration data recorded
simultaneously.
We also investigated involuntary eye movements using both the
retinal images and aberrations, by which the microsaccades and
drifts were confirmed. Furthermore, we measured the interframe
displacement of retinal images and found that during eye drift, the
displacement was approximately proportional to the residual low-
order aberration recorded simultaneously. The estimated duration of
drift was approximately 0.6 s and the cumulative amount of transverse
shift was approximately 37 µm or 7.5 arcminutes, which were within the
ranges measured by conventional methods. In this study, we are
interested in not onlyimprovingthe AO-SLOinstrumentsthemselves, but
also providing a new approach for involuntary eye movement
research.

59. Adaptive
Optics (AO)
Imaging
withScanningLaser
Ophthalmoscope
(SLO;AOSLO)
Adaptive Optics Ophthalmoscopy
Austin Roordaand Jacque L. Duncan | UCBerkeleySchool of Optometry; Departmentof Ophthalmology,Universityof California
Annual Review of Vision Science Vol. 1:19-50 (2015) . doi: 10.1146/annurev-vision-082114-035357
(a) Fluorescein angiography (FA) of a human retina with adaptive optics
scanning laser ophthalmoscopy (AOSLO). This particular implementation
employed oral fluorescein, offering a longer time course for imaging and
avoiding risks associated with injection. The use of AOSLO offers higher
contrast and higher resolution over conventional FA. (b) Image of the
microvascular structure of a human retina using offset-pinhole AOSLO.
Arrows point to purported individual mural cells that compose the arteriole
walls. - Chui et al. (2013). (c) Motion contrast image from split-detector
AOSLO recordings of a vessel and capillary network in a normal human eye. -
Sulai et al. (2014). (d ) A combination of confocal AOSLO motion contrast of
perfusion (magenta) and fluorescent AOSLO images of tagged pericytes
( green) in a mouse retina reveals thecolocation of these structures. - Schallek
etal.(2013)
Images of melanin pigment taken using five different imaging modalities.
Panels a–d show registered images from a single patient with idiopathic
macular telangiectasia type 2, panel e is cropped from the image of an age-
related macular degeneration (AMD) patient with geographic atrophy shown in
Figure 12. (a) Color fundus photo: pigment appears dark and brownish in color.
(b) Fundus autofluorescence from a Heidelberg SPECTRALIS: pigment
appears dark. (c) Adaptive optics scanning laser ophthalmoscopy: pigmentis
hyperreflective in near-infrared (NIR) (840 nm) light. (d) Optical coherence
tomography (OCT) b-scan (scan location indicated by the black line in panel c):
pigment is hyperreflective in NIR light. (e) Adaptive optics fundus
photography: pigmentishyporeflective. - Gocho etal.(2013).
(a) Confocal adaptive optics scanning laser ophthalmoscope (AOSLO) image
of a healthy human retina showing a complete mosaic of cones (large cells)
and rods (intervening smaller cells) at a location 10 deg temporal to the fovea. -
Cooperetal.(2011). (b) Split-detector AOSLO image of a healthy human
retina showing an array of cone inner segments at a location 10 deg from the
fovea. Owing to their small size, the rods, which fill the intervening space between
the cones at this location, are too small to be resolved. - Scolesetal. (2014). (c)
two-photon fluorescence AOSLO image of the retina of a macaque
monkey showing the array of photoreceptors (confirmed by taking a confocal
AOSLO image at the same location). The appearance of a mosaic in the two-
photon image indicates that the fluorophores are contained within the
photoreceptor. Image courtesy of J. Hunter, R. Sharma, and D. Williams [
Advanced Retinal Imaging Alliance, Rochester].

