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AO Instrumentation
Vision Science
Austin Roorda, PhDUniversity of California, Berkeley
CFAO Summer School 2007
IntroductionAs a vision scientist, you need first to concentrate on your science goals. These goals should drive your decision on the type of adaptive optics system you want to develop.
Basically, AO is an optical technique that offers microscopic optical access to living retina. Whether it is for stimulating a single cone, forming images of small features on the retina, measuring dynamic functional activity, or improving someone’s visual acuity to theretinal sampling limit will dictate exactly how and into what kind of system you implement adaptive optics.
In this talk, I will describe instrumentation that uses adaptiveoptics, from the simple to the most complex and describe the range of basic and clinical science applications associated witheach.
Once you’ve decided on your modality, then you need to know how to actually build the AO system…that’s Don Miller’s talk.
AO system for flood-illuminated imaging and
vision testing
imaging
wavefront sensing
wavefront correction
laser beacon
illumination
AO for AO for the eyethe eye
imagingvision testing
wavefront sensing
wavefront correction
laser beacon
illumination
AO for AO for the eyethe eye
No AO With AO
JW right eye1 deg eccentricity
image wavelength = 550 nm
Adaptive Optics Makes it Possible to See Microscopic Retinal Features
multiple AO frames
Courtesy of Heidi Hofer, Matt McMahon, David Williams
MD JP JC
YY
*
*
*
HS AP nasalAN
RS JW temporal BSJW nasal
AP temporal
5 arcmin Roorda and Williams, Nature, 1999Hofer et al, J Neurosci, 2005University of Rochester
Waveguide Properties of Cones
cone efficiency reduces with input angle
11
Light Delivery
EyeTranslating artificial pupil
Adaptive Optics Compensation
CCD
Light Delivery
EyeTranslating artificial pupil
Adaptive Optics Compensation
CCD
Light Delivery
EyeTranslating artificial pupil
Adaptive Optics Compensation
CCD
JP right eye1º eccentricity2 mm illumination pupil
T N
S
I
0,0
0.866,-1.5-0.866,-1.5
-1.73,0
-0.866,1.5 0.866,1.5
1.73,0
Cone Disarray Plot
T N
S
I
There is a systematic and measurable disarray among the photoreceptors …..
JP (275 cones) GY (200 cones)
Cone Disarray Projected into the Pupil PlaneCone Disarray Projected into the Pupil Plane
T N
S
I
1
2
0
1
2
0
…but all photoreceptors point in nearly the same direction
MM – all M conesNC – M cones absent
Has “normal” gene array Missing all L gene(s)
University of Rochester Carroll, Neitz, Hofer, Neitz, Williams, PNAS, 2004
Genotype-Phenotype comparisons
AO for Vision Testing
The eye may not prefer to have all of its aberrations corrected
Large stroke adaptive optics for abnormal eyes
52 channelDeformable mirror
WavefrontSensor
Eye
VisualStimulus
Customized Refraction Lab, Geunyoung Yoon, University of Rochester
Large stroke AO can provide high quality optics for both normal and keratoconic eyes.
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Totalaberration
HOaberration
Wav
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nt rm
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icro
ns)
Normal eyes
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Totalaberration
HOaberration
Keratoconic eyes
Without AO
With AO
With AO, visual acuity for keratoconic eyes was significantly worse than normal eyes.
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06 mm 2.5 mm 6 mm 2.5 mm
Pupil size
Visu
al a
cuity
(Log
MA
R) Visual acuity (Snellen)
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8.9
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20/Normal Keratoconus
NeurosensoryRetina
Choroid
Sclera
RodsCones
Horizontal Cells
Bipolar CellsAmacrine Cells
Ganglion Cells
Pigment Epithelium
Nerve Fibers
Inner Plexiform Layer
Outer Plexiform Layer
The Retina is a Thick, Multi-layered Structure~2
50 m
icro
ns
AO Scanning Laser Ophthalmoscopy
(AOSLO)
Adaptive Optics Scanning Laser Ophthalmoscope
In an SLO, AO improves:
• Throughput• Resolution• Contrast
1.2 degrees (~ 360 microns)
Confocal AOSLO
AOSLO confocalthrough-focus series
Dynamic Imaging: Leukocyte Velocity Measurement
• No fluorescent dyes required (safe, long term)• Identify ghost vessels (non-perfused capillaries)• 95% confident of velocity changes as small as 0.076 mm/sec
532 nm laser
Joy Martin
stabilized video difference video
Martin & Roorda, Ophthalmology 112(12): 2219-2224 (2005)
Imaging Function: Dynamic Stimulus Delivery
Allows for simultaneous measurement of optical andretinal limits to vision
Poonja et al, Journal of Refractive Surgery 21(5): 575-580 (2005)
Visual Acuity
No AO
AO 5.81mm
SnellenAcuity
20/1220/1120/1020/920/820/720/6
20/1320/13
20/515
20
25
30
35
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45
subject 1 subject 2 subject 3
MA
R (a
rc s
econ
ds)
Ethan Rossi
Optical Coherence Tomography(OCT)
http://www.fitzpatrick.duke.edu/biophotonics/research/OCT/slide_2.htm
OCT retinal cross section
Bigelow, Iftimia, Ferguson, Ustun, Bloom, Hammer. JOSA A, 2007
AO OFF->ON
AO Optical Coherence Tomography
courtesy of Jack Werner, UC Davis
cones
NFL
Focus at photoreceptors Focus at nerve fibers
1.25 degrees(375 microns)
AO-OCTB-scans at 2° retinal eccentricity
Courtesy of Donald Miller, Indiana UniversityZhang et al. Optics Express, 14(10): 4380-4394 (May 2006)
images courtesy of Robert Zawadzki, UC Davis
330 µm
ILM
IS/OS
Single frame
Avg of 60 frames
B-scan at 3° eccentricity with AO SD-OCT
38 µm
16 µm
16 µm 5 µm
Courtesy of Donald Miller, Indiana University
Resolution vs Contrast
• Optical resolution with AO is sufficient to resolve all cell bodies in the retina
• But most of the cells are transparent (fortunately for vision), so optical scattering methods are ineffective. In other words, the intrinsic contrast is low.
