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 Journal of Optics

J. Opt. 24 (2022) 054005 (12pp) https://doi.org/10.1088/2040-8986/ac5b51

Label-free Raman and fluorescence

imaging of amyloid plaques in human
Alzheimer’s disease brain tissue reveal
carotenoid accumulations
Loes Ettema1, Benjamin Lochocki1,∗, Jeroen J M Hoozemans2,
Johannes F de Boer1 and Freek Ariese1
1
LaserLaB, Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, The
Netherlands
2
Department of Pathology, Amsterdam Neuroscience, Amsterdam UMC—Location VUmc, Amsterdam,
The Netherlands

E-mail: ben.lochocki@vu.nl

Received 25 November 2021, revised 2 March 2022

Accepted for publication 7 March 2022
Published 1 April 2022

Abstract
Alzheimer’s disease (AD) is a neurodegenerative disease, characterized by the presence of
extracellular deposits (plaques) of amyloid-beta peptide and intracellular aggregates of
phosphorylated tau. In general, these hallmarks are studied by techniques requiring chemical
pre-treatment and indirect labeling. Imaging techniques that require no labeling and could be
performed on tissue in its native form could contribute to a better understanding of the disease.
In this article a combination of label-free and non-invasive techniques is presented to study the
biomolecular composition of AD human brain tissue. We build on previous research that already
revealed the autofluorescence property of plaque, and the presence of carotenoids in cored
plaques. Here, we present further results on cored plaques: showing blue and green
autofluorescence emission coming from the same plaque location. Raman microscopy was used
to confirm the presence of carotenoids in the plaque areas, with clear peaks around 1150 and
1514 cm−1 . Carotenoid reference spectra were recorded in hexane solution, but also adsorbed
on aggregated Aβ42 peptides; the latter agreed better with the Raman spectra observed in
plaques. From the six single carotenoids measured, lycopene matched closest with the peak
positions observed in the cored plaques. Lastly, stimulated Raman scattering (SRS) microscopy
measurements were performed, targeting the shift of the beta-sheet Amide I peak observed in
plaques. Employing SRS in the C–H stretch region we also looked for the presence of a lipid
halo around plaque, as reported in the literature for transgenic AD mice, but such a halo was not
observed in these human AD brain samples.

∗
Author to whom any correspondence should be addressed.

Original content from this work may be used under the terms
of the Creative Commons Attribution 4.0 licence. Any fur-
ther distribution of this work must maintain attribution to the author(s) and the
title of the work, journal citation and DOI.

2040-8986/22/054005+12$33.00 Printed in the UK 1 © 2022 The Author(s). Published by IOP Publishing Ltd
J. Opt. 24 (2022) 054005 L Ettema et al

Supplementary material for this article is available online

Keywords: Raman spectroscopy, amyloid, Aβ, lipid, protein, Alzheimer’s disease, plaques
(Some figures may appear in color only in the online journal)

