Viscoelasticity of amyloid plaques in transgenic mouse brain studied by Brillouin microspectroscopy and correlative Raman analysis amyloid plaques in transgenic mouse brain studied

Amyloidopathy is one of the most prominent hallmarks of Alzheimer's disease (AD), the leading cause of dementia worldwide, and is characterized by the accumulation of amyloid plaques in the brain parenchyma. The plaques consist of abnormal deposits mainly composed of an aggregation-prone protein fragment, (cid:1) -amyloid 1-40/1-42, into the extracellular matrix. Brillouin microspectroscopy is an all-optical contactless technique that is based on the interaction between visible light and longitudinal acoustic waves or phonons , giving access to the viscoelasticity of a sample on a subcellular scale. Here, we describe the ¯rst application of micromechanical mapping based on Brillouin scattering spectroscopy to probe the sti®ness of individual amyloid plaques in the hippocampal part of the brain of a (cid:1) -amyloid overexpressing transgenic mouse. Correlative analysis based on Brillouin and Raman microspectroscopy showed that amyloid plaques have a complex structure with a rigid core of (cid:1) -pleated sheet conformation ( (cid:1) -amyloid) protein surrounded by a softer ring-shaped region richer in lipids and other protein conformations. These preliminary results give a new insight into the plaque biophysics and biomechanics, and a valuable contrast mechanism for the study and diagnosis of amyloidopathy.


Introduction
Alzheimer's disease (AD) is the most common form of dementia, a®ecting approximately 47 million people worldwide with an incidence that is predicted to double every twenty years owing to ageing population. 1 Amyloidopathy, which is characterized by abnormal deposits of an aggregation-prone polypeptide, amyloid beta (A), within the parenchyma of AD brain, is one of the two most common hallmarks of AD, the other one being tauopathy. The A polypeptide is composed of 40-42 amino acids with prevalent -pleated sheet conformation; through hydrophobic interactions and hydrogen bonding, it tends to form a range of aggregates, from dimers to oligomers of various sizes, which can phase-separate from the aqueous medium of the intact brain and give rise to extracellular deposits or plaques'.
Amyloidopathy has been regarded, in the last 30 years, as the main underlying cause in the pathogenesis of AD, 2 and has been associated to alterations in cognitive and neurophysiological function, 3 both at a neural network 4 and single cell level. [5][6][7][8] Previous work using a combination of micro-Fourier Transform Infrared (FTIR) spectroscopic imaging, Raman microscopy and immunostaining has shown the complex structure and biochemistry of A plaques in the hippocampal Cornu Ammonis 1 (CA1) region of the brain in a genetically engineered mouse, the TASTPM model (F. Palombo et al., manuscript in preparation). This mouse presents, at the age of 9-12 months, severe accumulation of amyloid plaques. The plaque has a dense core of predominantly -pleated sheet conformation protein surrounded by a ring-shaped region richer in lipid esters and other protein conformations. While its origin is still debated, this lipid structure constitutes an amphiphilic`interface' between the essentially hydrophobic core and the hydrophilic medium surrounding the plaque.
