Biomedical Sensing Application of Raman Spectroscopy

Yukihiro Ozaki Kwansei Gakuin University

Ozaki Group: Molecular Spectroscopy Lab.

• Development of Instruments

ATR-FUV/DUV spectrometer, NIR imaging sytems, High

vacuum and low temperature tip-enhanced Raman

scattering system, SPR sensors in FUV and DUV, NIR

• Developments of Spectral Analysis Chemometrics, Two-dimensional correlation spectroscopy.

• Applications of Spectroscopy

Biomedical, Nanomaterials, Polymers.

Ozaki Group: Molecular Spectroscopy Lab.

• Far-ultraviolet (FUV) and deep-ultraviolet (DUV) spectroscopy

• Near-infrared (NIR) spectroscopy, NIR imaging

• Raman spectroscopy, Surface-enhanced Raman scattering and tip-enhanced Raman spectroscopy, Raman imaging

• Low-frequency Raman and terahertz spectroscopy

Mika Ishigaki, Kosuke Hashimoto, Naoya Ogawa, Kana Morimoto, Yukihiro Ozaki, Hidetoshi Sato

Kwansei Gakuin University

Study of mouse embryo by Raman spectroscopy

Health topics: Infertility

・ Infertility is a global public health problem.

Inability to become pregnant after 1 year of unprotected sexual intercourse. (WHO)

・ Japan: 10% couples are suffering from infertility.

・ In vitro fertilization (IVF) is performed to the couples.

Assessment of embryonic quality

World Health Organization www.who.int/topics/infertility/en/

・ The pregnancy rate of IVF remain about 30%. ・ Implantation and pregnancy rates after IVF have a relationship with visual inspection.

ovum collection

Ovum Sperm

fertilization incubation implantation

＜Flowchart of IVF treatment＞

blastocyst

Assessment of embryonic quality

Assessment of embryonic quality: Cleavage rates, morphological feature

• The grading method is actually used in clinical practice of IVF.

• Blastomere morphology is a predictive factor for embryonic quality.

• New evaluation techniques are needed in order to improve the success rate of IVF treatment.

Grade 1 Grade 2 Grade 3 Grade 4 Grade 5

uniform 0% 30-40%

uniform <10% 20-30%

non-uniform <10% 10-20%

non-uniform 10-50% <10%

non-uniform >50% ≈0%

BL:

FR: PR:

【 Grading method 】

（BL: blastomere, FR: fragment, PR: pregnancy rate）

Hum Report 1998; 13: 1003-1013

Evaluation of the metabolism of the embryo

Analysis of the culture medium the variation of pyruvic acid and glucose in the culture

• We want to assess the embryonic quality more directly, non-destructively, non-invasive

• Raman spectroscopy has the potential to assess the embryonic quality based on the molecular composition.

1. The relationship between the amount of Pyruvic acid intake and survival potential of human embryo.

2. The embryo with high grade intakes much glucose.

3. The metabolism evaluation through the culture medium by NIR and Raman spectroscopy.

Conaghan J. et al., J Assist Reprod Genet 1993; 10:21-30

Gardner DK. et al., Fertil Steril 2001; 76: 1175-80

Seli, Emre, et al. Fertility and Sterility 88.5 (2007): 1350-1357

Jcl:ICR

Mouse Embryo

2-cell 8-cell unfertilized pronuclear

0.5 day 1.5 day 2.5 day

4-cell

Embryonic development

【 Early mouse embryo 】

Objective lens +

Lens heater

CO2 gas

Electrical XY moving stage

Water bath (37℃)

Stage heater

Spectrometer (Nanofinder30; Tokyo instruments Inc.)

