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

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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

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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

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Cone Disarray Plot

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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

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…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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Normal eyes

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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

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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

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subject 1 subject 2 subject 3

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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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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

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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