In this work, we applied Brillouin microspectroscopy and correlative Raman scattering to investigate amyloid plaques in cryosections of TASTPM hippocampus, a brain area critically involved in memory encoding. Brillouin spectroscopy is an alloptical contactless technique for the viscoelastic characterization of biological materials. Micromechanical information is obtained by shining visible laser light onto a sample and measuring the inelastic light scattering from acoustic waves or phonons causing a shift in frequency of the light in the GHz region (hypersounds). Spatial resolution, which is di®raction limited to approximately 300 nm according to Abbe criterion using visible light, allows one to single out viscoelastic heterogeneities on a subcellular scale. We previously applied this technique to study the biomechanics of protein bers (collagen and elastin) of the extracellular matrix obtaining the full determination of their elastic constants, 9,10 and to map the elasticity of epithelial tissue biopsy in ex vivo sections of Barrett's oesophagus. 11,12 The same technique, through the use of single etalon (VIPA) approaches, has been applied to other clinically relevant samples such as keratoconus 13 and ageing crystalline lens, 14 bacterial meningitis in cerebrospinal°uid, 15 and atherosclerotic mouse carotid artery. 16 Traditionally, Brillouin spectroscopy has been performed using a scanning Fabry-Perot (FP) type interferometer, which gives very high contrast and spectral resolution at the expenses of the scanning time (sometimes minutes for a single spectrum). An angle-dispersive FP interferometer has also been used to demonstrate for the¯rst time a Brillouin imaging modality. 17 Alternative approaches to the use of the multipass FP interferometer for Brillouin scattering analysis have recently been developed based on multistage virtually image phased array (VIPA) spectrometers, 18 which enable rapid mapping at the expenses of achievable contrast which is key to analyze turbid media such as biomedical samples. Further advances in this direction are required to make Brillouin microspectroscopy a viable technology for rapid diagnostics in the context of healthcare and clinical applications. In those cases whereby high contrast and high resolution are required for a full viscoelastic characterization of a sample, multipass FP interferometers are still the preferred choice. Indeed, the Brillouin spectrum of biological matter derived from an FP-type spectrometer presents Brillouin bandshapes that are less a®ected by the interferometer's response function and can be reproduced by full viscoelastic functions 19,20 or, at least, by damped harmonic oscillator functions around the Brillouin peaks. 21,22 These high-contrast high-resolution spectra are better suited for accurate viscoelastic analysis of materials than those derived from VIPA-type spectrometers, hence making it possible to determine the acoustic wave attenuation and apparent viscosity (from the Brillouin linewidth).
Here, we present the detailed viscoelastic characterization of amyloid plaques in transgenic mouse brain by Brillouin microspectroscopy with a tandem multipass FP interferometer, and correlative micro-Raman analysis to provide the molecular structure and composition in correspondence to the mechanics of the plaques. Viscoelastic properties of Alzheimer's brain are expected to be di®erent from those of healthy brain, owing to biochemical and biophysical changes underlying neurodegenerative disease. Preliminary results of the correlative analysis based on site-matched Brillouin and Raman microspectroscopy presented in this work unveil changes in mechanical properties due to amyloidopathy which can become a contrast mechanism for diagnosis of neuropathology. This is the¯rst application of Brillouin scattering to dementia-related problems and will potentially pave the way to future developments in healthcare technology applied to the¯eld of neurophysiology.

Sample preparation
This work was carried out in accordance with the UK Home O±ce Guidelines and the University of Exeter Animal Welfare Ethical Review Board. 12-month old A-overexpressing TASTPM transgenic male mice were used in this study. TASTPM carries two mutations on the gene for the amyloid precursor protein (APPSwe K670N, M671L) and one on the presenilin 1 gene (M146V) that can be found in patients a®ected by familiar AD. 23,24 All animals were housed at room temperature under a 12/12 hour light/dark cycle, with free access to food and water ad libitum before being sacri¯ced. 7 The brain was rapidly removed and horizontal acute slices of 300 m thickness were cut in a vibratome and suspended in arti¯cial cerebrospinal°uid, before use for electrophysiological recordings. A number of slices of each brain containing hippocampus, striatum and cortex were retained for the present study. The slices were post-¯xed overnight with 4% formalin þ 0.1 M phosphate bu®er solution (PBS). This enabled to store the slices inde¯nitely while preventing deterioration from exposure to room temperature conditions. Note that, although this can a®ect the mechanical properties of tissues, the use of a¯xative is critical for structural and conformational analysis performed by Raman scattering here. Nevertheless, this e®ect acts uniformly on the tissue, changing in the same way the elastic properties of the healthy and the plaque zones. Previous work has shown that the cross-links that form in the process of formalin¯xation`lock in' the secondary structure of protein molecules; 25 therefore, proteins retain the secondary structure present before¯xation, i.e., the structure that confers them a particular rigidity (see below). The slices were rinsed twice with 0.1 M PBS (5 min each time) and stored in 30% (w/v) sucrose solution to inhibit subsequent formation of ice crystals, before being embedded in a water-soluble frozen section medium (NEG-50, Thermo Scienti¯c) and snap frozen. Sections of 20 m thickness were cut in a cryostat and left to rinse for at least 24 h in 0.1 M PBS. Sections were subsequently rinsed in distilled water and mounted onto calcium°uoride slides (Crystran). Two plaques within a brain hippocampal section of TASTPM mouse were analyzed, and a total of three microspectroscopic maps were obtained.