Polychromator +

CCD detector (-80℃)

Ti:S Laser (Ex. WL:785nm)

Beam expander

Long-pass filter

Excitation laser Raman scattering light

CO₂ incubator

Developed by H.Sato

Microscope Raman system

No

rmal

ize

d I

nte

nsi

ty

750 1800 1000 1200 1400 1600

Raman shift [cm-1]

unfertilized (n=15)

pronuclear (n=9)

2-celled (n=26)

8-celled (n=34)

4-celled (n=21)

Averaged Raman spectra

830 85

5 939

958

1034

10

48

1083

1128

1158

11

75

1211

1256

1343

1450

1274

1309

1544

1586

16

17

1659

Applied Spectroscopy Reviews, 42: 493-541, 2007

Phe. sym. ring breath 1003

amide III =C-H bend.

CH2def. CH2def.

amide I C=C str.

Tyr

Protein Lipid

DNA/RNA

PO43⁻ sym. Str.

PO2⁻str.

Subtraction spectra

750 1800 1000 1200 1400 1600

Raman shift [cm-1]

980 1048

1096

965

1463

Pro- UF

2 cell- UF

4 cell- UF

8 cell- UF

Secondary structure of protein Protein Lipid

DNA/RNA Protein Lipid DNA/RNA

DNA concentration increases after the fertilization.

UF Pro 2cell 4cell 8cell

UF Pro 2cell 4cell 8cell

α-helix β-sheet

938

965

Second derivative spectra

α-helix dominant

α ̸ β (938 ̸ 965 cm-1)

0

0,0005

0,001

0 1 2 3 4 5 6 UF Pro 2-cell 4-cell 8-cell

Secondary structure of protein

UF Pro 2cell 4cell 8cell

Secondary structure of protein

・ The ration (α ̸ β) increases as the embryonic development. ・ Secondary structure of protein becomes α-helix dominant from β-sheet. ・ Raman spectroscopy can monitor the secondary structural changes of protein with the embryonic development.

Tyrosine doublet (830 cm-1, 850 cm-1)

0

0,5

1

1,5

2

2,5

0 1 2 3 4 5 6 UF Pro 2-cell 4-cell 8-cell

UF

2-cell

4-cell 8-cell

Pro

UF Pro 2cell 4cell 8cell

Intensity ratio : RTyr=I850 / I830

Hydrogen bound behaves as drive force to form secondary structure of protein.

Rel

ati

ve in

ten

sity

Tyrosine ・ UF RTyr= 2.1 ・ Pro RTyr= 1.2 ・ 2-cell RTyr= 1.5 ・ 4-cell RTyr= 2.1 ・ 8-cell RTyr= 2.2

strong hydrogen bound acceptor

ionic state

strong hydrogen bound acceptor

Rtyr : sensitive to

OH hydrogen bound OH ionic state

The number variation of the ratio may relate with the change of the secondary stricture of proteins.

750 1800 1000 1200 1400 1600

Raman shift [cm-1]

Unfertilized

Pronuclear

8-cell

Unfertilized Pronuclear 8-cell

-10

-5

0

5

10

15

-10 0 10 20 30

PC1(69.4%)

PC

2(21

.1%

)

-12

-8

-4

0

4

8

12

-40 -20 0 20 40 60

PC1(97.0%)

PC

2(2.

6%)

-2

-1,5

-1

-0,5

0

0,5

1

1,5

2

-5 0 5 10 15

PC1(85.6%)

PC

2(5.

7%)

Score plot

Loading plot: PC1 lipid

good

no good

PCA: Each developmental stages

・ PCAs classify the dataset of each developmental stages into two groups by lipid component. ・ The concentration of lipid components of mouse embryo with not good morphological feature is high.