1. Introduction could lead to a method capable of diagnosing at an early stage

of the disease.
In Alzheimer’s disease (AD), neurons in the brain are being In this article, we confirm the previously obtained results
damaged or destroyed, resulting in a decrease in cognitive and extend the study with new results by performing a range
functioning [1]. AD makes up 60%–70% of all cases of of additional measurements:
dementia, making it the most common cause of dementia [2].
Furthermore, predictions are that the prevalence will continue
• This includes identifying which carotenoid(s) can be present
to increase worldwide [3, 4]; in 2050 it is estimated that 1 in
in plaque by measuring Raman spectra of six single caroten-
85 persons will be suffering from AD [3]. An important char-
oids in different micro-environments (dissolved in hexane
acteristic of patients with AD is the presence of the proteins
vs. adsorbed on Aβ42 aggregates) and additionally determ-
amyloid-beta and tau in the brain [5, 6], of which the former
ining whether the carotenoids could be responsible for the
is found extracellular and the latter is found intracellular [6].
green fluorescence observed.
Amyloid-beta is processed from the amyloid precursor protein
• Furthermore, in contrast to the blue excitation used in the
(APP). Cleavage of APP by two enzymes, beta-secretase and
mentioned study, a different study conducted by Thal et al
gamma-secretase, results in an amyloid-beta fragment which
[11] used UV light instead of blue light for excitation, lead-
is secreted by the cell in the extracellular space. There it
ing to blue light being emitted by the plaque. However,
accumulates and forms the amyloid plaque [6]. The peptides
in none of the studies both excitation wavelengths have
adopt a β-sheet conformation, which makes them poorly sol-
been used, and therefore a comparison between the observed
uble and neurotoxic [7]. In addition, amyloid plaques induce
green and blue fluorescence was not possible. We therefore
an immune response generating inflammatory molecules and
investigate, by using both UV and blue excitation, whether
cytokines [8] that contribute to the process of neurodegen-
the blue and green fluorescence occur at the same location
eration [1]. As the disease progresses, more plaques occur
or whether there are differences in the fluorescence areas,
and spread across the brain, starting in the neocortex [9].
indicating the presence of two fluorescent compounds at dif-
In our recently published article [10], we showed analyses
ferent locations in the plaque.
of snap-frozen human AD brain tissue with multiple label-
• Subsequently, at the plaque location, Raman spectra are
free and non-invasive techniques. These techniques included
recorded with specific attention to the peak position of the
autofluorescence microscopy, Raman microscopy and stimu-
Amide I protein band. Also, SRS measurements are per-
lated Raman scattering (SRS) microscopy. Among the differ-
formed to examine the β-sheet peak in more detail.
ent phenotypes of plaques, which can be characterized as cored
• Furthermore, recent publications from various research
or fibrillar, both showed green fluorescence when using blue
groups have shown that a lipid ring or halo is formed around
excitation. Subsequently, Raman microscopy measurements
an amyloid-beta deposit in transgenic mice [12–14]. The
were performed on those plaque locations, using a 532 nm
lipid halo was detected either with spontaneous Raman or
excitation laser. Different spectra were found for AD cases
SRS. Therefore, beside SRS measurements performed in
and the healthy control cases, with the main difference that
the fingerprint region, the study is extended by perform-
the spectra of cored plaque areas of AD cases showed addi-
ing measurements in the C–H stretch region to investigate
tional Raman peaks at wavenumbers characteristic of caroten-
whether a lipid halo is also present around a plaque in human
oids. Reference spectra of two carotenoid compounds, beta-
AD brain tissue.
carotene and lutein in hexane solution, showed Raman bands
quite close to (but not matching exactly) the spectra observed
in plaques. These carotenoids may have played a role as anti- 2. Materials and methods
oxidants fighting inflammation, but that needs to be further
investigated. Furthermore, shifts in the wavenumber of the 2.1. Brain tissue and preparation
Amide I peak were found, indicating a different protein con-
formation. To confirm this change in vibrational frequency, The post-mortem human AD brain tissue was provided by the
this protein peak shift was studied with SRS too. In summary, Netherlands Brain Bank (NBB, Netherlands Institute for Neur-
autofluorescence, Raman, and SRS microscopy, when applied oscience, Amsterdam). The research was conducted in accord-
to the same tissue section, can be combined to differentiate ance with the principles embodied in the Declaration of Hel-
between human AD tissue and healthy tissue by measuring sinki. The brain donor program of the NBB was approved by
their biomolecular composition directly in its native, unlabeled the local medical ethics committee of the Vrije Universiteit
state. Furthermore, they could potentially be used in-vivo, for Medical Center (Ref#2009/148). The collected brain tissue
example, by imaging the retina in a search for biomarkers. This was from donors with written informed consent for brain