60. Adaptive
Optics (AO)
Imaging
withOptical
Coherence
Tomography(OCT;
AO-OCT)
Photoreceptor-Based Biomarkers in AOSLO Retinal Imaging
Katie M. Litts; Robert F. Cooper;Jacque L. Duncan;JosephCarroll | Investigative Ophthalmology& Visual Science September 2017,
Vol.58, BIO255-BIO267. doi: 10.1167/iovs.17-21868
OCT B-scans of the retina obtained with different imaging
techniques. (Top) Clinical OCT acquired over 5 mm; (bottom left) AO
high-resolution spectral-domain OCT (0.5 mm scanning range) with
focus set at photoreceptor layer; (bottom center) enlarged area (0.5
mm) from the clinical OCT; and (bottom right) AO high-resolution
spectral-domain OCT (0.5 mm scanning range) with focus set at
ganglion cell layer. Wojtkowski etal. (2012)
A) Representative AO-OCT B-scan with the focus set to the photoreceptors recorded in a
healthy volunteer at 4.5 nasal eccentricity from the fovea (logarithmic intensity grey scale). B)
2x enlargement of the region marked with the white rectangle displayed on a linear intensity
scale (some inner and outer segments of cones are marked with red and yellow rectangles,
respectively) [Zawadzki etal. 2008]. The retinal layers are labeled as follows: RNFL retinal nerve fiber layer, GCL ganglion cell
layer, IPL inner plexiform layer, INL inner nuclear layer, OPL outer plexiform layer, ONL/HFL outer nuclear layer/Henle’s fiber layer, ELM external
limiting membrane, IS/OS junction between inner and outer segments of cone photoreceptors, COST cone outer segment tips, RPE retinal
pigment epithelium
Comparison between AO-OCT intensity images and AO-
OCTA (OCT Angiography) images extracted at different depths.
a-d) intensity images generated through depth integration. The
green arrow indicates an artifact caused by accommodation (and
according shift of focus position) of the subject. e-h)
Corresponding AO-OCTA images extracted at the same
locations as in a-d. (The red arrow in e) points to a vessel that
clearly shows increased contrast in this image compared to
the intensity image. The red arrow in h) indicates areas with low
signal intensity(Salaset al. 2017)

61. Adaptive
Optics
Provideground
truthsfordeep
learningsuper-
resolution
techniquesto
improveOCT
resolutionfromnon-
AOacquisitionas
well
Improving lateralresolutionand image quality ofoptical coherence tomography by the
multi-frame superresolution technique for 3D tissue imaging
KaiShen,HuiLu,SarfarazBaig,andMichaelR.Wang Biomed.Opt.Express 8(11)4887-4918(2017)
https://doi.org/10.1364/BOE.8.004887
Wide field-of-view high lateralresolutionoptical coherence tomography with3D image
stitching and superresolution
KaiShen;SarfarazBaig;GuominJiang;MichaelR.Wang. ProceedingsVolume10483,OpticalCoherence
TomographyandCoherenceDomainOpticalMethodsinBiomedicineXXII;104832R(2018);doi: 10.1117/12.2287480

62. Adaptive
Optics
Technology
Field-of-Viewvery
narrowstillwithAO
systems
Increasingthe fieldof view ofadaptive opticsscanninglaserophthalmoscopy
Marie Laslandes, MatthiasSalas, Christoph K. Hitzenberger, and Michael Pircher
Medical Universityof Vienna, CenterforMedical Physics and Biomedical Engineering
Biomedical OpticsExpressVol. 8, Issue 11, pp. 4811-4826 (2017)
https://doi.org/10.1364/BOE.8.004811
Typical AO systems support patches on the retina between
1°×1° and 2°×2° [Bedggoodetal.2008]. This limit is due to
anisoplanatism, which is a well known problem in AO systems.