Brillouin microspectroscopy
Micro-Brillouin maps were collected with an FPbased mapping system depicted in Fig. 1. A laser beam from a 532 nm single-mode solid state laser is focused onto the sample by a customized JRS Sci-enti¯c Instruments CM-1 Confocal Microscope. A polarizing beam splitter is placed in the beam path to transmit the depolarized backscattered light towards the spectrometer. Pinholes at the entrance of the (3þ3)-pass FP interferometer provide a confocal arrangement for microscopic mapping. A Mitutoyo long working distance 20Â (NA 0.42) objective is used for both focusing the laser beam on and collecting the backscattered light from the sample. A /4 wave plate is inserted upstream of the objective to switch from depolarized to unpolarized scattering con¯guration. The sample is mounted onto an xyz microtranslation stage for mapping acquisition.
The backscattered light is dispersed through a tandem (3þ3)-pass FP interferometer onto a single photon avalanche photodiode. Spectra were acquired in the range -30 to 30 GHz using a 90 s exposure time to achieve a reasonable signal-to-noise ratio, with a 5 mW laser power on the sample. GHOST software was used for data acquisition and processing. 26 Accurate focus adjustment of the objective was performed before each series of measurements. This gave approximately 2 m for the lateral and 8 m for the axial dimension of the scattering volume. The role of di®erent spatial scales involved in micro-Brillouin measurements is described in Sec. 2.2.1.
A typical spectrum obtained from a single micro-Brillouin measurement of TG mouse hippocampus (CA1 region) is reported in Fig. 1(b). This shows both (Stokes and anti-Stokes) symmetric branches of the Doppler-shifted Brillouin spectrum. The Brillouin peaks at approximately 16 GHz are due to longitudinal acoustic modes propagating within the sample at the position of the scattering volume shown in the photomicrograph ( Fig. 1(a)). Curve-¯t analysis based on a damped harmonic oscillator (DHO, Eq. (2) below) model was applied to the Brillouin peak to extract the values of frequency position (! b Þ and linewidth (À b Þ, which are related to the viscoelastic properties of the sampled volume, i.e., the real and imaginary parts of the longitudinal elastic modulus, respectively. 27 Viscoelasticity of soft matter in the GHz spectral range is brie°y recalled in Sec. 2.2.2.

Relevant spatial length scales for Bio-Brillouin scattering measurements
Brillouin scattering from inhomogeneous media such as biomedical samples is a quite complex matter because the presence of discontinuities can profoundly a®ect propagation and attenuation of both light and acoustic waves. Here, we outline some basic features to better understand the results obtained in this work and, more generally, to start a discussion that deserves the attention of the Bio-Brillouin community. Three di®erent (subcellular) spatial scales, covering about three orders of magnitude in length, regulate the results of Brillouin scattering experiments performed on inhomogeneous samples: (i) the smallest one (L 1 $ 0:1 m) is related to the wavelength of acoustic modes, (ii) the intermediate (L 2 $ 1 m) to the attenuation of acoustic modes, and (iii) the largest one (L 3 $ 10 m in micro-Brillouin measurements) to the size of the scattering volume (Fig. 2).