2
J. Opt. 24 (2022) 054005
Table 1. Demographics of AD brain donors.
AD Age Braak Amyloid PMD
donor Sex (years) [15] [15] Region (hours)
Case I F 90 5 C Middle 4 halide bulb (EL 6000) and the filter cubes A band-pass filter
frontal
gyrus
Case II M 84 4 B Middle 5:53
frontal
gyrus
autopsy and the use for clinical information and research pur-
poses. As described previously [10], the neuropathological
diagnosis was based on histochemical staining, while AD dia-
gnosis was based on Braak stages [15], Thal phases [9] and the
CERAD criteria [16].
The snap-frozen brain samples were cut in 20 µm sections
and mounted on CaF2 microscope slides without the use
of a cover slip. In this report, we focus on dense cored
plaques as they show the clearest differences with the sur-
rounding tissue in terms of Amide I shift and carotenoid
levels. The cored plaques usually have a size of 10–20 µm.
In total, we measured six different cored plaques from
three different donors. However, here we only show two
examples, for brevity called ‘case I’ and ‘case II’. Informa-
tion about these two cases can be found in table 1. Additional
information and spectra from the other four plaques can be
found in the supplementary information (available online at
stacks.iop.org/JOpt/24/054005/mmedia).
2.2. Chemicals, amyloid-beta and carotenoids
Aβ42 (amyloid β-protein (1-42)) was purchased from Bachem
(Basel, Switzerland); a 1% suspension was prepared by mix-
ing 1 mg with 100 µl isopropanol. Six carotenoid standards,
beta-carotene, lutein, lycopene, astaxanthin, zeaxanthin and
beta-cryptoxanthin were purchased from Sigma Aldrich. By
dissolving <1 mg in several ml of n-hexane with help of a
sonication bath, stock solutions were obtained, and UV–vis
and fluorescence emission spectra were recorded (not shown).
Raman spectra of the same hexane solutions were then recor-
ded under (pre) resonance conditions using 532 nm excitation
in a quartz cuvette.
In order to better mimic the micro-environment of a plaque,
the same carotenoid standards were measured after adsorption
on aggregated Aβ42 in the following way: 1 µl of the peptide
suspension was spotted on a stainless-steel polished substrate
and allowed to dry in air. Then the carotenoid stock solutions
in hexane were diluted (100 µl + 100 µl) with isopropanol
and concentrated by preferential evaporation of the hexane in
a gentle stream of nitrogen, adding more isopropanol, until
finally reaching the same concentration in a 100 µl volume in
pure isopropanol. Then 1 µl of that carotenoid solution in iso-
propanol was added to a dried peptide spot and allowed to dry
in air before recording (pre) resonance Raman spectra using
532 nm excitation.
3
L Ettema et al
2.3. Fluorescence microscopy
For the (auto-)fluorescence images of the freshly cut tissue,
a Leica DM2500 microscope with an external mercury metal
(BP) 340–380 nm, low-pass filter (LP) 425 nm) and I3 (BP
450–490 nm, LP 515 nm) were used. Images were acquired via
the Leica DFC450 color camera using 20× (NA 0.55) and 40×
(NA 0.8) Leica FLUOTAR objectives and the LAS X software.
The corresponding brightfield images were acquired using the
same microscope but using the white light source and no fil-
ters.
The images of the thioflavin-S stained tissue sections were
acquired using the same microscope but solely with filter I3.
Positively stained plaques appear as bright yellow areas within
the tissue.
2.4. Raman spectroscopy
Spontaneous Raman (SpR) measurements were recorded
with a Renishaw inVia spectrometer, using an excitation
wavelength of 532 nm. The spectral resolution of the instru-
ment when using the 1800 l mm−1 grating is better than
1 cm−1 (or 0.04 nm). The attached microscope was equipped
with a 50× objective (Leica HC PL FLUOTAR, 0.8 NA). The
excitation power was kept constant at 3 mW at the sample
plane, with a focal spot diameter of around 1 µm. Across the
plaques, mapping of 51 × 51 µm and 81 × 81 µm for case I
and II, respectively, was performed using a step size of 1 µm,
acquiring spectra in the fingerprint region (900–1900 cm−1 ).
In tissue, each spectrum was acquired with 1 s exposure time
and three accumulations. Measurements on the single caroten-
oids in hexane were performed by focusing the laser through
the window of a quartz cuvette. For each carotenoid, a spec-
trum (740–2383 cm−1 ) was recorded using 1 s exposure time
and ten accumulations, with an excitation power of 30 mW at
the sample plane.
Spectra of the carotenoids adsorbed on amyloid β-peptides
(Aβ42) were measured on polished stainless-steel slides.
Since some carotenoids proved to be very sensitive to pho-
todegradation when measured this way, the dried spots were
raster scanned in mapping mode and the spectra were obtained
after averaging over at least 100 locations. Typical measure-
ment conditions: 1.5 mW at the sample plane, 1 s exposure
times two accumulations.
2.5. SRS microscopy
Images via SRS were recorded with an in-house built pico-
second near-infrared (NIR) SRS system as described before
[17, 18]. In short, we used a combination of a Stokes beam
of 1064 nm, 80 MHz repetition rate and a pump beam tuned
from 901 to 907 nm (covering the Amide I band) with for
case I 30 and 60 mW at the sample plane, respectively, or
from 800 to 820 nm (C–H stretch), with for case I 15 and
60 mW at the sample plane, respectively. For case II, powers
of 25 mW and 50 mW at the sample plane, respectively, were