A technique to increase the FoV of AO instruments, known as
multi-conjugate AO (MCAO),wasdevelopedin astronomy[
Langloisetal. 2004]. MCAO can be applied to ophthalmic
imaging [Bedggoodetal.2006]. An AO flood illumination
funduscamerawith2 deformable mirrors (DM)wasdeveloped,
providing images of the retina on a 7°×7° FoV, for a 6 mm pupil
diameter [Thaunget al.2009]. This instrument uses five guide
stars to perform a uniform correction over the entire FoV. The
WFE is indeed measured on five points in the FoV by analyzing
the SH spots of these five reference sources on a single
detector.
Images recorded with our 2 DM set up (4°×4° FoV) are
compared with images obtained using only a single DM and
with images that were recorded on a small FoV (typical AO
mode). We also imaged a 10°×10° FoV by stitching 9
acquiredretinalimages.
Theoretically, the FoV of the instrument could be further
increased, still using the same concept. Currently, the size of
the second DM is a limitation as it has to accommodate the
entire scanned field. Thus, a larger DM diameter would be
requiredtosupportalargerFoV.
AO-SLO images of the model eye. (a) Image obtained without correction. (b)
Image obtained by correcting only with DM69 (Magnetic DM, Alpao). (c)
Image obtained with 2 DMs correction. The same grey scale is used for each
image.
Characterization oftheimagequalityofthethreeimages
from above. (a) Histogram of the pixel values. The
arrows indicate the maximum of the distributions. (b)
Verticalsectionoftheauto-correlationfunction.

63. Adaptive
Optics
Clinical
Applications
Review
Adaptive optics retinal imaging:
emerging clinicalapplications
Pooja Godara, AdamM. Dubis, Austin Roorda,
JacqueL. Duncan, and Joseph Carroll
OptomVisSci.2010Dec; 87(12): 930–941.
doi: 10.1097/OPX.0b013e3181ff9a8b | Cited by100
What does the future hold for retinal imaging with AO in
the clinic? While established as a powerful research
tool for nearly two dozen research groups worldwide,
AO has yet to achieve widespread clinical use. This is
not likely due to a lack of clinical utility, rather a lack of
clinical access and availability in addition to the time
required to obtain, process, and analyze the
images.
Adaptive optics retinal imaging–
clinical opportunitiesand
challenges
David B.Kay,DrewScoles,Alfredo Dubra &Marco Lombardo
CurrentEyeResearch Volume 38, 2013 - Issue 7
doi: 10.3109/02713683.2013.784792 | Cited by49
While AO is unquestionably a powerful research tool,
many clinicians remain undecided on the clinical
potential of AO imaging – putting many at a crossroads
with respect to adoption of this technology. This review
will briefly summarize the current state of AO retinal
imaging, discuss current as well as future clinical
applications of AO retinal imaging, and finally provide
some discussion of research needs to facilitate
more widespread clinicaluse.
Review ofadaptive opticsOCT
(AO-OCT): principlesand
applicationsforretinalimaging
MichaelPircherand RobertJ Zawadzki
Biomedical OpticsExpressVol.8, Issue5,pp. 2536-2562(2017)
https://doi.org/10.1364/BOE.8.002536
Application of AO-OCT for evaluating a patient with geographic
atrophy. Panel A is the color fundus photograph (CF) with the
multifocal electroretinogram (mfERG) traces and micro
perimetry (mP) sensitivity superimposed. Panel B shows the
mfERG response density map; panel C shows the mP
sensitivity map superimposed on the Fundus Auto
Fluorescense (FAF), and panel D is the FAF image. The three
numbered green lines in panel D correspond to the three B-
scan montages shown below. The magenta arrow in B-scan 1
shows the preferred retinal locus of the patient. The red, blue
and yellow bars on the B-scans correspond to ELM, IS/OS and
RPE loss, respectively. The magnified B-scan section shows
remaining RPE that corresponds to the location of the preferred
retinal locus [Panorgias et al.2013]

64. Adaptive
Optics
Clinical
Applications
Diabetic Retinopathy #1
Severalgroups have reported
on early and latechanges in the
retinal micro-vasculature using
OCTA[Freiberget al.2016; Hwang etal.2016;
Couturieret al.2015; Agemyetal.2015; Hwanget al.2015]
.
