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Nuclear analysis using Raman Spectroscopy
Literature seminar #32021.9.3
M2 Tamiko Nozaki
1Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.
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2
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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3
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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Raman sca,ering
4Bakthavatsalam, S.;…Sodeoka, M. RSC Chem. Biol. 2021. h@ps://sisu.ut.ee/heritage-analysis/book/32-raman-spectroscopy
Raman spectroscopy
, modified
ü Raman scattering is an inelastic process in which the molecule returns to a higher or lower vibrational state.
ü Raman scattering reveals the vibrational, rotational and other low frequency modes of molecules
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Raman spectra of biological molecules
5Shipp, D. W.; … NoOngher, I. Adv. Opt. Photonics 2017, 9, 315.
Raman spectroscopy
<Nucleic acid>
<Protein>
<Lipid>
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Raman imaging of living cells
6
Raman spectroscopy
Hamada, K;… Kawata, S.. J. Biomed. Opt. 2008, 13, 044027. Shipp, D. W.; … No:ngher, I. Adv. Opt. Photonics 2017, 9, 315.
Raman spectra obtained from the cytosol of a living cell
753 cm−1 : The pyrrole breathing mode= cytochrome c
1686 cm−1 : Amide-I vibration= protein β sheet
2852 cm−1 : C-C stretching vibration modes = lipid molecules
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Fluorescence microscopy vs Raman microscopy
7Kann, B.;… Otto, C. Adv. Drug Deliv. Rev. 2015, 89, 71.,
Raman spectroscopy
ü Images with very high spa0al resolu0on.ü The high temporal resolu0on.
<Raman microscopy>
<Fluorescence microscopy >
h@ps://www.chem-staOon.com/molecule/2009/01/green_fluorescent_protein_gfp.htmlDunst, S.; Tomancak, P. GeneEcs 2019, 211, 15.
p Labeling of the target biomolecules with fluorescent small molecules / gene0cally encoded fluorescent proteins.�Large enough to affect the biochemical/biophysical
proper0es of the target biomolecules.�They affect the na0ve cellular environment,
p Raman imaging has not yet achieved the spatiotemporal resolution of fluorescence imaging
= Raman signals are too weak
ü Raman imaging can be performed without labelling or with minimal labelling.
ü The obtained informa0on has molecular level detail.
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Surface-enhanced Raman spectroscopy (SERS)
8
Surface-enhanced Raman spectroscopy (SERS)
Ando, J.; Fujita, A, K. Rev. Laser Eng. 2010, 38 (6), 427.
Surface-enhanced Raman spectroscopy (SERS)
The Raman signal of the target molecule is enhanced when it is close to the surface of metals such as silver or gold.
p Since typical Raman scattering signals are compared to fluorescence yields.; The measurement of Raman spectra usually requires long exposure times, making
observations of living specimen difficult.
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Surface-enhanced Raman spectroscopy (SERS)
9
Surface-enhanced Raman spectroscopy (SERS)
! = 34%
& ' − &!& ' + 2&!
+0
Re[& ' ] = −2&!: Localized Surface Plasmon Resonance
P : polariza?on, ! " ∶ dielectric func?on!! : dielectric constant, E0 : external electric field
Ando, J.; Fujita, A, K. Rev. Laser Eng. 2010, 38 (6), 427.
ü Localized surface plasmon polaritons are generated due to oscillation of free electrons in the metal.
ü SERS can enhance the Raman signal on average by 104 to 108 times, and up to 1014-1015
times.
ü Optical detection and spectroscopy of single molecules have been achieved.
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10
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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SERS for nuclear analysis
11Wubben, J. M.;… Borg, N. A. Cells 2020, 9 (12), 1 Chen, Y.; … Wu, J. Int. J. Nanomedicine 2020, 15, 9407..
.
SERS for nuclear profiling
Nuclear localization signal peptides (NLS)
ü ConjugaOon with NLS allows the targeOng of NPs to the cell nucleus.
SV40 large T an?gen : GGVKRKKKPGGCHIV-1 Tat protein NLS : GCGYGRKKRRQRRRG
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SERS for label free Nuclear analysis
12
Label-free method
Huefner, A.;… Mahajan, S. Nano LeK. 2013, 13, 2463.
RNA peaks DNA/histone peaks; DNA packaged with chromosomal proteins in heterochromaOn
; looser packaging
UndifferenOated cell DifferenOated cell
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630. Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1
Kang, B.; …El-Sayed, M. A. Nano LeK. 2012, 12, 5369.
n Cell nucleus imaging during the cell cycle n IdenOfying cell types
n Intracellular monitoring of pH n MulO-targeOng SERS imaging
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AuNR-MPy-targeCng (AMT) pH nano-sensors
13
SERS for nuclear profiling
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.
The organelle-targeting peptides(nucleus targeting peptide (NLS))
<AuNR-MPy-targeOng (AMT) pH nano-sensors>
pH-sensiOve molecule
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AuNR-MPy-targeCng (AMT) pH nano-sensors
14
SERS for nuclear profiling
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.
The organelle-targeOng pepOdes(nucleus targe0ng pep0de (NLS))
<AuNR-MPy-targeting (AMT) pH nano-sensors>
ü AM-pH nano-sensor has a good response for pH
ü Most of ANT can locate in the nucleus or its adjacent area.
pH-sensiOve molecule
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Intracellular monitoring of pH fluctuaCons in organelles of cancer and normal cells
15
SERS for nuclear profiling
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.