Unfertilized Pronuclear 8-cell

958

1086 1272

1304

1443

1660

1741 837

Score plot Loading plot of PC1

837

872

952

1076

1121

1266 1301

1366

1438

1653

1741

lipid

・ PCA classifies the dataset into two groups by lipids. ・ The concentration of lipid components of mouse embryo with not good morphological feature is high. ・ The differences relating to the morphological features are bigger than the ones between developmental stages. ・ Lipid are considered to be energy source. ・ Hyperlipidaemic condition reduce embryonic quality. Human reproduction 25.3 (2010): 768-778

BMC cell biology 11.1 (2010): 38

Principal Component Analysis (PCA): all stages

good

no good

【 Morphological feature 】

-3

-2

-1

0

1

2

3

-4 -2 0 2 4 6

UF

Pro

2-cell

4-cell

8-cell

PC1(52.4%)

PC

2(2

7.2

%)

Score plot

Principal Component Analysis (PCA)

・ PC1: lipid component changes in a cyclic way ・ PC-2 classify all stage data into two groups: unfertilized and fertilized

・ In the loading plot of PC-2:

939 cm-1: α-helix 976 cm-1: β-sheet

The secondary structure of protein is changed from β-sheet to α-helix after the fertilization.

fertilized

unfertilized

750 1800 1000 1200 1400 1600

Raman shift [cm-1]

PC1

PC2 836

894

1137

1302 1438

1463 1674

871

1087

1302

1444

1659

1750

lipid

939

β-sheet C-C str.

976

C-C str. α-helix

-2

-1,5

-1

-0,5

0

0,5

1

1,5

2

UF

Pro

2-cell 4-cell

8-cell

Average of PC1 score

Analysis of mouse embryo on early stages by Raman spectroscopy

（unfertilized・pronuclear・ 2-cell・ 4-cell・ 8-cell）

The variation of inner material with the embryonic development was detected.

Morphological features (good morphology or not) relates with the lipid concentration.

PCA result show the secondary structural changes of protein:

from β-sheet to α-helix

The structural changes of protein is also indicated by the change of tyrosine doublet ratio.

Raman spectroscopy has the potential for the in-situ monitoring of the embryonic development and to assess the embryonic quality.

Acknowledgement: MEXT KAKENHI Grant Number 25560212

Summary

A promising tool for non-invasive, multiplexed measurement of blood constituents

KGU-MIT Collaboration

We propose a novel analytical framework

• Vibrational spectroscopy has emerged as a promising tool for non-invasive, multiplexed measurement of blood constituents – an outstanding problem in biophotonics. Here, we propose a novel analytical framework that enables spectroscopy-based longitudinal tracking of chemical concentration without necessitating extensive a priori conocentration information. The principal idea is to employ a concentration space transformation acquired from the spectral information, where these estimates are used together with the concentration profiles generated from the system kinetic model.

Our approach exhibits a 35% reduction in error over partial least squares regression

• Using blood glucose monitoring by Raman spectroscopy as an illustrative example, we demonstrate the efficacy of the proposed approach as compared to conventional calibration methods. Specifically, our approach exhibits a 35% reduction in error over partial least squares regression when applied to a detaset acquired from human subjects undergoing glucose tolerance tests. This method offers a new route at screening gestational diabets and opens doors for continuous process monitoring without sample perturbation at intermediate time

A Schematic Illustration of the Raman Spectroscopic Measurement Process for in vivo

Continuous Glucose Monitoring

Representative Raman Spectrum Acquired from a Human Subject Undergoing OGTT

Plot of Prospective Prediction and Reference Glucose Concentration for a Representative

Human Subject

Blood Glucose Predictions of the iCONIC Model for the Complete Human Subject Dataset

Acknowledgement

Hidetoshi Sato (Kwansei Gakuin Univ.)

Mika Ishigaki (KGU)

Nicolas Spegazzini (KGU-MIT)

・ Dr. Yasuhiro Maeda (Riken Center, Japan) ・ Dr. Ryu Ishihara (Osaka Medical Center, Japan) ・ Dr. Bibin B. Andriana (KGU) ・ Akinori Taketani (KGU) ・ Kosuke Hashimoto (KGU) ・ Naoya Ogawa (KGU)

Prof. Aritake Mizuno (JikeiUniversity)

Kanet Wongravee (Chulalongkorn

Univ. KGU)

Thank You Very Much!!