Visualizationof micro-capillaries
using opticalcoherence
tomography angiography with
and without adaptive optics
MatthiasSalaset al. (2017)
Biomedical OpticsExpressVol. 8, Issue 1, pp. 207-222
https://doi.org/10.1364/BOE.8.000207
OCTA and AO-OCTA images of a patient with
diabetic retinopathy. (A) Overview OCTA image
recorded with a commercial instrument (AngioVue,
Optovue, AngioPlex OCT; the red square indicates the
region of interest that has been imaged using AO-
OCTA). (B) Enlarged region of interest (indicated by
the red square in (A) depth integrated over the anterior
layers (region 1 and 2 in Fig. 2). (C) The same region as
in (B) but depth integrated over deeper retinal layers
(region 3 and 4 in Fig. 2). (D) OCT B-scan recorded at
the center of the region of interest shown in (A). (E)
En-face AO-OCTA image depth integrated over the
region between the green lines shown in (G). (F) En-
face AO-OCT intensity image depth integrated over
the same region as in (E). (G) AO-OCTA B-scan
showing the microaneurysm (H) AO-OCT intensity
image at same location asin (G).
The red arrows in the images show the location of a
microaneurysm. The green arrows point to a hard
exudate. The blue arrows indicate a small capillary
that appears to perform a twisted loop and is
embedded in highly scattering media. The
increased contrast provided by AO-OCTA
enables the visualization of a small capillary (yellow
arrow) that is not visible in the AO-OCT intensity
image

65. Adaptive
Optics
Clinical
Applications
Diabetic Retinopathy #2
In addition to vasculature, cone
properties can be used as a
biomarker for DR progression
Analysisof Cone Mosaic Reflectance Properties
inHealthy Eyesand in EyesWith Nonproliferative
Diabetic Retinopathy Over Time
LetiziaMariotti; NicholasDevaney;Giuseppe Lombardo;MarcoLombardo
AppliedOptics Group,School ofPhysics,National University ofIreland,Galway,Ireland; Istitutoper i Processi Chimico-
Fisici; Vision Engineering Italy srl; Fondazione G.B.Bietti IRCCS
Investigative Ophthalmology& Visual Science August 2017, Vol.58, 4057-4067
doi: 10.1167/iovs.17-21932
While the spatial arrangement of the cones still is the most studied
property of the cone mosaic
their light-reflecting properties recently
have become the subject of an increasingly number of
studies. Nonetheless, the investigation of the light-reflecting properties
of cones in retinal diseases still is limited, even if in some clinical cases
the cone reflectance has been the only apparent feature of the cones
that distinguished a healthy cone mosaic from a mosaic with altered
functionality.
The study of cone reflectance deserves attention, as it potentially could
lead to a deeper understanding of the cone cell physiology or
pathophysiology, which cannot be inferred by their spatial distribution
alone. In this view, the development of quantitativemetrics, whichcan be
automated for extending the benefits of high-resolution retinal imaging
to large populations, is expected for capturing clinically valuable
information (Agurtoetal. 2011).
In conclusion, we observed significant differences in cone mosaic
reflectance properties between healthy eyes and eyes with mild
NPDR, in its spatial organization and in its intensity, especially
between directionalandnondirectionalbackscattering.
Illustration of the method used for the evaluation of the
contribution of the inner retina on the cone mosaic intensity.
Sample areas of the cone mosaic and of the image focused on the inner
retina (200 × 200 μm)areselected on thesameretinallocation. Theimage
of the inner retina is low pass filtered, to exclude all frequencies that
correspond to the cones (Yellott's ring) or smaller features. The low pass
filtered imagethen issubtracted fromtheconelayerimage.
