Nonlinear Optics and Quantum Optics, Vol. 40, pp. 241–251
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Luminescence and Two-Photon Absorption
Cross Section of Novel Oligomeric
Luminescent Conjugated Polythiophenes for
Diagnostics of Amyloid Fibrils
Mikael Lindgren1 , Eirik Glimsdal1 , Andreas Åslund2 ,
Rosalyn Simon2 , Per Hammarström2 and K. Peter R. Nilsson
1 Department
of Physics, Norwegian University of Science and Technology,
7491 Trondheim, Norway
E-mail: [email protected]
2 Department of Biochemistry, Linköping University, 581 83 Linköping, Sweden
Received: July 21, 2009. Accepted: November 7, 2009.
Here we present the TPA cross section and quantum efficiencies of a
series of novel oligomeric luminescent conjugated polythiophenes used
for detection and spectral diagnostics of amyloid protein aggregates of the
amyloid-beta peptide associated with Alzheimer’s disease. Specifically,
these probes consist of pentameric or heptameric thiophenes derivatives
with carboxylic substituents attached onto various thiophene rings. The
probes absorbs over a broad range approx. 400–500 nm with quantum
efficiency of approx. 20% in at neutral pH conditions, and also showed
TPA cross sections of 5–50 GM in the range 700–840 nm, in the same
order of magnitude as commonly used fluorescein derivatives. Importantly, the multiphoton excitation capabilities of LCPs provided excellent
performance when compared to imaging using conventional “single photon” excitation. It is also demonstrated their utilization in both one- and
two-photon excitation laser scanning microscope spectral imaging for
diagnostics of Alzheimer disease pathology in ex vivo histological sections.
Keywords: Fluorescent dyes, thiophene oligomers, amyloid detection, amyloid
spectroscopic diagnostics, TPA cross section, quantum efficiency.
INTRODUCTION
Over the past two decades, the two-photon absorption (TPA) process has been
exploited and used in several laser based applications, such as fluorescence
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microscopy and spectroscopy [1], optical power limiting [2, 3], singlet oxygen
production in photo-dynamic therapy [4, 5], two-photon light harvesting [6],
and many other. Recently, we reported on luminescent conjugated polythiophenes (LCP) and showed that these have large TPA cross-sections promising
for use both in vitro and in vivo imaging of amyloid protein aggregates, by
employing two-photon excitation (TPE) laser scanning microscopy (LSM) [7].
Here, it is given a brief introduction to the use of LCP for diagnostics of
amyloid proteins associated to diseases such as Alzheimer’s disease (AD)
along with results on novel oligomeric LCPs, with particular emphasis on
TPA cross-sections and relevant photo-physical characterization.
The detailed mechanisms behind self-aggregatiton of native proteins into
amyloid fibrils are still a mystery, even though the fibrillar deposits have
many structural properties in common, such as the β-sheet morphology [8].
The common fibrillar structures have also the ability to bind small molecules
such as thioflavin T (ThT) [9] and Congo red [10], followed by changes
in their luminescent and optical properties. Early studies of fibril formation
utilized turbidity, sedimentation combined with ThT binding provided data
on appearing high molecular weight aggregates or disappearance of soluble low-molecular peptides. Moreover, small soluble precursor aggregates
known as “oligomers” or “proto-fibrils” are generally believed to be very
important as being involved in the early stages of disease progression [11,12].
The structures of such oligomeric aggregates for a variety of proteins are of
great interest in order to understand amyloid formation, cell toxicity and disease. [13,14]. Using combinations of size exclusion chromatography, electron
microscopy and quasielastic light scattering spectroscopy it was possible to
distinguish intermediate structures in terms of dimers and protofibrils of typical sizes 2 and 100–200 nm, respectively [15]. Small molecular dyes such
as (4-(dicyanovinyl)-julolidine) (DCVJ), as well as derivatives of amino-8naphtalene sulphonate (ANS, Bis-ANS) have been used for amyloid fibril
detection being known to bind to the fibrillar or pre-fibrillar states with dissociation constant in the µM range [16]. Derivatives of thioflavins, Congo red
derivatives (e.g. X-34) and oxazine-derivatives (e.g. Nile red) typically bind
amyloid fibrils in the nM-µM range with multiple binding sites. For more
details on early work it is referred to recent reviews on the topic [17,18].