used. In the NIR range the spectral resolution of the setup
J. Opt. 24 (2022) 054005
is determined by the 0.1 nm bandwidth of the lasers, which
corresponds to 2 cm−1 . The tissue was scanned with a water
immersion 32× objective (0.85 NA) and a constant pixel dwell
time of 177.32 µs with constant lock-in amplifier settings. The
obtained images are an average of two (C–H stretch region) or
either two or four (Amide I band) repetitive measurements, for
case II and I, respectively.
2.6. Thioflavin-S staining
After the SpR and SRS measurements were completed, the tis-
sue was stained with thioflavin-S to confirm plaque presence
and location. The actual staining procedure is described in
detail in [10]. Thioflavin-S is a commonly used stain/dye that
binds to amyloid [13, 19–21], which is the beta-pleated con-
formation of stacked amyloid-beta and can be found in most
plaque phenotypes.
2.7. Data processing
The acquired spectral data were processed using an in-house
written chemometric Matlab GUI, allowing for pre-processing
and various subsequent data analysis routines [22]. The raw
data were processed by smoothing (using the locally weighted
nonlinear least squares regression function ‘rlowess’ and a
window size of 7), baseline removal (using arPLS [23]), and
vector normalization and subsequently clustered using ver-
tex component analysis (VCA) [24]. The presented spectral
figures were eventually composed in OriginPro 2020b (Ori-
ginLab, USA). The carotenoid mapping data from the peptide
spots were averaged and then processed.
3. Results
Following our recently developed procedure, amyloid plaques
were located by using the auto-fluorescence properties of
amyloid accumulations as location indicator [10]. In brief: the
freshly cut and mounted tissue section was examined under
the fluorescence microscope using filter cubes A and I3. Dense
cored plaques could be located due to their bright emission in
the blue and green, respectively. The plaque positions were
marked by making scratches close to the plaque using the tip
of a needle. Subsequently, the tissue was placed under the SpR
and SRS microscopy setups, where the plaque areas could eas-
ily be located using the brightfield view.
3.1. Spectroscopic results on tissue
In figure 1, results of the fluorescence imaging of two
cored plaques are presented. Both plaques appear as dense
spots, showing blue (>425 nm) and green (>515 nm) auto-
fluorescence, depending on whether the sample is illuminated
with UV (340–380 nm) or blue light (450–490 nm), respect-
ively. It should be mentioned that the green emission light
was better visible under the microscope by eye than it is in
the pictures. The presence of a plaque at the location of blue
or green emission is confirmed by the pictures on the right,
obtained after staining the tissue sections with thioflavin-S.
4
L Ettema et al
Figure 1. Images of two cored plaques. From left to right:
brightfield, auto-fluorescence for UV and blue excitation, and
thioflavin-S fluorescence images, all the same plaque area. In the
UV and blue excited auto-fluorescence images, plaques appear in
blue and green, respectively. The white and yellow spots in the
auto-fluorescence images depict lipofuscin. The plaques are
confirmed afterwards by thioflavin-S staining (right column, yellow
areas). Case I size: 51 × 51 µm, case II size: 81 × 81 µm. Scale
bars: 20 µm.
This fluorescent compound binds to amyloid, resulting in the
plaques appearing as yellow areas in the fluorescence images.
After localizing and imaging the plaques using auto-
fluorescence, the same plaque locations were measured with
SpR. For case I, results of those measurements are depic-
ted in figure 2. The top graph (figure 2(b)) depicts Raman
spectra for four components, with the graph representing the
plaque consisting of three peaks at wavenumbers characteristic
of carotenoids, namely around 1003, 1151, and 1514 cm−1 .
Very similar Raman spectral images were obtained from four
other plaque areas. The results are shown in the supplementary
information.
The first carotenoid peak overlaps with that of other tis-
sue components at this same wavenumber, due to the pres-
ence of phenylalanine. The Raman signal of the amino acid
phenylalanine is relatively strong and sharp, and therefore it is
often prominent when scanning tissue components that con-
tain protein [25]. However, the peaks at wavenumbers 1151
and 1514 cm−1 are unique to the spectrum of plaque and match
the major Raman peaks of carotenoids, see further below. The
colors of the spectra correspond to the colors in the cluster
image (figure 2(a), center), showing the location from where
the plaque spectrum was obtained in orange. Comparison with
the auto-fluorescence image on the left of figure 2(a) shows
that this location is indeed the plaque location.
A second difference between the spectrum of the plaque
and the other spectra can be found in the peaks around
wavenumbers 1440 and 1658 cm−1 . The bottom graph
(figure 2(c)) zooms in on this region. The peaks at lipid
(1440 cm−1 ) and protein (1658 cm−1 ) wavenumbers are
slightly shifted towards the higher wavenumbers in the spec-
trum of the plaque, compared to the other spectra from the
tissue. The shift in protein wavenumber (Amide I) has been
visualized in the top right picture (figure 2(a), right). Here,
we took the already processed hyperspectral Raman map data
and confined the wavenumber range to the Amide I band
J. Opt. 24 (2022) 054005 L Ettema et al