Marginal mean variogram curves for the
study (red curve) and control (blue curve)
groups with ±1 SD (dashed curves). ost of
the NPDR cases showed variogram curves
having a shape not leveling to a maximum
value, but rather some showed a peak and
then decreased again, while others showed
two peaks
Variograms of cone intensities for two
subjects (C2 and NPDR3), before and after
the subtraction of the low frequency
intensities as measured from the inner
retina images. The subtraction process
causes a flattening of the curves and a
steepening of the slope at the origin.
Where present, the peaks also are
suppressed.

66. Adaptive
Optics
Clinical
Applications
Age-related Macular
Degeneration
ConeStructureImagedWithAdaptiveOptics
ScanningLaserOphthalmoscopyinEyes WithNon-
neovascularAge-RelatedMacularDegeneration
Shiri Zayit-Soudry; JacqueL.Duncan; ReemaSyed; Moreno Menghini; Austin J. Roorda
Department ofOphthalmology,University ofCaliforniaat San Francisco
Investigative Ophthalmology& Visual Science August 2013, Vol.54, 7498-7509.
doi: 10.1167/iovs.13-12433 | Cited by40 articles
Adaptive optics scanning laser ophthalmoscopy provides adequate
resolution for quantitative measurement of cone spacing at the margin of GA
and over drusen in eyes with AMD. Although cone spacing was often normal
at baseline and remained normal over time, these regions showed focal areas
of decreased cone reflectivity. These findings may provide insight into the
pathophysiology of AMD progression. (ClinicalTrials.gov number,
NCT00254605.)
Appearanceofmedium–largedrusenandreticular
pseudodrusenonadaptiveoptics inage-related
maculardegeneration
Giuseppe Querques, CynthiaKamami-Levy, Rocio Blanco-Garavito, Anouk Georges, Alexandre Pedinielli,
VittorioCapuano, Fanny Poulon, Eric HSouied. | Departmentof Ophthalmology, Centre Hospitalier Intercommunalde
CreteilUniversityParisEst Creteil,Creteil, France;Institutd'Optique GraduateSchool, Palaiseau,France
Br JOphthalmol (2014). doi: 10.1136/bjophthalmol-2014-305455
AO allows differences in reflectivity between medium–large drusen
and reticular pseudodrusen to be appreciated. The cone mosaics may
be detected as continuous ‘bright’ hyper-reflective dots overlying/on the
border of drusen and pseudodrusen deposits, and possibly as continuous
‘dark’hyporeflective dotsoverlyingdrusenonly
Multimodalimagingofsmallhardretinal
druseninyounghealthy adults
HildeR Pedersen, StuartJ Gilson, AlfredoDubra, IngerChristineMunch,
Michael Larsen, RigmorCBaraas UniversityCollege of Southeast Norway,
Kongsberg,Norway; Department of Ophthlmology,StanfordUniversity; Department of
Ophthalmology,ZealandUniversity; Universityof Copenhagen /Rigshopsitalet,Copenhagen,
Denmark
Investigative Ophthalmology& Visual Science August 2013,
Vol.54, 7498-7509. doi: 10.1167/iovs.13-12433 | Cited by40
articles
Multimodal image montage of a small hard drusen (49 µm) in left eye of a
female aged 19 years (participant 5007). Colour fundus photograph (A)
show small hard drusen (arrows). SD-OCT B-scans were taken along the
yellow line on the infrared image (B). The white box represents the area of the
AO flood image (C). Segment (D) show the drusen located within the retinal
pigment epithelium (RPE) complex at 2° temporal/4° superior retinal
eccentricity. Line plots represent reflectivity profiles through the corresponding
OCT image at the location of drusen (black arrow in (A), solid yellow arrow and
black line in (D)) and control area (dashed arrow and dashed line in (D)). The
gap in the interdigitation zone band (IZ: red arrow) corresponds with the
hyporeflective area in the cone mosaic in the AO flood image (C). ELM, external
limitingmembrane;EZ, ellipsoidzone;ILM, internal limiting membrane.