LCPs, in contrast to sterically more well-defined amyloidotropic dyes (such
as ANS, DCVJ, thioflavins and Congo red), contain a twistable thiophene
backbone whose π-conjugation framework changes the luminescent properties [19–22]. The spectroscopic fingerprint of the LCP bound to a protein or
protein aggregate reflects the conformational differences of the protein state.
By employing this new probe technology it was possible to discriminate conformational heterogeneities in A-beta amyloid plaques in Alzheimer disease
mouse models and, morphologically different amyloid deposits in systemic
AL amyloidosis [23,24]. We here present the spectroscopic and two-photon
absorption characterization of novel conjugated luminescent oligothiophenes
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FIGURE 1
Schematic drawing of the chemical synthesis of LCOs and examples of LCOs: a) Schematic
drawing of the synthesis of the iodinated trimeric thiophene precursor. b) Examples of pentameric
(LCO1 and LCO2) and heptameric LCOs (LCO3). All LCO are anionic at pH 7.5 as R=COOH.
(LCOs). The Molecular structures to be discussed along with their abbreviations are depicted in Fig. 1. For further details on the synthesis of the
molecular structures and results concerning more biologically relevant issues
can be found elsewhere [25].
EXPERIMENTAL
Spectroscopy and TPA cross-sections
Luminescence and time-resolved measurements were performed employing a
Jobin-Yvon IBH FluoroCube photon-counting spectrometer. A 200 fs pulsed
Ti:Sapphire laser (Coherent MIRA 900-F) and/or the IBH NanoLEDs light
laser-diodes were used as excitation sources. For two-photon excitation measurements the laser was tuned in the 700–860 nm range. For single photon
excitations the laser beam was frequency doubled using a SHG crystal (Inrad
Ultrafast Harmonic Generation System, Model 5-050). A pulse picker (Coherent 9200) is placed after the laser (76 MHz) to control the pulse repetition
frequency (prf). This was found to be especially important if long lived
excited states are present [Glimsdal, 2007]. For the measurements reported
here a prf of 4.75 MHz prf was found appropriate. Measurements of steady
state optical absorption spectra were recorded using an Agilent 8456E UV-vis
spectroscopic system.
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Fluorescence decay times were measured in time-correlated single photon
counting (TC-SPC) mode, with a time resolution down to 0.014 ns. An optical trigger (IBH TB-01) was used as time-reference using a thin glass wedge
to take out a small part of the fundamental. Emission spectra were recorded
by scanning the monochromator in front of the PMT. Fluorescence quantum
yields were calculated from absorption and fluorescence measurements at five
different concentrations with peak absorbance below 0.1 OD. The absorbance
at the excitation wavelength and the spectrally corrected integrated fluorescence is collected at each concentration. The relative quantum yield of two
samples is then calculated from the equations given by Williams et al. [26].
Using a reference sample with known quantum yield, the absolute quantum
efficiency of the new sample is determined. The two-photon absorption (TPA)
cross-section can be found from two-photon excitation induced fluorescence,
as outlined by Webb et al. [27–28]. The integrated fluorescence was collected
by two-photon excitation using typically 50 µM concentrated samples. The
relative TPA cross section is then found when the quantum efficiency of both
samples is known, and other experimental parameters either are determined, or
identical. Albota et al. give expressions suitable for numerical calculation. Utilizing one sample with known quantum efficiency and TPA cross section, the
absolute value of the cross section for the new sample is calculated [28]. Here,
fluorescein in NaOH was used as reference material due to its well-known
high quantum yield and suitable TPA cross section.
