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1PIBM Sept 05 Andy Harvey
Spectral Imaging of the Retina
Andy R.Harvey, Ied Abboud, Alistair Gorman, Andy I.McNaught*
School of Engineering & Physical Sciences,
Heriot Watt University, Edinburgh, UK
*Eye Unit,Cheltenham General Hospital,
Cheltenham, UK
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2PIBM Sept 05 Andy Harvey
Outline
• What is spectral imaging?• Spectral retinal imaging
• Why?• Spectral time-sequential spectral imaging
• For flexibility and research
• 2D snapshot• For real-time, high throughput screening
• Conclusions
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3PIBM Sept 05 Andy Harvey
Conventional Spectral Imaging
RGB Image Spectrally classified image
Dysplasticcell
Superficialsquamouscell
Intermediatecell
Lymphocyte
PMN
Courtesy CRI
Classificationspectra
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4PIBM Sept 05 Andy Harvey
Imaging the eye
Sclera
Cornea
Iris
Lens
Retina
Macula
Vitreous humour
Light
Anterior chamber (full of aqueous humour)
Optic nerve
Posterior chamber
Choroid
Optic disc
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5PIBM Sept 05 Andy Harvey
The Role of Spectral Retinal Imaging
• By 2020 there will be 200 million visually-impaired people world wide• Glaucoma, diabetic retinopathy, ARMD• 80% of those cases are preventable or
treatable • Screening and early detection are
crucial • Can spectral imaging offer
enhancements to current screening techniques ?
• Spectral imaging is non-invasive and safe• cf. fluorescein angiogram
• Spectral imaging can enable imaging of • Retinal biochemistry
• Blood oximetry• Diabetic retinopathy, glaucoma
• Lipofuscin etc• Age-related macula degeneration
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6PIBM Sept 05 Andy Harvey
Spectral Imaging:Traditional approaches
And Fourier-transform equivalents
N(t)
NxNy
N
NxNy(t)
Time-sequential spectral multiplex
Time-sequential spatial multiplex
• Limitations
• Optically inefficient
• 2D time-varying scenes
• 2D snapshot
• required for:• Retinal
imaging• in vitro, in
vivo imaging
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7PIBM Sept 05 Andy Harvey
Spectral Fundus Camera
• Source filtering by LCTF incorporated into COTS fundus camera• 10 nm spectral width• 20 msec random access
• Images captured using a cooled, low-noise CCD camera
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8PIBM Sept 05 Andy Harvey
1.00
10.00
100.00
1000.00
10000.00
200 400 600 800 1000
Wavelength (nm)
Abs
orpt
ion
Coe
ffici
ent (
cm-1
)
Absorption Coefficient for HbO2 (cm-1)
Absorption Coefficient for Hb (cm-1)
Isobestic point
Coregistered Spectral Images of a Healthy Retina
800nm
Images translationally and rotationally coregistered
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9PIBM Sept 05 Andy Harvey
Spectral angle map of healthy and diabetic retina
• Shading indicates similarity of each pixel spectrum with artery and vein spectra• Qualitative oxymetry
Normal Retina
Diabetic Retina
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10PIBM Sept 05 Andy Harvey
Supervised spectral classifiaction
• Implicit calibration based on spectral signatures within the eye• Classification possible
without absolute calibration
• Clear distinction between veins/arteries, on/off optic disc • Spectra depends on
local environment
• Inversion of data to calculate biochemical concentrations (eg oxygenation) requires a model of light propagation and scattering in the retina to remove environmental effects
• Monte Carlo, Kubelka Monk, Transfer equation
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11PIBM Sept 05 Andy Harvey
Requirements for a snapshot technique: retinal imaging
• Improved calibration
• Patient patience
• Remove imperfect coregistration
• due to Variations in imaging distortion between images
• Similar issues with other in vivo applications
• Imaging internal epithelial cancers
• Eg gastrointestinal
PC15
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12PIBM Sept 05 Andy Harvey
Image Replication Imaging Spectrometer: IRIS
Snapshot image• zero temporal misregistration
• ‘100%’ optical efficiency• World’s only snapshot, 2D
spectral imager (almost !)• Conceptually related to Lyot
filter
Large formatdetector
SpectralDemultiplexor
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13PIBM Sept 05 Andy Harvey
Lyot filter: principle of operation
n=1 � l Cos2@pîDDCos2@pîDDCos2@2pîDDCos2@pîDDCos2@2pîDDCos2@4pîDDCos2@pîDDCos2@2pîDDCos2@4pîDDCos2@8pîDD
PolariserWaveplate
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14PIBM Sept 05 Andy Harvey
