BM3A.4.pdf
Optics in the Life Sciences 2015 © OSA 2015
Polarization Properties of Amyloid Beta in the Retina of the Eye as a Biomarker of Alzheimer’s Disease Melanie C.W. Campbell1,2,3, David DeVries1, Laura Emptage1, Chris Cookson1, Marsha Kisilak1,2, Juan M. Bueno4, Francisco J. Avila1,4 1
Physics and Astronomy and 2School of Optometry and Vision Science, University of Waterloo, Waterloo, Canada, N2L3G1 3 Guelph Waterloo Physics Institute, Waterloo, Canada, 4 Laboratorio de Óptica, Universidad de Murcia, Campus de Espinardo (Ed. 34), 30100 Murcia, Spain Author e-mail address: (
[email protected])
Abstract: Polarimetry was performed on amyloid deposits (thioflavin S positive) in human postmortem retinas of those with a diagnosis of Alzheimer's disease. Mueller matrix properties and polarization contrast of this biomarker show promise as a non-invasive diagnostic. OCIS codes: (170.4580) Optical diagnostics for medicine; (330.7327) Visual optics, ophthalmic instrumentation; (110.5405) Imaging systems: Polarimetric imaging.
1. Introduction The retina, with its neural tissue, also acts as a window to the brain. We are using this property to develop a novel, non-invasive, and less expensive diagnostic for Alzheimer’s disease. Alzheimer’s disease is a neurodegenerative disease, characterized by the formation of insoluble fibrils (plaques) composed of amyloid beta proteins. Alzheimer’s disease is currently only definitively diagnosed after death from an analysis of deposits of amyloid beta in the brain. Retinal function has been reported to be directly affected by Alzheimer’s disease and neurotoxic effects of amyloid have been demonstrated in the retina. However, amyloid had not been found reliably in the retinas of those diagnosed with Alzheimer’s disease before the work of our group and one other [1-5]. In the past, amyloid beta has been imaged with the dye Congo Red, which gives a unique birefringence signature when illuminated under cross polarization [6]. The retina has polarization properties which change in some diseases [7]. Ours are the first measurements of the polarization properties of unstained presumed amyloid beta deposits in neural tissue [1-3]. Unstained presumed amyloid beta deposits also have polarization properties which can be quantified via Mueller matrix polarimetry [2]. We have previously used a combined Atomic Force Microscope (AFM)/fluorescence microscope to study fluorescently labeled structures near the retinal surface in some retinas, positive for AD or dementia. Simultaneous imaging in fluorescence and AFM allowed direct image overlay of fluorescence, characteristic of deposits and structural measurements [1]. Co-located AFM images confirmed that some structures positive for thioflavin S fluorescent were located close to the surface of the retina adjacent to or within neural cell layers. These showed beta sheet structure consistent with the protein, amyloid beta. We have also imaged amyloid beta plaques in non-stained human retinas samples with multiphoton microscopy [5]. We have shown that some polarization properties of presumed amyloid beta deposits and of pure amyloid beta differ from surrounding areas [2]. The neural retina is optically accessible so polarization properties could enable non-invasive detection of amyloid beta deposits in the living eye. Here we wished to characterize which polarization properties of amyloid deposits in the retinas of humans with Alzheimer’s disease are the best candidates to be used as part of a polarization based diagnostic. Polarimetry could then form the basis for a non-invasive method of imaging amyloid deposits in the retina and could allow objective, longitudinal tracking of Alzheimer’s disease and its treatments. Here we report on properties in addition to diattenuation, polarizance, birefringence and depolarization, reported previously [2, 3]. 2. Methods Retinas were dissected from eyes obtained following informed consent under the auspices of the Eye Bank of Canada (Ontario) and fixed in paraformaldehyde for patients with a diagnosis of AD and age matched normals without AD or glaucoma. Post dissection, retinas were flat mounted and stained with 0.1 % thioflavin-S [8] and then studied using fluorescence microscopy and a polarimeter on the same microscope. Thioflavin stains amyloid proteins and amyloid beta deposits were expected in neural layers of the retina, close to the anterior surface of the retinas of those with Alzheimer’s disease.