The amyloid fibrils were visualized using a Zeiss 510-LSM equipped with
a META spectral detector. Excitation was performed using a 488 nm argon
laser and a Ti:Sapphire fs laser tuned to 800–830 nm used for two-photon
excitation, for further details, see [7,23,24].
Synthesis of LCOs
The detailed synthesis of the LCOs will be reported elsewhere [25]. Briefly,
all of the LCO were synthesized by iodination of a trimeric thiophene precursor [22,25]. The iodinated trimer was further converted to pentamers
or heptamers by addition of suitable thiophene monomers through Suzuki
coupling.
Preparing Abeta samples
Recombinant Abeta 1–40 was obtained from rPeptide (GA, USA), as a
lyophilized powder from hexafluoroisopropanol. The peptide was dissolved at
1 mg/ml in 2 mM NaOH and was stored at −20◦ C until further use. Amyloid
fibrils were generated by dissolving 10 µM Abeta 1–40 in 10 mM phosphate
buffer pH 7.5 in 100 µL samples, containing 0.3 µM LCO, in 96 well plates
(Corning) that were subjected to 8 min cycles of vigorous shaking 12 min
resting at 37◦ C in a Saphire2 (Tecan, Switzerland) fluorescence plate reader
for 20 hrs. Emission and excitation spectra in 3D format were obtained using
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5 nm slits. The wavelengths used were 400–490 nm excitation and 500–750
nm emission for LCO2 and LCO3.
RESULTS AND DISCUSSIONS
LCO2
LCO2/A-Beta
20000
Emission (counts)
Excitation (counts)
Spectroscopic characterization
Representative excitation and emission spectra of LCO2 are shown in Fig. 2,
upper panels, both for the dye in neutral pH buffer before and after the addition
of amyloid-beta fibrils. As shown in the spectra there is a dramatic change of
appearance both for the excitation and emission spectrum, with a considerable
shift of the excitation spectrum. Thus, the dye gives very good contrast when
using the common excitation wavelength around 488 nm for the Ar-laser line,
and virtually no background is observed (more below).
We also demonstrate the length and chemical substituent differences of
two of the LCOs in the optical output as shown in the contour plots of combined excitation and emission (Fig. 2, lower panels) upon binding to amyloid
15000
10000
5000
0
350
400
450
Wavelength (nm)
15000
10000
460
440
420
5000
0
500
500
550
600
Wavelength (nm)
650
LCO3
480
Excitation (nm)
Excitation (nm)
20000
LCO2
480
400
500
25000
460
440
420
550
600
650
Emission (nm)
700
750
400
500
550
600
650
Emission (nm)
700
750
FIGURE 2
Upper panels: Excitation and emission spectra of LCO 2 at pH 7.5 with and without amyloid beta
fibrils. Lower panels: Contour plots of excitation and emission spectra of LCO1 and LCO3 with
amyloid beta fibrils.
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30000
20000
10000
0
0.00
Fluorescein (ref = 0.9)
LCO1 (0.22)
LCO2 (0.21)
LCO3 (0.28)
0.05
0.10
Absoption (OD)
0.15
TPA cross section (GM)
Fluorescence counts
246
Fluorescein (ref)
LCO1
LCO2
LCO3
100
75
50
25
0
700
750
800
Wavelength (nm)
850
FIGURE 3
QE (left) and TPA (right) for the LCOs. The determined quantum efficiencies are given in the
inset (left panel). Fluorescein in 0.1 M NaOH was used as reference.
beta fibrils. As can be seen, there is a considerable red-shift in the excitation spectrum of the heptamer, compared to the excitation spectrum of the
pentamer.