Exploded view of N Wollaston prisms N wave plates
2N spectral images at detector Field
stop
Input polarizer
• Wollaston prism polarisers replicate images• Each Wollaston prism-waveplate pair provides both cos2 and sin2 responses
• All possible products of spectral responses are formed at detector
)(sin
)(cos2
2
)2(sin
)2(cos2
2
)4(sin
)4(cos2
2
IRIS snapshot spectral imager: principle of operation
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15PIBM Sept 05 Andy Harvey
Spectral transmission
cos2
sin2
cos2(cos2(2)
sin2 (
cos2 (2)
cos2(sin2(2)
sin2(sin2(2)
cos2(cos2(2)cos2(4) cos2(sin2(2)cos2(4)
cos2(cos2(2)sin2(4) cos2(sin2()sin2(4)
sin2(cos2(2)cos2(4) sin2(sin2(2)cos2(4)
sin2(cos2(2)sin2(4) sin2(sin2()sin2(4)
Wollaston/waveplateassembly
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16PIBM Sept 05 Andy Harvey
Spectral responses
• Bands are overlapping bell shapes• Choose cost function to minimise sidelobes
• Small (~5%) reduction in spectral separation• Cut-off filters used to define spectral range
Theoretical system response
0
20
40
60
80
100
450 500 550 600 650 700 750 800 850
Wavelength (nm)
Res
po
nse
(%
)
•8 channel visible-band system
•520nm820m
•3 Quartz retarders
•32 channel, visible-band system
•520nm 720nm
•5 Quartz retarders
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17PIBM Sept 05 Andy Harvey
Optical scaling laws
Hamamatsu
ORCA-ER
Inputs:
FoV
Sub image size on CCD
CCD pixel size
Primary lens magnification & F#
Collimating lens back focal distance, focal length & front element diameter
Prism birefringence
Outputs:
Field stop size
Collimating lens rear element diameter
Splitting angles, apertures & depths of prisms
Apertures of retarders, polarisers and filters
Imaging lens focal length & front element diameter
Field stopCollimating
lens
Bandpass
filter
Imaging
lens
Camera
Polariser, retarders & Wollaston prisms
(index matched)Primary lens
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18PIBM Sept 05 Andy Harvey
Components & Assembly
• 8 channel system• 520nm to 820nm• 3 Quartz retarders• 3 Calcite Wollaston prisms
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19PIBM Sept 05 Andy Harvey
Spectral Retinal Imaging • Difficult imaging conditions render application of traditional HSI
techniques problematic• IRIS enables real-time and snapshot spectral imaging
Canon CR4-45NMCR4-45NM
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20PIBM Sept 05 Andy Harvey
Blood oximetry
• Optimal spectral band for retinal oximetry• Vessel thickness ~ optical depth• 570-615 nm• Eight bands approximately equally spaced
0
2
4
6
8
10
12
14
16
18
20
565 575 585 595 605 615 625
Wavelength (nm)
Tra
nsm
issi
on (
%)
40
20
80
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21PIBM Sept 05 Andy Harvey
Video sequence recorded with bandpass filtered inspection lamp
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22PIBM Sept 05 Andy Harvey
Retinal image recorded with flash illumination
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23PIBM Sept 05 Andy Harvey
574581585592595603607613
Coregistered and PCA images
PC1PC2PC1 & PC2
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24PIBM Sept 05 Andy Harvey
Summary
• Spectral imaging of the retina shows promise for non-invasive detection of retinal disease• Clinical trials on-going
• LCTF-based, time-sequential spectral filtering enables rapid and flexible 2D spectral retinal imaging• Flexible data acquisition• Pulse and other motion artefacts limit accuracy
• Snapshot spectral imaging in 2D (IRIS) promises high-performance real-time multi-spectral imaging• Ideal for in vivo imaging• No temporal misregistration
• Absolute, quantitative data requires a model of light interaction within the retina
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25PIBM Sept 05 Andy Harvey
• Wollaston prism polarisers replicate images• Each Wollaston prism-waveplate pair provides both cos2 and sin2 responses
• All possible products of spectral responses are formed at detector
Exploded view of N Wollaston prisms N wave plates
2N spectral images at detector Field
stop
Input polarizer
IRIS snapshot spectral imager
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26PIBM Sept 05 Andy Harvey
Measured & predicted spectral responses
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27PIBM Sept 05 Andy Harvey
Absolute total transmission
• Bandpass filter & polariser dominate losses
• Improved system: T>80%
• Theoretical throughput is 2n times higher than for spatial/spectral multiplexed techniques!