L 1 : Wavelength of acoustic modes
In backscattering geometry ( Fig. 2(a)), the wavelength of acoustic modes Ã is related to the wavelength of the excitation laser through the relation: Ã ¼ =2n $ 0:18 m, where n is the refractive index of the sample material. Inhomogeneity on a length scale which is much smaller than this is hidden to the acoustic¯eld and hence an e®ective homogeneous medium is revealed, with average elastic constants. 28,29 However, the case of inhomogeneity on a length scale approaching L 1 /10 is the most challenging one to deal with, since acoustic scattering e®ects can become dominant, giving rise to anomalous dispersion and attenuation of acoustic modes (see, for instance, Refs. [30][31]). Analysis of these phenomena is beyond the aim of the present work.
For inhomogeneous media on length scales larger than L 1 , an important role is played by L 2 and L 3 , which can induce an inhomogeneous broadening of Brillouin peaks that should be distinct from homogeneous e®ects in order to gain the viscoelastic characterization of the sample.

L 2 : Attenuation of acoustic modes
The Brillouin doublet is due to light scattered by longitudinal acoustic phonons, i.e., density°uctuations q ðtÞ propagating with wavevector q which, in backscattering geometry, is given by q ¼ 2nk i , where k i is the wavevector of the incident light and n the refractive index of the sample. Some contribution can be expected also from lower q values, due to thē nite aperture of the objective, as previously reported for Brillouin imaging 32 and explained below for the present experiments. In the case of simple homogeneous liquids, the linearized hydrodynamic equations, neglecting the thermal di®usion mode and its contribution to the acoustic damping, give: where is the static mass density, M 0 ¼ c 2 0 is the adiabatic longitudinal modulus, c 0 the adiabatic sound velocity, and 0 the static longitudinal viscosity, responsible for the damping of acoustic waves.
Looking for harmonic solutions, q ðtÞ / exp(i!tÞ, the presence of a complex elastic modulus Mð!Þ ¼ M 0 þ i! 0 becomes apparent in Eq. (1). Though more sophisticated models for Mð!Þ should be applied in the case of viscoelastic media, as shown in Sec. 2.2.2, this simple approximation for Mð!Þ is able to describe propagation and attenuation of density°uctuations in liquids (simple hydrodynamics), solids (Voigt model of viscoelasticity 33,34 and even in general viscoelastic media, provided that a narrow frequency region around the Brillouin peaks is analyzed 35 and apparent, !-dependent values of Mð! b Þ and ð! b Þ are de¯ned. In this condition, the spectrum of scattered light is that of a damped harmonic oscillator (DHO): where ! b and À b approximately corresponds to the frequency position and full width at half maximum of the Brillouin peak. These parameters are related to the (apparent) longitudinal modulus and (apparent) viscosity through: Internal friction can also be obtained from these parameters through the inverse of the quality factor of the Brillouin peak, Q À1 ¼ À b =! b . The quality factor can be intuitively de¯ned as the number of oscillations of the system before their amplitude decreases by a factor e, so that the characteristic propagation length of acoustic phonons, their mean free path, can be estimated as L 2 $ 2Q=q 36 For the spectrum in Fig. 1 Alternatively, a Lorentzian function can be used to¯t Brillouin peaks. In that case, a further function must be added to the Lorentzian to account for the asymmetry and the frequency shift of the peaks. 37 Instead Eq. (2) includes all these e®ects and needs no additional manipulation.