The quantum efficiencies (QE) for the three LCOs were measured by
recording the corresponding absorption spectra and plot the integrated fluorescence yield vs. absorbance for a fixed excitation wavelength (443 nm),
see Fig. 3, left panel. By comparing with a well known reference sample, in this
case fluorescein in 0.1 mM NaOH, the QE is obtained. For all LCOs the QE was
found to be in the range 20–30 % at pH 7.5. Using a similar fluorescence based
technique the TPA cross section can be obtained by measuring the integrated
fluorescence upon two-photon excitation keeping all beam focussing parameters identical (see [3] for details). Typically 50 µM of solution was used, and
the values were found to be in the range 5–40 in the excitation wavelength
range 725–840 nm, i.e. wavelengths typically used for multiphoton excitation
imaging using Ti:Sapphire based lasers.
The fluorescence quantum yield for a series of LCPs at different pH was
previously determined, giving quantum efficiencies in the range 5–10 % [7].
This was found much lower than for conventional molecular high efficiency
fluorescence systems based on e.g. fluorescein. The QE reported here are 3–4
times larger indicating that self-quenching due to aggregation or exciton migration is limited in the oligomeric compounds, resulting in a better fluorescence
contrast in imaging applications. The TPA cross sections are here reported
to be comparable to fluorescein for the LCO2, whereas the two other LCOs
were found to have much lower values. Tentatively, we ascribed this to a more
planar conformational structure for LCO2 due to the charged COO- groups
at the terminals. It can be anticipated that the repulsion between the central
COO- groups in LCO1 and LCO3 will render these in a twisted conformation
being free in solution. The nonlinear properties in terms of TPA cross-section
was for the LCP case found to be in the range of 400–1000 GM [7], being
approximately two orders of magnitude better than fluorescein and the LCOs
presented here, based on TPA per molecule, but as pointed out earlier [7],
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FIGURE 4
Emission images of A: one- and B: two-photon absorption excitation, respectively, of amyloid
beta protein aggregates using LCO3 as fluorescent stain.
compensating for the molecular size and normalizing to molecular weight
the TPA cross section would be lower in a comparison. Interestingly, it was
also found that multi-photon excitation schemes in some instances gave complementary spectral signatures in terms of polarization and spectral shifts in
addition to results obtained using single photon excitation of the very same
LCP stained samples [23,24]. We foresee similar differences in TPA spectral emission and efficiency when comparing the same LCO when bound to
different amyloid deposits.
Spectroscopic imaging of amyloid deposits
The progress in the chemical and biological analyses from molecular events to
pathologic conditions is leveraged by the development of new and improved
existing optical probes and markers for in vitro and in vivo applications. In
addition, the increasing interest in spectral imaging is constantly being spurred
by innovative optical designs, rapid advances in detector array and computer
technologies. Obviously, the fluorescent probes discussed so far are readily
used in conventional confocal microscopy, although it is sought for methods
that allow also in vivo studies of animal models, and potentially also humans.
The most widely used nonlinear effect in multiphoton microscopy is twophoton absorption. Because of the nonlinear response, the excitation volume
will be confined to the focal point, so in principle the method is “self-confocal”
in contrast to the conventional confocal fluorescence microscopy that uses a
pin-hole to reject unwanted scattered light. Special sensitizers are normally
developed in order to utilize TPA.
Molecular probes based on fluorescein derivatives are commonly used
since they have a high quantum yield in addition to a good two-photon absorption (TPA) cross-section (this holds also true for LCPs and the LCOs to be
discussed below). Moreover, since they allow NIR light as excitation that
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ex 458 nm
ex 830 nm
1.0
Emission (a.u.)
0.8
0.6
0.4
0.2
0.0
500
550
600
650
Wavelength (nm)
FIGURE 5
Spectral emission content of emission images of A: one- and B: two-photon absorption excitation
of A-beta protein amyloid aggregates using LCO3.
interacts only weakly with biological materials, the effects of photobleaching
and photo-damage above and below the focus are reduced. Because of the
much larger TPA cross-section of the used LCP dyes compared to the nonlinear response of the surrounding tissue, a good signal-to-noise ratio is achieved
and the LCP signal can easily be distinguish from the auto-fluorescence that is
known to hamper image quality in conventional fluorescence microscopy. TPA
has been used in studies of many different tissue types including brain [29–
30]. Progress towards the in vivo imaging of AD lesions in transgenic mice
has been made with the development of imaging agents that enter the brain
and target amyloid deposits such as methoxy-X04 [31].