0
25
50
Re
sp
on
se
(%
)
Absolute response curves in polarised light
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28PIBM Sept 05 Andy Harvey
Application to microscopy:Imaging of multiple fluorophors
• IRIS fitted to conventional epi-fluorescence microscope
• Germinating spores of Neurospora crassa stained with• GFP – nucleii fluoresce at 510 nm• FM4-64 – membranes fluoresce at >580 nm0
25
50
Re
sp
on
se
(%
)
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29PIBM Sept 05 Andy Harvey
Principle component decomposition
PC1
PC15
• Artery structure is a pulse artefact
• Very difficult to co-register by image processing means
• Snapshot technique desirable
PC3
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30PIBM Sept 05 Andy Harvey
Conclusions
• IRIS is a new spectral imaging technique that enables snapshot spectral imaging in 2D• No rejection of light• No data inversion
• Highest-possible signal-to-noise ratios• Simple logistics
• Inherently compact and robust• Simply fitted to conventional imaging systems
• Birefringent materials exist for applications from 0.2m to 12 m
• Applications• In vivo, in vitro imaging
• Retinal imaging• Microscopy
• Multiple fluorophors• Quantum dots
• Surveillance• Remote sensing• Etc.
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31PIBM Sept 05 Andy Harvey
• Optical depth of Hb & HbO2 dominates variation of penetration with
• Tissues vary between highly turbid and transparent• Blue light images retinal surface• Light at ~600 nm enables spectral oximetry within retinal blood vessels
• optical depth of HbO2 > vessel thickness so vessels translucent
• optical depth of Hb < vessel thickness so vessels are opaque
• Light > 640 nm penetrates to coroid
BlueGreenRed
1.00
10.00
100.00
1000.00
10000.00
200 400 600 800 1000
Wavelength (nm)
Abs
orpt
ion
Coe
ffici
ent (
cm-1
)
Absorption Coefficient for HbO2 (cm-1)
Absorption Coefficient for Hb (cm-1)
Isobestic point
Spectral Characteristics of the Retina
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32PIBM Sept 05 Andy Harvey
Issues for Spectral Retinal Imaging
• Calibration• Components of interest within a
complex turbid medium• Patient tolerance
• Using current technology, time-sequentialspectral bandpass offers• Optimal SNR• Reduced light intensity at the retina• Agile selection of spectral bands (data efficient)
• Issues• Coregistration• Calibration
± 100 pixels
±2º
• Spectral imaging of static scenes is relatively ‘easy’
• Spectral imaging of the retina encounters
• Imaging through an erratically moving, low-quality f/6 eye-lens system
Solutions: 2D snapshot spectral imaging
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33PIBM Sept 05 Andy Harvey
The End
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34PIBM Sept 05 Andy Harvey
1D image x path difference
Fixedmirror
Scanning mirror
Detector array
N
NxNy(t)
N
NxNy(t)
FTFT
N(t)
NxNy
N
NxNy(t)
Direct Imaging Spectrometry (Fourier) Transform Imaging SpectrometryT
emp
oral
ly s
can
ned
Sn
apsh
ot/f
ull
y st
arin
g
N(t)
NxNy
FT
N
NxNy
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35PIBM Sept 05 Andy Harvey
Why another spectral imaging technique?
• Traditional approaches• Time sequential spectral multiplex
• Monochromatic two-dimensional image in snapshot• Time sequential spatial multiplex
• One-dimensional spectral image in a snapshot• (and Fourier-transform equivalents)
• Problems• Cannot record two-dimensional spectral images of time-varying
scenes• Optically inefficient
• Time-resolved (snapshot) spectral imaging is required for• Dynamic scenes
• In vitro, in vivo imaging and microsocopy• Combustion dynamics, surveillance…
• Irregular motion between scene and imager• In vivo imaging• Ophthalmology• Remote sensing, airborne surveillance, industrial inspection…