It is worth noting that¯tting the experimental spectra to Eq. (2) gives the correct values of ! b and À b , provided that spurious broadening e®ects are adequately addressed. Firstly, Eq. (2) requires convolution with the instrumental function, i.e., the measured spectrum of monochromatic light, to yield the experimental spectral band-shape. Moreover, the asymmetric broadening generated by the¯nite angle of collection of scattered light ( in Fig. 2(a)) needs to be taken into account. Though this e®ect may be quite complex, 38 in the backscattering geometry used in Brillouin microscopy the broadening is minimized and the collected q values are mainly ranging between q M ¼ 2 nk i and q m ¼ 2 nk sin [( À Þ/2], neglecting the contribution from incident light far from the optical axis. The induced broadening of Brillouin peaks can approximately be estimated by the relationship In our case, having used an objective with NA 0.42 gives a $ 2% broadening, a rather small e®ect which can be included into the convolution process as a small enlargement of the instrumental function and a $ 1% reduction of the average q value. In the case of larger numerical apertures or scattering geometries di®erent from the backscattering, this contribution may become dominant and the¯t to a single DHO function must be replaced by a distribution of functions, with the appropriate weight in q. 38 Other spurious e®ects which can distort the Brillouin lines are the absorption in strongly opaque media, which can induce an indetermination in the value of q, and multiple scattering from turbid media, which is a source of inelastic scattering at smaller q values and gives a broadening of the measured Brillouin peaks towards lower frequencies. A detailed analysis of these e®ects is beyond our aims. The e®ects of multiple scattering in Brillouin spectra from biological matter is discussed in a further work (M. Mattarelli et al., manuscript in preparation).

L 3 : Scattering volume
The scattering volume L 3 may be larger than L 2 , being a potential origin for inhomogeneous broadening of Brillouin lines.
In micro-Brillouin measurements, the size of the scattering volume is determined both by the shape of the incident light beam and by the diameter of the pinhole that de¯nes the confocal condition. The scattering volume can operatively be de¯ned as the region of the enlightened sample from which scattered light enters the pinhole and, passing through the interferometer, then reaches the photo-detector.
It can be approximated to an ellipsoid, several times as long as it is wide. In our set-up, the width is approximately 2 m and the length is 8 m (S. Caponi et al., manuscript in preparation). This is the smallest portion of the sample that can be investigated, de¯ning the granularity of the¯nal Brillouin map. By increasing the NA and reducing the diameter of the pinhole, L 3 can be reduced by almost one order of magnitude 39 at the expenses of reducing the scattered light intensity by nearly 2-3 orders of magnitude, at a¯xed power density of the incident light. Note that the value of the power density cannot be arbitrarily increased, being limited to the amount that starts causing photo-damage to the sample.
Within a single scattering volume, a distribution of homogeneous sub-regions larger than L 2 can be present, each one characterized by a given elastic constant. In this case, the measured spectrum is the sum of Brillouin peaks originating from the di®erent sub-regions. If their spectral spacing is larger than their width (after convolution with the instrumental function), their contribution can be singled out and their relative scattering intensity can give information on the relative volume of the sub-regions. Otherwise, a heterogeneous broadening is measured and ! b is an average of those of the subunits. This may be the origin of the uncorrelated (broad) peak width and frequency shift observed (see below) in the region of the extracellular matrix surrounding the lipid ring.

Viscoelastic behavior of biological samples
When mapping viscoelastic materials such as cells and tissues, di®erent values of probed Brillouin frequencies cannot immediately relate to stronger or weaker interactions between molecules, i.e., to genuine changes in elastic properties of the sample, as a potential increase or decrease in viscosity should also be taken into account. In fact, small changes of, e.g., humidity in di®erent parts of the sample can give appreciable di®erences in acoustic wave velocity, mainly due to a change of viscosity and of the corresponding structural relaxation time, rather than to a change in bonds strength. Here, some basic properties of viscoelasticity are recalled, which can help the interpretation of viscoelastic maps of biological samples.