Multiphoton excitation imaging by using various LCP was previously
demonstrated in [7,23,24]. As shown in Fig. 4, two-photon excitation images
of A-beta amyloid aggregates stained by LCO3. The corresponding emission spectra making averages of 5 ROIs is shown in Fig. 5. As discussed
in the previous section, LCPs in contrast to conventional stains provide
variable spectral signatures of amyloid aggregates in vitro and in histological sections, depending on the type of amyloid/prion deposit it interacts
with [32]. Thus, LCP luminescent staining technology constitutes an important new tool for the analysis of amyloid and prion complexes when analyzed
with modern high-resolution spectral imaging techniques. Importantly, the
multiphoton excitation capabilities of a few LCPs provided excellent performance when compared to imaging using conventional “single photon”
excitation. The possibility of combining different lengths of LCOs which
provide variable conjugation lengths further emphasize the possibility of multiple labelling schemes using either the same or variable excitation sources to
image differences in the amyloid structure (e.g. Fig. 2, lower panel).
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SUMMARY AND CONCLUSIONS
The TPA cross-section and QE of a series of the novel LCOs for detection
and spectroscopic diagnostics of amyloid protein are here reported. The TPA
crcoss-section of the best LCO was in the same order of magnitude as fluorescein, and the fluorescence quantum efficiency was found to be in the
range 20–25 % in pH 7.5 buffer solutions. The novel oligomeric thiophene
fluorescent dyes showed large differences in excitation and emission spectra
upon binding to amyloid beta protein fibrils. Notably, one of the variants, LCO
showed very good contrast using one-photon excitation using the conventional
488 nm excitation wavelength. It was also demonstrated the use of the dyes in
multiphoton excitation imaging using a laser scanning microscope. There are
still a number of aspects of thiophenes based derivatives well worth further
examination e.g., 1) to what extent will the new spectroscopic probes allow
discrimination of different amyloid fibrils based on different protein native
structures and classes, 2) can the spectroscopic properties of an LCO bound to
a pre-fibrillar oligomer be used as a marker for an on or off pathway species?
If the spectroscopic property (i.e. conformation) of the LCO mimics that of
the mature fibril is it likely that the building block is conformationally related,
i.e. a building block for the mature fibril. If on the other hand the oligomer
require a substantial conformational change prior to formation of an amyloid
fibril the spectral property will be different. Markers of this type will certainly
be imperative for both basic understanding of pathways (intermediates) of
amyloid fibril formation but also for imaging purposes so the long sought for
oligomeric states can be ascribed a pathological role also in vivo. Work along
these lines is currently in progress.
ACKNOWLEDGEMENTS
The work is supported by the Swedish Foundation for Strategic Research
(P:N, R.S. PH), the Knut and Alice Wallenberg foundation (P.N., PH) and the
Swedish Foundation for International Cooperation in Research and Higher
Education (P.N., R.S.). A generous gift from Astrid and Georg Olsson is also
gratefully acknowledged.
REFERENCES
[1] Zipfel, W. R., Williams, R. M. and Webb, W. W. Nonlinear magic: Multiphoton microscopy
in the biosciences. Nature Biotech. 21(11) (2003), 1369–1377.
[2] Spangler, C. W. Recent development in the design of organic materials for optical power
limiting. J. Mater. Chem. 9 (1999), 2013–2020
[3] Glimsdal, E., Carlsson, M., Eliasson, B., Minaev, B. and Lindgren, M. Excited states and
two-photon absorption of some novel thiophenyl Pt(II)-ethynyl derivatives. J. Phys. Chem.
A 111 (2007), 244–250.