BM3A.4.pdf
Optics in the Life Sciences 2015 © OSA 2015
We studied the polarization properties of the amyloid deposits found in the ex vivo retinas. We enumerated the fluorescing deposits with and without polarization contrast. We also searched for locations showing polarization contrast without fluorescence. For each location, in red light, 16 images were examined using 4 positions of each of the generator and analyzer as well as images in crossed polarization. The apparatus consisted of a modified, inverted Nikon microscope, incorporating a polarimeter to obtain the spatially resolved Mueller matrix of the samples. The polarization state generator and analyzer units were each composed of a fixed linear polarizer plus a rotating quarterwave plate, symmetrically arranged. 16 polarimetric images corresponding to 4 independent combinations of polarization states of the generator and analyzer were obtained. These states were generated by setting the axes of both quarter-wave plates at -45º, 0º, 30º and 60º while keeping the linear polarizers fixed. Mueller matrices were then calculated for areas with deposits and for control areas. From the spatially resolved Mueller matrices, many polarization properties were calculated as a function of position. Properties were compared between deposits and surrounding retina and between deposits in retinas from those with Alzheimer’s disease and areas in control retinas of age matched subjects without the disease or any dementia. 3. Results The majority of thioflavin positive deposits showed polarization contrast. Not all thioflavin positive deposits showed identical polarization contrast but there was at least one deposit showing polarization contrast in each of the retinas from those with positive staining. All retinal regions positive for polarization contrast were positive for thioflavin staining. Mueller matrix results were similar to those reported previously by us for human retinas with a diagnosis of Alzheimer’s disease [2, 3]. For a number of polarization properties, the average values across of the deposits differed significantly from those of the surrounding retinal tissue and from those of retinal tissue in control retinas. However, there were some deposits for which the average values of polarization properties did not differentiate the deposit from the surround. In many of these cases, there was still polarization contrast present from values within the deposit that were both above and below those of the surrounding tissue. We assume that the varying orientation of fibrils within the deposits produces this signature. For most deposits, polarization properties derived from interactions with linearly polarized light were sufficient to produce image contrast but interaction with circularly polarized light sometimes improved contrast or sensitivity. We present combinations of properties which give the best differentiation of presumed amyloid beta deposits from the surrounding tissue. 4. Conclusions From the knowledge that we have gained of the polarization properties of presumed amyloid beta in the retinas of those with Alzheimer’s disease, we are designing an in vivo, non-invasive method of imaging the retina to detect amyloid deposits in association with the disease. Polarization imaging will provide contrast of amyloid deposits against the surrounding retina with high sensitivity and specificity. 5. Acknowledgements This research was supported by CIHR and NSERC Canada. Vince Choi assisted in the development of some of the methods described here. 6. References [1] M. C. W. Campbell, L. Gowing, Y. Choi, Z. Leonenko “Imaging of amyloid-beta deposits in the post-mortem retina in Alzheimer’s disease.” Invest. Ophthalmol. Vis. Sci. 50, Abstract 5778 (2010). [2] M. C. W. Campbell, F. Avila, L. Emptage, M. L. Kisilak, and J. M. Bueno, “Polarimetry in ex vivo retina from donors with Alzheimer’s disease,” Frontiers in Optics, paper FW5F.4 (2013). [3] L. Emptage, M. L. Kisilak, M. Wilson, Z. Leonenko and M. C. W. Campbell, “Sensitivity and specificity of fluorescence and polarimetry of the retina in Alzheimer’s disease,” ARVO Meeting Abstracts 55, 3366 (2014). [4] M. Koronyo-Hamaoui, Y. Koronyo, A. V. Ljubimov, C. A. Miller, M. K. Ko, K. L. Black, M. Schwartz, and D. L. Farkas, “Identification of Amyloid Plaques in Retinas from Alzheimer’s Patients and Noninvasive In Vivo Optical Imaging of Retinal Plaques in a Mouse Model.” Neuroimage. 54, S204-S217 (2011). [5] F. J. Avila, L. Emptage, R. Palacios, M. L. Kisilak, P. Artal, M. C.W. Campbell and J. M. Bueno, “Two-photon excitation fluorescence microscopy of -amyloid deposits in retinal tissues affected by Alzheimer disease,” ICO-23 Meeting, pg. 60 (2014). [6] P. Divry, C. R. Florkin, “Sur les pro- priétées optiques de l'amyloide.” C R Soc Biol. 97, 1808-1810 (1927). [7] N. T. Choplin, D. C. Lundy, A. W. Dreher “Differentiating patients with glaucoma from glaucoma suspects and normal subjects by nerve fiber layer assessment with scanning laser polarimetry.” Ophthalmology 105, 2068-2076 (1998). [8] S. Perez et al. “Beta-amyloid deposition and functional impairment in the retina of the APPswe/PS1DeltaE9 transgenic mouse model of Alzheimer's disease,” Invest. Ophthalmol. Vis. Sci. 50, 793-800 (2009).