Figure 2. Spontaneous Raman scattering results for case I after

Figure 3. Spontaneous Raman scattering results for case II after
VCA clustering. (a) Images of the plaque location with a size of
VCA clustering. (a) Images of the plaque location with a size of
51 × 51 µm are shown. In addition to the visualization of the plaque
81 × 81 µm are shown. In addition to the visualization of the plaque
in the fluorescence image, the plaque is also visible in the cluster
in the fluorescence image, the plaque is also visible in the cluster
image after VCA. Furthermore, a visualization of the protein peak
image after VCA. Furthermore, a visualization of the protein peak
has been depicted. At the plaque location, this protein peak is
has been depicted. At the plaque location, this protein peak is
shifted towards the higher wavenumbers. In (b) the full fingerprint
shifted towards the higher wavenumbers. In graph (b), the full
spectrum after VCA clustering is depicted, showing carotenoid
fingerprint spectrum after VCA clustering has been depicted,
peaks at 1151 and 1514 cm−1 in the plaque spectrum. In the
showing carotenoid peaks at 1150 and 1514 cm−1 in the plaque
zoomed-in (1400–1750 cm−1 ) graph (c), shifts in the lipid and
spectrum. In the zoomed-in (1400–1750 cm−1 ) graph (c), a
protein peak wavenumbers of the plaque curve (around 1440 and
broadening of the lipid curve and a shift in the protein peak
1658 cm−1 ) are visible. The colors of the curves in the graphs
wavenumber of the plaque curve (around 1439 and 1658 cm−1 ) are
correspond to the colors in the cluster image after VCA.
visible. The colors of the curves in the graphs correspond to the
colors in the cluster image after VCA.