• There are a host of methods to improve contrast of targeted features in the retina
Improving Contrast:
Fluorescence
AOSLO Fluorescein Angiography(macaque retina)
Registered frame
University of RochesterGray et al, Optics Express 14, 7144-7158 (2006)
AOSLO movie
In vitro
Fluorescently labeled ganglion cell bodies and dendrites taken with a confocalmicroscope
Retrograde rhodamine labeled ganglion cells in the living macaque taken with AOSLO
Images courtesy of: Dan Gray, Bill Merigan, David Williams, University of Rochester
In vivo
Improving Contrast:
Autofluorescence
Autofluorescence image of the RPE mosaic in a macaque retina
100µm Courtesy of Morgan, Gray, Merigan, Williams, U Rochester
Courtesy of Heidi Hofer, Matt McMahon, David Williams100µm
Flood-illuminated image of the cone mosaic in a human retina
Improving Contrast:
Polarization
Deselecting polarized light removes specular reflections
patient with epiretinal membrane
raw SLO image depolarized component
AO permits PS-OCT to probe smaller structures.
900 µm
34 dB
0 dB
0°
40°
Henle’s fiberlayer induces
phase retardation.
Intensity
Double pass phase retardation
0°
40°0dB
34dB
~490
µm
Higher lateral resolution and smaller speckle size reduce spatial averaging in the polarization analysis.
Cense et al., Indiana University
Improving Contrast:
Wavelength
830 nm
drusen
543 nm
Retinal vessels
488 nmMacular pigment, nerve fiber layer
633 nm
Some nerve fiber layer
514 nm
Some macularpigment, melanin
Elsner et al., Vision Research, 1996
Courtesy of Steve Burns and Ann Elsner, Indiana University
Red vs Green AOSLO video
660 nm light 532 nm light
Improving Contrast:
Motion Contrast
Imaging Blood Flow
R. Ferguson, D. Hammer, A. Elsner, R. Webb, S. Burns, and J. Weiter, Opt. Express 12, 5198-5208 (2004)
Functional Imaging
• Imaging the anatomy on a microscopic scale with AO can only tell us so much
• There have been many efforts in the last few years to make structure function relationships
Imaging Retinal Function
Intrinsic Retinal Signals
Cone Scintillation Indicates Visual Activity
control
test
Don Miller, Indiana University
Functional Imaging via Reflectance Changes
infrared imaging beam
red stimulus beam
Kate Grieve, UC Berkeley
Origin of Cone Signals
Kate Grieve, UC Berkeley
Imaging Retinal Function
Eye Tracking
Eye Movement Tracking
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-10
-5
0
5
10
15
0 0.25 0.5 0.75 1 1.25 1.5Time (seconds)
Posi
tion
(arc
min
utes
)
horizontalvertical
960 Hz eye trace, accurate to one image pixel (0.14 minutes of arc)
original video stabilized video
Scott Stevenson, Houston; David Arathorn, Curt Vogel, Al Parker, Qiang Yang, MSU
The fixation point is displaced about 10’ of arcfrom the point of maximum cone density
What is the fovea?
50µm
Putnam et al, Journal of Vision 5(7):632-639 (2005) )University of Rochester
Visual Psychophysics
saccades pursuit
Where does the eye place the image immediately after a saccade?
Where does the eye place an image that it is pursuing?
Scott Stevenson, Girish Kumar, University of Houston
What is the fovea?
Scott Stevenson, Girish Kumar, University of Houston
Where am I looking? (WAIL)
Scott Stevenson, Girish Kumar, University of Houston
Imaging Retinal Function
Small Spot Stimulation
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1 arcminute
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Without adaptive optics With adaptive optics
Adaptive optics increases the fraction of light absorbed by a single cone
6 mm pupil 550 nm
Courtesy: Heidi HoferHofer et al. University of Rochester
Delivery of AO-corrected Stimuli
All stimuli seen Some stimuli not seen
Makous and Carroll, University of Rochester
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Log Luminance
Prop
orti
on o
f D
etec
ted
Flas
hes
normals: open symbols
AO-Microperimetry confirms cone drop-out
Makous et al, IOVS 47(9): 4160-4167 (2006)University of Rochester
Hofer et al. J Vis. 2005 May 19;5(5):444-54.
AOSLO - Stabilized Stimulus Delivery
Future Applications
• novel anatomical imaging– 2-photon– phase contrast imaging
• novel functional imaging– activity dependent dyes – combined AO imaging and electrophysiology
• novel applications– targeted photodynamic therapy– microsurgery
Combined Stimulus Delivery and Electophysiology