“NLOQO” — “nloqo_is17” — 2010/3/19 — 11:53 — page 249 — #9
250
M. Lindgren et al.
[4] Frederiksen, P. K., McIlroy, S. P., Nielsen, C. B. , Nikolajsen, L., Skovsen, E., Jørgensen,
M., Mikkelsen, K. V. and Ogilby, P. R. J. Am. Chem. Soc. 127 (2005), 255–XXX.
[5] McIlroy, S. P., Cl’o, E., Nikolajsen, L., Frederiksen, P. K., Nielsen, C. B., Mikkelsen, K. V.,
Gothelf, K. V. and Ogilby, P. R. J. Org. Chem. 70 (2005), 1134–XXX.
[6] Picot, A., Malvolti, F., Le Guennic, B., Baldeck, P. L., Williams, J. A. G., Andraud, C. and
Maury, O. Inorg. Chem. 46 (2007), 2659–XXX.
[7] Stabo-Eeg, F., Lindgren, M., Nilsson, K. P. R., Inganas, O. and Hammarstrom, P. Quantum
efficiency and two-photon absorption cross-section of conjugated polyelectrolytes used for
protein conformation measurements with applications on amyloid structures. Chem. Phys.
336(2–3) (2007), 121–126.
[8] Selkoe, D. J. The molecular pathology of Alzheimer’s disease. Neuron 6 (1991), 487–498.
[9] Naiki, H., Higuchi, K., Hosokawa, M. and Takeda, T. Fluorometric determination of amyloid
fibrils in vitro using the fluorescent dye, thioflavine T Analytical Biochemistry, 177(2)
(1989), 244–249.
[10] Klunk, W. E., Pettergrew, Abraham, D. J. Quantitative evaluation of congo red binding to
amyloid-like proteins with a beta-pleated sheet conformation. J. Histochem. Cytochem. 37
(1995), 1273–1281.
[11] Selkoe, D. J. Translating cell biology into therapeutic advances in Alzheimers disease.
Nature Suppl. 399, A23-A31; Yanker, B. A. (1996) New clues to Alzheimer’s disease:
Unraveling the roles of amyloid and tau. Nature Medicin 2 (1999), 850–852.
[12] Sörgjerd, K., Klingstedt, T. Lindgren, M. Kågedal, K. and Hammarstrom, P. Prefibrillar
transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell
culture. BBRC 377 (2009), 1072–1078.
[13] Zako, T., Sakono, M., Hashimoto, N., Ihara, M. and Maeda, M. Bovine insulin filaments
induced by reducing disulfide bonds show a different morphology, secondary structure, and
cell toxicity from intact insulin amyloid fibrils. Biophys. J. 96 (2009), 3331–3340.
[14] Frare, E., Mossuto, M. F., de Laureto, P. P., Tolin, S., Menzer, L., Dumoulin, M., Dobson,
C. M. and Fontana, A. Characterization of oligomeric species on the aggregation pathway
of human lysozyme. J. Mol. Biol. 387(1) (2009), 17–27.
[15] Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M. and Teplow, D. B. Amyloid
b-protein fibrillogenesis – Detection of a protofibrillar intermediate. J. Biol. Chem. 272(35)
(1997), 22364–22372.
[16] Lindgren, M., Sörgjerd K. and Hammarström, P. Detection and characterization of
aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence
spectroscopy. Biophys. J. 88 (2005), 4200–4212.
[17] Sipe, J. D. and Cohen, A. S. Review: History of the Amyloid Fibril. J. Struct. Biol. 130
(2000), 88–98.
[18] Nilsson, K. P. R. Small organic probes as amyloid specific ligands – Past and recent
molecular scaffolds. FEBS Letters, 2009, DOI: 10.1016/j.febslet.2009.04.016.
[19] Nilsson, K. P. R., Rydberg, J., Baltzer, L. and Inganäs, O. Self-assembly of synthetic peptides control conformation and optical properties of a zwitterionic polythiophene derivative.
Proceedings of Natl. Acad. Sci. U.S.A. 100 (2003), 10170–10174.
[20] Nilsson, K. P. R., Herland, A., Hammarström, P. and Inganäs, O. Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation.