(1650–1670 cm−1 ). Subsequently, we obtained the peak pos-

ition of each hyperspectral pixel and computed the peak map.
Afterwards, we smoothed the image with a 2D Gaussian filter.
At the plaque location, higher wavenumbers are more common
than at other locations in the sample.
The same results are found after processing the SpR
data for case II. In the top graph (figure 3(b)), the two
characteristic carotenoid peaks are present at wavenumbers
1150 and 1514 cm–1 . In contrast to the wavenumber shifts
observed in case I, for case II the wavenumber shift at the lipid
wavenumber is less pronounced. The protein shift, however,
is clearly present in the bottom graph (figure 3(c)). The shift
towards higher wavenumbers is visible in the spectrum of the
plaque, as well as at the plaque location in the visualization of
this protein peak (figure 3(a), right).

5
J. Opt. 24 (2022) 054005
Figure 4. SRS spectra and images of case I. (a) SRS scan across the
protein peak from 1633 to 1681 cm−1 , indicating a peak shift at the
plaque location. (b) SRS image results for four different
wavenumbers. Inset shows thioflavin-S staining to confirm plaque
presence. Scale bar: 50 µm.
In both cases the expected protein peak shift (Amide I) can
be observed, transitioning from an α-helix to β-sheet struc-
ture, when the plaque spectrum is compared to the surrounding
tissue [26–28]. In the Raman spectra the Amide I band (1640–
1680 cm−1 ) was rather strong and showed clear wavenumber
shifts for different tissue regions. Such shifts were much less
obvious for the weaker Raman peaks in the Amide II (1480–
1575 cm−1 ) or Amide III (1200–1300 cm−1 ) ranges and there-
fore we concentrated on the Amide I band.
The protein peak shifts observed in both cases have been
examined in more detail with SRS microscopy. Measurements
have been performed around the plaque locations in the range
from 1633 to 1681 cm−1 in 3 cm−1 steps (by tuning the optical
parametric oscillator (OPO) output wavelength, with smaller
step sizes around the 1660 cm−1 region). The results, presen-
ted for case I in figure 4(a) and for case II in figure 5(a),
6
L Ettema et al
Figure 5. SRS spectra and images of case II (a) SRS scan across the
protein peak from 1633 to 1681 cm−1 , indicating a peak shift at the
plaque location. (b) SRS image results for four different
wavenumbers. Inset shows thioflavin-S staining to confirm plaque
presence. Scale bar: 50 µm.
clearly show a shift of the protein peak at the plaque location
towards 1666 cm−1 , compared to the lower peak wavenum-
ber of 1657 cm−1 observed at the other locations. In addi-
tion to the graph, figures 4 (for case I) and 5 (for case II)
show images taken at wavenumbers corresponding to this pro-
tein peak shift (1666 cm−1 ), lipids (2850 cm−1 ), protein/lip-
ids (2930 cm−1 ), and unsaturated lipids (3019 cm−1 ) [12–
14, 29]. The thioflavin-S staining images confirm that meas-
urements have been performed at and around the plaque loca-
tions. Looking at the same plaque locations in the images taken
at 1666 cm−1 , the plaque has a higher intensity than the tissue
surrounding it, which corresponds to the observation from the
graph.
However, the signal in the Amide I region is low compared
to that in the C–H stretch region. In practice, the visualization
J. Opt. 24 (2022) 054005
Figure 6. (a) Normalized C–H stretch 2800–3100 cm−1 in 5 cm−1
steps for case II. The image (b) highlights the points where the
spectral data were extracted from. Scale bar: 50 µm.
of plaque could therefore be more convincing in the C–H
region.
Furthermore, since in several studies a lipid halo has been
observed around the plaques in brain samples of transgenic
mice [12–14], additional measurements have been performed
on case I and case II to examine the possible presence of such a
lipid halo in human AD brain tissue. Lipids show a strong char-
acteristic Raman peak in the C–H stretch region at 2850 cm−1
due to the abundant CH2 groups. This is illustrated for case II
in figure 6, with in figure 6(a) the normalized intensities of the
averaged spectra obtained at four different locations in the tis-
sue. Within this C–H stretch region, the spectrum of the plaque
(orange) shows a low intensity compared to the other graphs
at the lipid wavenumber (2850 cm−1 ) and has its peak value
at 2935 cm−1 . Figure 6(b) shows for each spectrum where the
data was extracted from.
When looking at the images in figures 4(b) and 5(b) taken
at 2850 cm−1 , no lipid halo is visible around the plaques. Also,
7
L Ettema et al
around the plaque area from case I (using SRS at six wavenum-
bers, results not shown) we did not observe such a halo. These
results in human brain tissue contradict the findings of the
above-mentioned publications on transgenic mice [12–14].
3.2. Analysis of carotenoids and Aβ42
In order to find out which carotenoid(s) may be present in
human AD brain tissue; SpR measurements have been per-
formed on six pure carotenoids, of which five are reported to
be common in human brain tissue [30].
This has first been done by measuring spectra of the
carotenoids dissolved in hexane, and later by measuring spec-
tra of the same carotenoids adsorbed on amyloid-β peptides,
to mimic more closely the conditions found in plaques.
In figure 7(a), the plaque spectra of cases I and II are shown
for comparison with the spectra recorded of the pure caroten-
oids in hexane, which are presented in figure 7(b). The spec-
tra recorded after the carotenoids were mixed with Aβ42 are
shown in figure 7(c).
The corresponding peaks fitted with a Gaussian are indic-
ated in table 2. For comparison, table 2 gives an overview
of the wavenumber peaks found for the single carotenoids in
both hexane and amyloid-β environment, and of the wavenum-
bers found in the two cases. Comparing the values of the pure
carotenoids in hexane with the values found in human AD
brain tissue shows that, although values differ between the
pure carotenoids (especially peak 2 around 1520 cm−1 ), all
values are higher than the values found in the two human cases.
Simulating a chemical environment better matching the biolo-
gical conditions, peak values were found to decrease to the
values shown in the second and fourth column of the table by
the amounts indicated in brackets. The shifts towards lower
wavenumbers result in values much closer to the values found
in tissue. Among the single carotenoid spectra obtained in an
amyloid-β environment, lycopene approaches the values in tis-
sue most closely.
4. Discussion
The applied label-free imaging techniques show promising
results for the characterization and determination of the bio-
molecular composition of the examined human AD brain tis-
sue. With the help of our developed technique, we were able
to further add to our previous reported results on the autofluor-
escence properties of cored plaques [10]. Here, we present
new auto-fluorescence images of plaques using two different
excitation wavelengths measuring two different emission out-
comes. The observation of two different fluorescence emis-
sions at the same plaque location suggests that at least two
different molecules are present [11]. Unfortunately, we could
not identify their origin, although the weak green emission
could be due to carotenoids; weak fluorescence spectra with
broad bands around 540 nm (not shown) were recorded in our
lab for all six carotenoids in n-hexane solution. Literature also
suggests that amyloid fibrils might be one of the underlying
causes [32]. A more thorough study with various excitation
J. Opt. 24 (2022) 054005 L Ettema et al

