Nuclear analysis using
Raman Spectroscopy
Literature seminar #3
2021.9.3
M2 Tamiko Nozaki

Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.

1

ContentsØ 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

Ø Mini-proposal
Ø Summary2

ContentsØ 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

Ø Mini-proposal
Ø Summary3

Raman spectroscopy

Raman sca,eringü 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 ofmolecules

Bakthavatsalam, S.;…Sodeoka, M. RSC Chem. Biol. 2021.

h@ps://sisu.ut.ee/heritage-analysis/book/32-raman-spectroscopy 4, modiﬁed

Raman spectroscopy

Raman spectra of biological molecules<Nucleic acid><Protein><Lipid>Shipp, D. W.; … NoOngher, I. Adv. Opt. Photonics 2017, 9, 315.

5

Raman spectroscopy

Raman imaging of living cells
Raman spectra obtained from the cytosol of a living cell

753 cm−1 : The pyrrole breathing mode
= cytochrome c1686 cm−1 : Amide-I vibration= protein β sheet
2852 cm−1 : C-C stretching vibration modes= lipid molecules

Hamada, K;… Kawata, S.. J. Biomed. Opt. 2008, 13, 044027.

Shipp, D. W.; … No:ngher, I. Adv. Opt. Photonics 2017, 9, 315.

6

Raman spectroscopy

Fluorescence microscopy vs Raman microscopy<Fluorescence microscopy >ü Images with very high spa0al resolu0on.
ü The high temporal resolu0on.
p Labeling of the target biomolecules with ﬂuorescent small
molecules / gene0cally encoded ﬂuorescent proteins.
Large enough to aﬀect the biochemical/biophysicalproper0es of the target biomolecules.
They aﬀect the na0ve cellular environment,<Raman microscopy>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 withminimal labelling.
ü The obtained informa0on has molecular level detail.
Kann, B.;… Otto, C. Adv. Drug Deliv. Rev. 2015, 89, 71.,
Dunst, S.; Tomancak, P. GeneEcs 2019, 211, 15.

h@ps://www.chem-staOon.com/
molecule/2009/01/green_ﬂuorescent_protein_gfp.html

7Surface-enhanced Raman spectroscopy (SERS)

Surface-enhanced Raman spectroscopy (SERS)
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.

Surface-enhanced Raman spectroscopy (SERS)The Raman signal of the target molecule is enhanced when it is close to the surfaceof metals such as silver or gold.

Ando, J.; Fujita, A, K. Rev. Laser Eng. 2010, 38 (6), 427.

8

Surface-enhanced Raman spectroscopy (SERS)

Surface-enhanced Raman spectroscopy (SERS)3 & ' − &!
!=+4% & ' + 2&! 0
P : polariza?on, ! " ∶ dielectric func?on!! : dielectric constant, E0 : external electric ﬁeld

Re[& ' ] = −2&!
: Localized Surface Plasmon Resonanceü 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.

Ando, J.; Fujita, A, K. Rev. Laser Eng. 2010, 38 (6), 427.

9

ContentsØ 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

Ø Mini-proposal
Ø Summary
10

SERS for nuclear proﬁling

SERS for nuclear analysis

Nuclear localization signal peptides (NLS)
SV40 large T an?gen : GGVKRKKKPGGC
HIV-1 Tat protein NLS : GCGYGRKKRRQRRRG

ü ConjugaOon with NLS allows the targeOng of NPs to the cell nucleus.
Wubben, J. M.;… Borg, N. A. Cells 2020, 9 (12), 1.

Chen, Y.; … Wu, J. Int. J. Nanomedicine 2020, 15, 9407..

11

Label-free method

SERS for label free Nuclear analysis
n Cell nucleus imaging during the cell cyclen IdenOfying cell types
UndiﬀerenOated cell

RNA peaks; looser packaging

n Intracellular monitoring of pHKang, B.; …El-Sayed, M. A. Nano LeK. 2012, 12, 5369.
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.

DiﬀerenOated cell

DNA/histone peaks; DNA packaged with chromosomal
proteins in heterochromaOnn MulO-targeOng SERS imaging

Huefner, A.;… Mahajan, S. Nano LeK. 2013, 13, 2463.
Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 112

SERS for nuclear proﬁling

AuNR-MPy-targeCng (AMT) pH nano-sensors<AuNR-MPy-targeOng (AMT) pH nano-sensors>
pH-sensiOve molecule

Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.

The organelle-targeting peptides(nucleus targeting peptide (NLS))

13SERS for nuclear proﬁling

AuNR-MPy-targeCng (AMT) pH nano-sensors<AuNR-MPy-targeting (AMT) pH nano-sensors>
The organelle-targeOng pepOdes

pH-sensiOve molecule(nucleus targe0ng pep0de (NLS))ü AM-pH nano-sensor has agood response for pH
ü Most of ANT can locate in thenucleus or its adjacent area.

Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.

14

SERS for nuclear proﬁling

Intracellular monitoring of pH ﬂuctuaCons in organelles of cancer and normal cells

8.2 7.9

7.8 7.5
6.5 4.7

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 tothat in the normal cells.
Shen, Y.;…; Xu, W., Nanoscale 2018, 10, 1622–1630.

