1. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk1 Spectral Imaging In a Snapshot Andrew R Harvey*, David W Fletcher-Holmes, Alistair Gorman School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh, UK Kirsten Altenbach, Jochen Arlt and Nick D Read COSMIC, The University of Edinburgh, Edinburgh, UK *a.r.harvey@hw.ac.uk

2. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk2 Presentation outline Why another spectral imaging technique? IRIS:image replication imaging spectrometry Design issues Example applications Retinal imaging Microscopy Conclusions

3. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk3 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…

4. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk4 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 Spectral imaging provides a non-invasive route to monitoring retinal biochemistry Blood oximetry, lipofuscin accumulation Diabetic Retina Normal Retina

5. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk5 Requirements for a snapshot technique: retinal imaging Improved calibration Patient patience Remove misregistration artefacts; imperfect coregistration arises due to Distortion of eye ball with pulse Variations in imaging distortion between images Similar issues with other in vivo applications Imaging epithelial cancers PC15

6. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk6 Image Replication Imaging Spectrometer: IRIS Snapshot image zero temporal misregistration 100% optical efficiency Conceptually related to Lyot filter Large format detector Spectral Demultiplexor

7. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk7 Lyot filter: principle of operation Polariser Waveplate

8. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk8 Wollaston prism polarisers replicate images Each Wollaston prism-waveplate pair provides both cos 2 and sin 2 responses All possible products of spectral responses are formed at detector IRIS snapshot spectral imager:

9. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk9 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 8 channel visible-band system 520nm 820m 3 Quartz retarders 32 channel, visible-band system 520nm 720nm 5 Quartz retarders

10. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk10 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 stop Collimating lens Bandpass filter Imaging lens Camera Polariser, retarders & Wollaston prisms (index matched) Primary lens

11. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk11 Modelling and ray-tracing 15mm prisms 20mm prisms 25mm prisms 30mm prisms 16mm lenses 25mm lenses35mm lenses 50mm lenses 8 channel system

12. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk12 Components & Assembly 8 channel system 520nm to 820nm 3 Quartz retarders 3 Calcite Wollaston prisms

13. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk13 Measured & predicted spectral responses

14. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk14 Absolute total transmission Bandpass filter & polariser dominate losses Improved system: T>80% Theoretical throughput is 2 n times higher than for spatial/spectral multiplexed techniques! 0 25 50 Response (%) Absolute response curves in polarised light

15. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk15 Blood oximetry Optimal spectral band for retinal oximetry Vessel thickness ~ optical depth 570-615 nm Eight bands approximately equally spaced 40 20

16. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk16 Spectral Retinal Imaging Difficult imaging conditions render application of traditional HSI techniques problematic IRIS enables real-time and snapshot spectral imaging Canon

17. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk17 Video sequence recorded with bandpass filtered inspection lamp

18. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk18 Retinal image recorded with flash illumination

19. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk19 574581 585 592595 603 607 613 Coregistered and PCA images PC1PC2PC1 & PC2

20. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk20 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 nm 0 2525 5050 Response (%)

21. Photonics West, 2005 Andy Harvey: a.r.harvey@hw.ac.uk21 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 0.2 m to 12 m Applications In vivo, in vitro imaging Retinal imaging Microscopy Multiple fluorophors Quantum dots Surveillance Remote sensing Etc.