In molecular liquids and soft matter, density°u ctuations may be coupled with molecular internal degrees of freedom and the structural relaxation (or -relaxation) process, giving rise to dispersion and absorption of acoustic modes. 19,40 These relaxation e®ects which are responsible for the order of magnitude di®erence between elastic moduli probed at Brillouin frequencies and in quasi-static conditions can be taken into account by a generalization of the hydrodynamic equations, introducing a complex !-dependent elastic modulus Mð!Þ ¼ M 0 ð!Þ þ iM 00 ð!Þ which can be expressed as where M 1 ¼ c 2 1 is the high-frequency (solid-like) longitudinal modulus and 1 is the high frequency longitudinal viscosity. ÁMð!Þ can be arbitrarily complex, depending on the number and nature of the relaxation processes that are coupled with density°uctuations. The simplest possible scenario is depicted in Fig. 3: it shows a single relaxation, the -relaxation, which is present in all viscoelastic materials and is usually described by a Cole-Davidson relaxation function 41 where is the characteristic relaxation time and is the stretching parameter, describing the deviation from a single exponential behavior (see Ref. 27 and references therein). Note that in the ! ( 1 limit (corresponding to, e.g., low frequency phonons or high temperature or high hydration of the sample), it results that recalling the Maxwell model for stress relaxation. This limit matches with the simple hydrodynamics Mð!Þ ¼ M 0 þ i! 0 expressed by Eq. (1), corresponding to the low frequency part of Fig. 3. If the Brillouin peak lies in this region, an increase of the acoustic mode velocity can occur with an increase of static viscosity 0 or relaxation time (-relaxation moving towards lower frequencies in Fig. 3) due to, e.g., a reduction of humidity in the sample. In this case, an increase of frequency shift correlates with an increase of linewidth of Brillouin peaks. We have found such a behavior in highly hydrated tissues, namely cornea samples (unpublished results).
The intermediate condition, the central region in Fig. 3, is typical of Brillouin scattering from highly viscous materials. If ! b lies in this intermediate region, as depicted in the¯gure, an increase of frequency shift would correlate with a decrease of linewidth of the Brillouin peak, and vice versa.
This is the case of the lipid ring in the TASTPM sample (see Fig. 4). As a consequence, the lipid ring appears as a quite homogeneous part of the sample, at least on the L 2 scale, with a reduced static viscosity, whose origin is still debated and will be the focus of further investigation.

Raman microspectroscopy
Micro-Raman maps were collected with a Renishaw inVia Raman microscope using a near-infrared 830 nm laser and a Leica long working distance 50Â (NA 0.50) objective. The backscattered light from the sample was analyzed by a spectrometer comprising a 600 gr/mm grating and a Renishaw CCD camera. Raman maps were acquired in streamline mode with an exposure time of 50 s per point in the 2356-384 cm À1 spectral region. WiRE 4.0 software was used for acquisition and manipulation of the data.
Spectral maps were corrected via cosmic ray removal with a nearest neighbor routine and then analyzed by Principal Component Analysis (PCA) using 10 components, spectrum centring þ normalization (SNV) pre-processing. In this manner, the¯rst principal component (PC1) corresponds to the average spectrum of the sample in the mapped region. Results were reported as obtained, without any further modi¯cation including baseline correction. Figure 4(a) shows a photomicrograph of a TASTPM mouse brain hippocampal section containing two plaques. The plaques are visible as a dark core (rich in A polypeptide) separated from the normal tissue by a lighter lipid ring. The molecular composition of the plaques was investigated through correlative chemical mapping based on Raman microscopy (see below).

Results and Discussion 3.1. Micro-Brillouin mapping
A¯rst series of Brillouin spectra was collected along a 50 m line scan starting from the periphery through to the center of a plaque (yellow line in Fig. 4(a)).
Plots of the frequency and linewidth of the Brillouin peak derived from¯t analysis (to Eq. (2) in Sec. 2.2.1) of the spectra extracted from the line scan are reported in Figs. 4(b) and 4(c). The heterogeneous nature of the sample is evidenced by the position-dependent mechanical properties, highlighting the appropriateness of the microspectroscopic approach to investigate the mechanical di®erences between plaque, lipid ring and normal tissue. The Brillouin frequency shift (Fig. 4(b)) shows a decrease when going from the normal tissue to the lipid ring and a marked increase going towards the core of the plaque. To better understand the nature of this variation, it is useful to compare this behavior with the linear map of Brillouin linewidth reported in Fig. 4(c). Indeed it can be seen that À b has a maximum (broader peak) in the same region where ! b has a minimum (smaller shift), corresponding to the lipid ring in Fig. 4(a). Such correlation between À b and ! b is the characteristic signature of a viscoelastic e®ect, as detailed in Sec. 2.2.2 and previously observed in epithelial tissue, Barrett's oesophagus. 11 Conversely, ! b appears to be quite uncorrelated from À b in correspondence of the normal tissue, suggesting a heterogeneous origin for the broadening of Brillouin peaks therein.