ü The lysosome is acidic, and nucleus and mitochondria are neutral or slightly alkaline.ü The pH of the subcellular organelles in the cancer cells is more acidic as compared to
that in the normal cells.
7.9 7.5
4.7
8.2 7.8
6.5
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16
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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SERS imaging of the Cme-dependent changes of cell nuclei during apoptosis
17Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1
SERS for nuclear imaging
Rane
Early apoptosis stage: diminishing nuclei and slight condensaOon of chromaOn
Late apoptosis stage :: chromaOn decomposiOon and nuclearmembrane rupture The high-resoluOon SERS imaging of HeLa was constructed
from the total SERS intensity between 490 and 1630 cm-1.
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Multi-targeting SERS imaging of cell
18
MBA-coated AuNPs
CVa-coated AuNPs
CV-coated AuNPs
Chen, Y.; …Hu, J.. Sci. Rep. 2016, 6, 1. Liu, X.; …Wang, Z.. Nanoscale 2017, 9, 15390.Caires, A. J.;… Ladeira, L. O. J. Colloid Interface Sci. 2015, 455, 78.
SERS for nuclear imaging
HS
OH
O
N
NN
O
N
H2N NH
Three different Raman-acOve molecules was inserted into the gap of the Au@Au core-shell structure.
<Raman-dyes as SERS reporters>
label-free AuNPs (black)
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SERS imaging of a cell treated with Raman-dye coated AuNP
19
1078 1175 595
Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1
SERS for nuclear imaging
SERS DAPI
ü The membrane- or nuclear-targe1ng AuNPs were found inside the nucleus and surrounding the cells, respec1vely.
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MulC-targeCng SERS imaging using coated AuNPs in the same single cell
20Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1
SERS for nuclear imaging
ü The positions of the nucleus and membrane can be clearly distinguished.
ü The multi-targeting SERS imaging of various dye-AuNPs in the same cell.
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21
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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Alkyne-tag Raman imaging (ATRI) for nuclear imaging
22
Labeling method
Yamakoshi, H.; …S.; Sodeoka, M.. J. Am. Chem. Soc. 2012, 134, 20681. Palonpon, A. F.; S.; Fujita, K. Nat. Protoc. 2013, 8, 677.
AltQ2
ü Alkynes have strong Raman signals in the cellular silent region and can be excellent tags.
ü Multi-color imaging with ATRI was achieved due to the narrow line width of alkyne peak.
Silent region
O
OMeO
MeOPh
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Histone imaging with Raman tag incorporated via geneCc code expansion
23
Histone imaging using ATRI
Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685. Bakthavatsalam, S.; ….; Sodeoka, M. RSC Chem. Biol. 2021
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IncorporaCon of a Raman tag via geneCc code expansion
24
Histone imaging using ATRI
Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685.
� Expression of tRNA synthase
� The incorporaOon of UAA
ü A clear image of the Histone3.3 protein in the nucleus was observed @2135 cm−1.ü The vibraOonal signal of alkyne was very weak.
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IncorporaCon of a Raman tag via geneCc code expansion
25
Histone imaging using ATRI
Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685.
in fixed cell
ü In the nucleus a strong Raman signal was observed @2225 cm-1.
��W�� !"#$%&'()*+,-./
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26
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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EdU as a nucleus localization interior label for SERS detection
27
SERS detecAon of nucleus with ATRI-tag
Zhang, J.; …Xu, W. Anal. Bioanal. Chem. 2018, 410, 585.
Silent region
Alkyne group
EDU (+)
EDU (-)
EdU (ATRI-tag) was adopted as an internal label to locate the nuclear region accurately.
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EdU as a nucleus localiza/on interior label for SERS detec/on
28
SERS detec)on of nucleus with ATRI-tag
Zhang, J.; …Xu, W. Anal. Bioanal. Chem. 2018, 410, 585.
Alkyne group
ü EdU as a nucleus localization interior label can allow us to realize accurate location of nuclei.→ Obtaining precise SERS spectra of nucleus to investigation the biomolecules from the cell
nucleus by SERS.
SERS spectra of presen?ng SERS signals at 1920 and 2265 cm−1 are from the nuclear region.
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33
Ø Introduc)on
- Raman spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)
Ø Label-free method
-SERS for nuclear profiling
-SERS for nuclear imaging
Ø Labeling method
-Alkyne-tag Raman imaging (ATRI), Histone imaging using ATRI
-SERS detecAon of nucleus with ATRI-tag
ØMini-proposal
Ø Summary
Contents
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Summary
34
ü Raman imaging can be performed without labelling or with minimal labelling and obtain informaOon in molecular detail level.
ü SERS enhance the Raman signal of the target molecule when it is close to the surface of metals such as silver or gold.
ü SERS can be applied for nuclear analysis and the dynamics tracking of physiological processes.
ü ATRI realized the mulO-color imaging of biological molecules using alkyne funcOonalgroup.
ü CombinaOon of ATRI and SERS realized the detecOon of precise nuclear signal.
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Appendix
35
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SRES
36h@p://home.sato-gallery.com/research/JEMEA_databaseWG_metal_yudenritsu.pdf
h@ps://ir.library.osaka-u.ac.jp/repo/ouka/all/34490/26976_��.pdf
Surface-enhanced Raman spectroscopy (SERS)
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