Table 2. Overview of the peak wavenumbers (C–C and C=C

modes) of the pure carotenoids, dissolved in hexane and after
mixing with Aβ42, based on figure 7.

Pure Carotenoid Pure Carotenoid

carotenoid mixed with carotenoid mixed with
peak 1 Aβ 42; peak peak 2 Aβ 42; peak 2
(cm−1 ) 1 (cm−1 ) (cm−1 ) (cm−1 )

Astaxanthin 1157.3 1155.4 (−1.9) 1517.5 1513.9 (−3.6)

Beta-carotene 1157.1 1154.7 (−2.4) 1522.6 1517.1 (−5.5)
Beta- 1157.0 1155.2 (−1.8) 1522.6 1520.0 (−2.6)
cryptoxanthin
Lutein 1157.3 1156.3 (−1) 1525.6 1521.5 (−4.1)
Lycopene 1160.0 1152.2 (−7.8) 1518.8 1514.5 (−4.3)
Zeaxanthin 1157.5 1156.2 (−1.3) 1523.2 1515.0 (−8.2)
in tissue in tissue
(cm−1 ) (cm−1 )
Case I x 1151 x 1514
Case II x 1150 x 1514
All peaks were obtained by a Gaussian fit and compared (grey rows) with
the peak wavenumbers found in cored plaques in tissue of case I
(figures 2(b) and (c)) and case II (figures 3(b) and (c)), see orange spectral
lines. Peaks around 1003 cm−1 are not discussed due to the strong overlap
with phenylalanine.

detection will be required to investigate the molecular nature

Figure 7. (a) Averaged carotenoid spectra found in both AD plaques
from both cases. (Orange spectrum shown in figures 2 and 3).
(b) Vector normalized spectra of pure carotenoid compounds
dissolved in hexane, in good agreement with the data of [31].
(c) Vector normalized spectra of carotenoids mixed with Aβ42.
Peak analysis of the 6 carotenoids was done using a Gaussian fit
(not shown).
and emission filters, or the use of a confocal fluorescence
microscope with spectral detection would help to differenti-
ate and separate the location of the emission in a more precise
way. In addition, micro-extraction followed by liquid chroma-
tography (LC) with fluorescence and mass spectrometry (MS)
of the fluorescence in plaques in more detail.
After areas of interest were identified via the autofluores-
cence images, we mapped areas of plaque locations using SpR
and SRS microscopy and in particular studied the Amide I
peak position in the SpR spectra in more detail. With both
instruments we were able to confirm the expected β-sheet
shift [33, 34] at the protein peak location. When measur-
ing at the exact plaque locations the Amide I peak shifts
towards longer wavenumbers (∼1666 cm−1 ), indicating a β-
sheet conformation. The Amide I band in the surrounding
tissue peaks around 1657 cm−1 , maintaining its predomin-
antly α-helix structure. Misfolding of a protein can be caused
by different factors, such as ‘mistakes in biogenesis, disease-
causing mutations, and physiological stressors’ [28]. By chan-
ging its secondary structure from α-helix to β-sheet the protein
becomes insoluble and neurotoxic [7]. The shift in wavenum-
ber corresponding to this change in conformation is in accord-
ance with literature [26, 27], and can clearly be seen in
the orange spectral curves of the SpR results (figures 2(c)
and 3(c)), and in the SRS results (figures 4(a) and 5(a)).
In addition, the computed images in figures 2(a) and 3(a)
depict the peak shift in a novel way at the locations where
amyloid deposits are present. The dense cored plaques show
a strong β-sheet shift signal compared to other, more dis-
perse and diffuse plaques [10]. These results provide further
proof that vibrational spectroscopy is capable of label-free
characterization of the biomolecular composition of areas of
interest.
Besides the clear indication of the described protein peak
shift, the presence of carotenoids at the plaque location was
observed using SpR. Clear spectral peaks associated with

