Two-photon Microscopy to Visualize Amyloid Plaques in Unstained Retinas from Alzheimer’s Disease

Abstract

Purpose: Alzheimer’s disease (AD) is a neurodegenerative process characterized by the formation of insoluble plaques of amyloid β (Aβ) proteins in brain tissues. Classical microscopy techniques have shown Aβ deposits in stained retinal tissues of human donors and dogs, often used as animal models of AD. Although these Aβ deposits can be seen in unstained retinas using polarized light (Campbell et al., 2013), alternative non-invasive methods to image Aβ deposits in unstained retinal tissues are of interest. We explored here two-photon microscopy as a new tool to visualize Aβ deposits.

Methods: A research two-photon microscope (Bueno et al., 2010) was used to record two-photon excitation fluorescence (TPEF) images originating from flat-mounted, fixed, unstained retinas of human donors with a diagnosis of AD. Another set of retina samples from dogs suffering progressive cognitive impairment that is paired with Aβ accumulation (Mutsuga et al., 2012) was also examined. Different areas across the retina containing presumed Aβ deposits (first identified with polarized light) were imaged. At each location a Z-scan motor coupled to the microscope objective allowed optical sectioning of the samples.

Results: Aβ plaques provided a stronger TPEF signal than the surrounded retinal structures. Deposits could easily be distinguished and were found to be lying in front of nerve fiber layer and slightly penetrating into it. 3D volume renderings of the imaged areas were reconstructed from the set of images. This provided valuable and detailed information on the shape and distribution of deposits within the retina.

Conclusions: Two-photon microscopy is a useful alternative method to visualize Aβ deposits within unstained retinas. This technique offers high resolution imaging and avoids photo-damage. Optical sectioning allows accurate Aβ depth location tracking, as well as detailed information on morphology and geometrical characterization. Possible future clinical implementations of this technique in vivo might help to establish early diagnoses of AD and to quantify the progression the disease.