A two-dimensional Brillouin map gives further evidence to these observations.   Figure 5(b) shows a clear maximum in ! b (higher rigidity) at the core of the plaque, approximately 10% larger than average, corresponding to $20% increase in elastic modulus (assuming constant density and refractive index). Distinct mechanical properties in correspondence to di®erent types of protein secondary structure and conformational disorder have previously been identi¯ed. 42 Secondary structures of proteins follow the increasing rigidity order: random coil < -helix < -sheet. 43 Hence, the rigidity of the plaque core observed here can be attributed to the deposition of aggregates of -amyloid protein in -pleated sheet conformation and to the exclusion of hydration water from this highly hydrophobic region.
Moving radially away from the core of the plaque towards the periphery, the Brillouin map of frequency shift shows (Fig. 5(b)) a decrease of ! b , with a minimum in the region of the lipid ring. It would be unwary to attribute this e®ect entirely to a reduction of sti®ness, since a viscoelastic e®ect can also contribute to a decrease in apparent elastic modulus. In fact, in this area, the reduction of ! b correlates quite strictly with an increase of À b (Fig. 5(c)), which can be explained in terms of a reduction of static viscosity and a blue-shift of the -relaxation process (see Fig. 3). Note that this counterintuitive e®ect, i.e., broadening of the Brillouin peak in correspondence to a decreasing static viscosity, requires a full-viscoelastic approach and cannot be accounted for neither in the liquid-like (simple hydrodynamic) or in the solid-like (Voigt) limit. A possible origin for the reduction of viscosity in the lipid ring can be the retention of hydration water within this amphiphilic region. Further investigation will be required to causally relate this mechanical behavior with the molecular composition of the ring.
The top left side of the maps in Figs. 5(b) and 5(c) corresponds to the normal tissue's extracellular matrix. In this region, À b markedly changes from one point to another showing no apparent correlation with ! b . In this case, heterogeneous broadening e®ects may be the cause for this behavior, as explained in Sec. 2.2.1. Heterogeneities in tissue can indeed be expected due to the presence of cell bodies (astrocytes and microglia) in the area surrounding the plaque -stratum radiatum of the CA1 sub¯eld of the hippocampus.
The Brillouin peak frequency for the normal extracellular matrix (ECM) observed here (Figs. 4(b) and 5(b)), approximately 16 GHz, is lower than the average frequency of the¯brous matrix of human epithelium biopsy (ca. 18 GHz), 11   order-of-magnitude increase of the elastic moduli measured in the GHz range with respect to those measured by quasi-static techniques. Though some works (see for instance Ref. 44) have reported the existence of a correlation between high and low frequency values of the moduli, this is a topic that deserves an in-depth phenomenological and theoretical investigation. Figure 6 shows the results of correlative Raman mapping on the same A plaque analyzed by micro-Brillouin scattering.

Micro-Raman mapping
Principal Component Analysis applied to the micro-Raman map provided the distribution of the plaque core (PC2; Fig. 6(b)) distinct from the lipid loaded ring (PC4; Fig. 6(c)). (Note that PC1 yielded the average spectrum of the specimen; data not shown.) Corresponding loading plots for these PCs are shown in Fig. 7.