8
J. Opt. 24 (2022) 054005
carotenoids around 1150 and 1514 cm−1 [31, 35] are measured
and their spectrum can easily be distinguished from spec-
tra obtained from surrounding brain tissue. Carotenoids can-
not be synthesized by humans and must be obtained from
diet [36]. Carotenoids are known for their anti-inflammatory
and antioxidant effects [37, 38] and potentially fight oxidat-
ive damage [38–41]. Their presence, directly at the plaque
location confirms our previous results, suggesting an ongoing
inflammatory process associated with AD [42, 43]. This could
explain the elevated level of carotenoids found at the plaque
location.
Since we could confirm our previous findings in addi-
tional cored amyloid plaques, it would contribute to the fur-
ther understanding of the disease progression to know which
of them are present at the plaque location. However, there are
more than 600 known carotenoids [44, 45]. We recorded ref-
erence SpR spectra of six common carotenoids, of which five
had been found in human brain extract samples (but not spe-
cifically in plaque) [30]. The two prominent carotenoid peaks,
around 1156 and 1521 cm−1 , were not matching exactly the
carotenoid peaks found in human plaques. However, the lab-
environment did not fit the environment found in AD brain tis-
sue since the carotenoids were dissolved in hexane. To mimic
the micro-environment of plaques more closely, we also meas-
ured these carotenoids after adsorption on aggregated Aβ42
protein. The results, presented in figure 7, show a peak shift
towards the lower wavenumbers for carotenoids measured
in the simulated plaque environment. This suggests that the
carotenoid peak positions depend on their micro-environment.
These new peak maxima are in much better agreement with the
carotenoid peaks found in human amyloid-beta plaques (com-
pare the spectral peaks in figures 7(a) and (c)). Based on the
Raman spectra measured after adsorption on Aβ42 aggregates,
lycopene appears to match the spectra obtained from plaque
areas most closely (see table 2). This carotenoid has previ-
ously been mentioned as possibly important in the prevention
of cognitive misfunctioning [46, 47] although no significant
correlation was observed. Of the carotenoids measured in this
study, lycopene is among the least soluble carotenoids in water
due to the absence of a hydroxyl group. This property matches
the hydrophobic nature of the amyloid plaque and could thus
explain co-aggregation. However, although the results suggest
lycopene might be present in plaque, we can only conclude
so with tentativeness. In addition, if lycopene turns out to be
present, it is uncertain whether lycopene is the only carotenoid
or the major carotenoid present in plaque. Our finding would
therefore require confirmation with an independent chemic-
ally specific technique such as LC-MS. To the best of our
knowledge, similar experimental results have not been repor-
ted before.
In the current study, the spectrum of carotenoids could
only be measured with the 532 nm excitation source in the
SpR system. Our current SRS system is not capable of ima-
ging the presence of carotenoids. The reason is the absorbance
band of the carotenoids which peaks around 450 and 478 nm
[10, 48]. The excitation source of the SpR system is near to
those peaks and offers therefore (pre) resonance enhancement
which increases the overall signal detection and selectivity
9
L Ettema et al
over other tissue components. Our current SRS system, how-
ever, operates in the NIR region which is further away from the
carotenoids absorbance peak and therefore does not offer any
resonance enhancement. In our previous publications, we were
not able to measure any carotenoid associated peaks at plaque
locations using a 785 nm excitation source [10, 49] with our
SpR system.
A SpR or VIS-SRS system with a 450–500 nm excita-
tion source better matching the absorption band of caroten-
oids would most likely provide more pronounced results and
better signal-to-noise ratio (SNR) [50, 51]. Eventually, that
would allow faster image acquisition and reduced power
options, further reducing potential photo-damaging effects.
It would also allow to investigate different plaque pheno-
types which are less dense and therefore potentially lack a
strong carotenoid signal, if any at all. Currently, the load
of pathology in AD brain is assessed with (immuno-) his-
tology and different staging methods according to Braak,
CERAD and Thal [52]. Assessment and staging of patho-
logy without labeling would offer an advantage for dia-
gnostics. Furthermore, the carotenoid analysis could provide
additional information on the pathological structures used
for pathological staging and therefore provide more insight
in the disease process. Since carotenoids fight inflamma-
tion, further research will help to understand if carotenoids
only appear when amyloid(-β) is already accumulated or if
they are already present when the first amyloid fibrils are
occurring.
Recent publications report on the presence of a distinct
lipid halo around amyloid deposits in Alzheimer mouse mod-
els. Since there is no similar analysis reported yet on human
AD brain tissue, we employed SRS in the C–H stretch region.
During this study, we focused on dense cored plaques since
they appear in a similar manner as plaques in the transgenic
mice. There, the amyloid accumulation is very dense, and the
plaque size is rather compact and usually below 20 µm. Using
the wavenumbers suggested in the literature for lipids [12–14],
we imaged human AD plaques. As shown in the SRS images
of figures 4 and 5, we could not observe a pronounced lipid
ring around the dense cored plaques of our cases. Apart from
the ‘raw’ wavenumber images presented here, we also applied
a simple linear decomposition algorithm to increase differ-
entiation [18]. The resulting images were higher in contrast
but still did not show any lipid-associated ring or halo around
the plaque. We hypothesize that the reported lipid ring/halo
in AD mice might be a side effect of artificially introduced
amyloid and can therefore only be found in transgenic mice.
The ring might emerge due to the lack of protein in the near
surrounding tissue area since it is rapidly aggregated in the
artificially induced protein accumulation. That could lead to
an increased relative occurrence of lipid in the surrounding
ring. The absence of a lipid ring in our data is an important
reminder that cell type differences in animal models compared
to human tissue can be extensive and could lead to differ-
ent results [53]. However, a direct comparison study between
transgenic AD mice and human tissue, using the same SRS
device, would help to clarify these potentially contradictory
results.
J. Opt. 24 (2022) 054005
SpR has the advantage of recording a full spectrum per
pixel but at the cost of extended acquisition time. If one wants
to investigate only specific wavenumbers, as e.g. in our case
the distinct carotenoid peaks or the beta-sheet shift, SRS out-
performs SpR by at least two-three orders of magnitude in
mapping speed, and apart from shortening the overall meas-
urement time this can also be used for imaging a larger tissue
area.
5. Conclusion
In this study, we performed fluorescence and Raman micro-
scopy on unlabeled amyloid plaques in human AD brain tis-
sue. The fluorescence images obtained from two cored plaques
show the autofluorescence property of plaque, with the plaque
emitting blue and green fluorescence when excited in the UV
(340–380 nm) and blue (450–490 nm), respectively. Compar-
ison of the location and shape of the fluorescent spots indic-
ate that the two components responsible for the fluorescence
observed are largely co-located. SpR measurements across the
same location within the tissue sections reveal the presence of
carotenoids in amyloid-beta cored plaques, by showing clear
peaks at 1150/1151 and 1514 cm−1 .
Furthermore, the plaque spectra show shifts in the
wavenumbers of the lipid and protein (Amide I) peaks, com-
pared to the spectra from the surrounding tissue. Further
investigation on the protein shift with SRS microscopy indic-
ates a peak value of 1666 cm−1 in plaque, compared to a
peak value of 1657 cm−1 in the rest of the tissue. This shift
in wavenumber can be explained by the change in protein
structure, namely from an α-helix or random conformation
into a β-sheet structure, when amyloid-beta peptides accu-
mulate into plaque. Additional SRS measurements have been
performed in the C–H stretch region. However, the presen-
ted results, obtained from human AD tissue, do not show a
pronounced presence of lipids around the plaques. This sug-
gests that the presence of a lipid ring as reported for trans-
genic mice, might be a side-effect of the transgenic mouse
model. Lastly, SpR measurements on six carotenoids have
been performed under pre-resonance conditions, both with the
carotenoids dissolved in hexane, as well as with the caroten-
oids in a mixture with amyloid-beta peptides. The results show
that the micro-environment of the carotenoids influences the
vibrational frequencies, leading to different peak wavenum-
bers. We note that the measurements performed on the protein
aggregates give peak wavenumbers more similar to the val-
ues found in the human AD brain sections. Of the six caroten-
oids measured, lycopene comes closest to the values found in
the two presented human plaque cases. However, the caroten-
oids’ exact molecular identification would require confirma-
tion with an independent, chemically specific technique such
as LC-MS.
Data availability statement
The data that support the findings of this study are available
upon reasonable request from the authors.
10
L Ettema et al
Acknowledgments
The authors would like to thank R W Schmidt for providing
the chemometric software GUI. We would also like to thank
Tjado H J Morrema for technical assistance.
This research is supported by the Dutch Technology Found-
ation STW (Grant Number 13935, I-READ), which is part of
the Netherlands Organization of Scientific Research (NWO),
and which is partly funded by the Ministry of Economic
Affairs.
ORCID iDs
Benjamin Lochocki  https://orcid.org/0000-0002-4060-
3944
Jeroen J M Hoozemans  https://orcid.org/0000-0002-2888-
8967
Johannes F de Boer  https://orcid.org/0000-0003-1253-
4950
Freek Ariese  https://orcid.org/0000-0002-8756-7223
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