Biochemistry 44 (2005), 3718–3724.
[21] Nilsson, K. P. R., Olsson, J. D. M., Stabo-Eeg, F., Lindgren, M., Konradsson, P.
and Inganas, O. Chiral recognition of a synthetic peptide using enantiomeric conjugated
polyelectrolytes and optical spectroscopy. Macromolecules, 38(16) (2005), 6813–6821.
[22] Åslund, A., Herland, A., Hammarström, P., Nilsson, K. P. R., Jonsson, B-H. and Konradsson, P. Studies of luminescent conjugated polythiophene derivatives: enhanced spectral
discrimination of protein conformational states. Bioconjug. Chem. 18 (2007), 1860–1868.
“NLOQO” — “nloqo_is17” — 2010/3/19 — 11:53 — page 250 — #10
Luminescence and Two-Photon Absorption
251
[23] Nilsson, K. P. R., Hammarstrom, P., Ahlgren, F., Herland, A., Schnell, E. A. and Lindgren,
M., et al. Conjugated polyelectrolytes – Conformation-sensitive optical probes for staining
and characterization of amyloid deposits. Chembiochem, 7(7) (2006), 1096–1104.
[24] Nilsson, K. P. R., Aslund, A., Berg, I., Nystrom, S., Konradsson, P. Herland, A., Inganas, O.,
Stabo-Eeg, F., Lindgren, M. and Westermark, G. T. Imaging distinct conformational states of
amyloid-beta fibrils in Alzheimer’s disease using novel luminescent probes. ACS Chemical
Biology 2(8) (2007), 553–560.
[25] Åslund, A., Sigurdson, C. J., Klingstedt, T., Grathwohl, S., Bolmont, T., Dickstein, D. L.,
Glimsdal, E., Prokop, S., Lindgren, M. and Konradsson, P., et al. ACS Chemical Biology
4 (2009), 673–684.
[26] Williams, A. T. R., Winfield, S. A. and Miller, J. N. Relative fluorescence quantum yields
using a computer-controlled luminescence spectrometer. Analyst 108 (1983), 1067–1071.
[27] Xu C., Webb, W. W. Measurement of two-photon excitation cross sections of molecular
[fluorophores with data from 690 to 1050 nm. J. Opt. Soc. Am. B 13 (1996), 481–491.
[28] Albota, M. A., Xu, C. and Webb, W. W. Two-Photon fluorescence excitation cross sections
of biomolecular probes from 690 to 960 nm. Appl. Opt. 37 (1998), 7352–7356.
[29] Helmchen, F., Denk, W. Deep tissue two-photon microscopy. Nature Mehods 2 (2005),
932–940.; Theer, P., Hasan, M. T. and Denk, W. Two-photon imaging to a depth of 1000
microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Optics Letters 28
(2003), 1022–1024.
[30] Jung, J. C., Mehta, A. D., Aksay, E., Stepnoski, R. and Schnitzer, M. J. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. Journal of
Neurophysiology 92 (2004), 3121–3133.
[31] Klunk, W. E., Bacskai, B. J., Mathis, C. A., Kajdasz, S. T., McLellan, M. E., Frosch,
M. P., Debnath, M. L., Holt, D. P., Wang, Y. and Hyman, B. T. Imaging Abeta plaques
in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically
administered Congo red derivative. J. Neuropathol. Exp. Neurol. 61 (2002), 797–805.
[32] Sigurdson, C. J, Nilsson, K. P. R., Hornemann, S., Manco, G., Polymenidou, M., Schwarz,
P., Leclerc, M., Hammarstrom, P-, Wüthrich, K. and Aguzzi, A. Prion strain discrimination
using luminescent conjugated polymers. Nature Methods 12 (2007), 1023–1030.
“NLOQO” — “nloqo_is17” — 2010/3/19 — 11:53 — page 251 — #11
“NLOQO” — “nloqo_is17” — 2010/3/19 — 11:53 — page 252 — #12