The PC2 loading plot refers to the spectrum of the plaque core ( Fig. 6(b)), with distinctive protein bands. The single Raman peak at 1663 cm À1 can be attributed to the amide I mode of protein with a -pleated sheet conformation, which indeed is concentrated in the plaque core ( Fig. 6(b)). Other notable bands are at 1441 (CH 2 bending) and around 1000 cm À1 (Phenylalanine). Instead, the PC4 spectrum presents a derivative-type signal in the amide I region (with minimum at 1668 cm À1 and maximum at 1650 cm À1 Þ which indicates that PC4 is a linear combination of spectra where positive signals corresponds to essentially lipids and other protein conformations, e.g., -helix which resonates at lower frequency than -sheet in the amide I region. Other bands at 1435, 1294 and 1059 cm À1 can be assigned to the CH 2 deformation in protein and lipid, CH 2 deformation in lipids, and skeletal C-C stretching in lipids, respectively, in analogy with the assignment in Ref. 45. The absence of the Phenylalanine peak in PC4 indicates a di®erent protein primary structure than PC1. Indeed, the PC4 loading plot relates to the lipid-rich layer surrounding the plaque core (Fig. 6(c)).
Therefore, correlative Brillouin-Raman mapping enables us to conclude that, in TASTPM mouse brain hippocampus, the A plaque core has high rigidity identi¯ed by a marked increase in Brillouin peak frequency associated with the abnormal deposition of -amyloid protein, which is a typical pathological hallmark of Alzheimer's disease. The surrounding tissue presents high viscoelasticity in correspondence of a lipid-rich layer around the dense core of the plaque and high heterogeneity in the external tissue, plausibly due to the presence of cell bodies (i.e., astrocytes and microglia).

Whole plaque viscoelasticity
At a visual inspection, di®erent plaques can show quite di®erent morphologies due to their structural complexity. Therefore, we can expect that the viscoelastic behavior is also re°ecting this heterogeneity. Figure 8(a) shows the microphotograph of a plaque from a di®erent area of the same mouse brain specimen. The maps of Brillouin frequency shift and linewidth are reported in Figs. 8(b) and 8(c). Also, here the core of the plaque is characterized by a distinctive maximum in ! b (higher rigidity), approximately 10% larger than average, whilst the region around the plaque core shows a decrease of ! b , with a minimum (cyan-blue region in Fig. 8(b)) which correlates with a maximum in À b (red-yellow region in Fig. 8(c)), although this is less apparent than in Fig. 5(c).
For this plaque, the region of the lipid ring is visible but less de¯ned than in the previous plaque (Fig. 5), hence indicating a di®erential distribution in plaque composition, structure and biomechanics across the TASTPM hippocampus.
Although an accurate evaluation of elastic modulus maps requires a point-by-point determination of density and refractive index n of the sample, it can be noticed that M 0 ¼ ! 2 b =q 2 ¼ ð=2nÞ 2 where is the Brillouin frequency shift, and is the wavelength of laser light. Here the ratio =n 2 is constant, with good approximation, far from electronic resonances, 44 so that the relative variation of 2 gives the correct estimation of the variation in M 0 . In our case, assuming n ¼ 1:36 46 and $ 1:0, and taking into account the 1% frequency shift due to the spread in q (see Sec. 2.2.1), we can calculate the mean variation of the elastic modulus for the probed plaques: it ranges between 9 and 12 GPa, for the measured frequency shifts of $15.4 GHz in the lipid ring and $17.9 GHz in the core (Figs. 5 and 8).

Conclusion
We have shown that Brillouin microscopy is a sensitive tool to investigate the micromechanics and viscoelasticity of A plaques (a major hallmark of AD) within the brain in a mouse model of the disease. Amyloidopathy is characterized by abnormal deposition of A plaques, which have a rigid core rich in A protein with a -pleated sheet conformation, a viscoelastic lipid-rich layer around the core, and a surrounding heterogeneously composed extracellular matrix, presumably disseminated of glial cell bodies. Correlative micro-Raman analysis of the plaques gives the chemical speci¯city to identify the molecules responsible for the biomechanical response, hence being able to relate high rigidity to the A plaque core and low rigidity to the lipid ring. This multimodal approach is key to investigate complex phenomena such as the viscoelasticity of tissues which can provide an invaluable contrast mechanism for the diagnosis of Alzheimer's disease.