15

Contents
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

Ø Mini-proposal
Ø Summary16

SERS for nuclear imaging

SERS imaging of the Cme-dependent changes of cell nuclei during apoptosis

Rane
Early apoptosis stage: diminishing nuclei and slightcondensaOon of chromaOn
Late apoptosis stage :: chromaOn decomposiOon and nuclear
membrane rupture

Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1

The high-resoluOon SERS imaging of HeLa was constructedfrom the total SERS intensity between 490 and 1630 cm-1.

17

SERS for nuclear imaging

Multi-targeting SERS imaging of cellThree diﬀerent Raman-acOve
molecules was inserted into thegap of the Au@Au core-shellstructure.
<Raman-dyes as SERS reporters>
OOH

MBA-coated AuNPs

HSN

CVa-coated AuNPs

H 2NONH

N

CV-coated AuNPs
label-free AuNPs (black)N

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.

N18

SERS for nuclear imaging

SERS imaging of a cell treated with Raman-dye coated AuNPSERS

DAPI

1078

1175595

ü The membrane- or nuclear-targe1ng AuNPs were found inside the nucleus andsurrounding the cells, respec1vely.
Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 1

19

SERS for nuclear imaging

MulC-targeCng SERS imaging using coated AuNPs in the same single cell

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.

Chen, Y.; …; Hu, J.. Sci. Rep. 2016, 6, 120

ContentsØ 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

Ø Mini-proposal
Ø Summary21

Labeling method

Alkyne-tag Raman imaging (ATRI) for nuclear imaging

Silent region

Oü Alkynes have strong Raman signals in the cellular silent region and canbe excellent tags.

MeOPhMeOO

AltQ2

ü Multi-color imaging with ATRI was achieved due to the narrow line
width of alkyne peak.
Yamakoshi, H.; …S.; Sodeoka, M.. J. Am. Chem. Soc. 2012, 134, 20681.

Palonpon, A. F.; S.; Fujita, K. Nat. Protoc. 2013, 8, 677.

22

Histone imaging using ATRI

Histone imaging with Raman tag incorporated via geneCc code expansion

Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685.

Bakthavatsalam, S.; ….; Sodeoka, M. RSC Chem. Biol. 2021

23

Histone imaging using ATRI

IncorporaCon of a Raman tag via geneCc code expansion

 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.
Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685.

24

Histone imaging using ATRI

IncorporaCon of a Raman tag via geneCc code expansion
in ﬁxed cell× W !"#$%&'()*+,-./

ü In the nucleus a strong Raman signal was observed @2225 cm-1.
Zhang, J.;… Wang, P. J. Phys. Chem. LeK. 2018, 9, 4679–4685.

25

ContentsØ 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

Ø Mini-proposal
Ø Summary26

SERS detecAon of nucleus with ATRI-tag

EdU as a nucleus localization interior label for SERS detection
EdU (ATRI-tag) was adopted as an internal label to locate the nuclear region accurately.

Silent region
Alkyne group

EDU (+)EDU (-)

Zhang, J.; …Xu, W. Anal. Bioanal. Chem. 2018, 410, 585.

27

SERS detec)on of nucleus with ATRI-tag

EdU as a nucleus localiza/on interior label for SERS detec/on
Alkyne group

SERS spectra of presen?ng SERS signals at 1920
and 2265 cm−1 are from the nuclear region.
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.
Zhang, J.; …Xu, W. Anal. Bioanal. Chem. 2018, 410, 585.

28

ContentsØ 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

Ø Mini-proposal
Ø Summary33

Summaryü Raman imaging can be performed without labelling or with minimal labelling andobtain informaOon in molecular detail level.
ü SERS enhance the Raman signal of the target molecule when it is close to the surfaceof metals such as silver or gold.
ü SERS can be applied for nuclear analysis and the dynamics tracking of physiologicalprocesses.
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.

34

Appendix
35

Surface-enhanced Raman spectroscopy (SERS)

SRES×W8TdJ5 Y< =`X "IiJK@Q[20ZF")Q[7C #o[q εmaEWo[1O ε(ω)%5;8TdrB UAω =[KM]/H`O.
g[KE0 ,!$#K@
8TdrB_g[ B t%Gj*i6 P o3$#^DdrBfn.g[ K4k>[K%bP# fn[- o3$#
ci6K(Ed)Ed = -4πLP

L=1/3

aE N9 
L : ci6:O, drB;L+U5;rBK@

o[VWi6[-  #[K?s#ep "'&(lR !
" = $ % & = & + 4)*
 *=! " #$%&

E : hEW[K, D : [Sm\, P : i6, ε(ω) : o[1O(1.1)

drB_g[KE.g[KE0ci6[KEd& = &! + &"

!

(1.2)
$(1.1), (1.2) "o[q εmaEW #i6* = %&*=

3 $ % − $(&!
4) $ % + 2$(! "#$!#! "$'#$!#&!

h@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

L=1/3

36