Biomedical Sensing Application of Raman Spectroscopy. Yukihiro Ozaki Kwansei Gakuin University

Size: px

Start display at page:

Download "Biomedical Sensing Application of Raman Spectroscopy. Yukihiro Ozaki Kwansei Gakuin University"

Annabel Mitchell
5 years ago
Views:

1 Biomedical Sensing Application of Raman Spectroscopy Yukihiro Ozaki Kwansei Gakuin University

2 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.

3 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

4 Study of mouse embryo by Raman spectroscopy Mika Ishigaki, Kosuke Hashimoto, Naoya Ogawa, Kana Morimoto, Yukihiro Ozaki, Hidetoshi Sato Kwansei Gakuin University

Health topics: Infertility Infertility is a global public health problem.

(WHO) Japan: 10% couples are suffering from infertility.

Ovum <Flowchart of IVF treatment> Sperm World Health Organization www.who.

5 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. Ovum <Flowchart of IVF treatment> Sperm World Health Organization Assessment of embryonic quality blastocyst ovum collection fertilization incubation implantation The pregnancy rate of IVF remain about 30%. Implantation and pregnancy rates after IVF have a relationship with visual inspection.

Assessment of embryonic quality Grading method Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 BL: FR: PR: uniform 0% 30-40% uniform <10% 20-30% non-uniform <10% 10-20% non-uniform 10-50% <10% (BL:

6 Assessment of embryonic quality Grading method Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 BL: FR: PR: uniform 0% 30-40% uniform <10% 20-30% non-uniform <10% 10-20% non-uniform 10-50% <10% (BL: blastomere, FR: fragment, PR: pregnancy rate) non-uniform >50% 0% Hum Report 1998; 13: 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.

7 Evaluation of the metabolism of the embryo Analysis of the culture medium the variation of pyruvic acid and glucose in the culture 1. The relationship between the amount of Pyruvic acid intake and survival potential of human embryo. Conaghan J. et al., J Assist Reprod Genet 1993; 10: The embryo with high grade intakes much glucose. Gardner DK. et al., Fertil Steril 2001; 76: The metabolism evaluation through the culture medium by NIR and Raman spectroscopy. Seli, Emre, et al. Fertility and Sterility 88.5 (2007): 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.

8 Mouse Embryo unfertilized Early mouse embryo pronuclear 2-cell 4-cell 8-cell 0.5 day 1.5 day 2.5 day Embryonic development Jcl:ICR

+ Lens heater CO 2 gas Developed by H.Sato Ti:S Laser (Ex.

9 Microscope Raman system Spectrometer (Nanofinder30; Tokyo instruments Inc.) CO₂ incubator Polychromator + CCD detector (-80 ) Long-pass filter Objective lens + Lens heater CO 2 gas Developed by H.Sato Ti:S Laser (Ex. WL:785nm) Beam expander Water bath (37 ) Stage heater Electrical XY moving stage Excitation laser Raman scattering light

10 Normalized Intensity Protein Lipid DNA/RNA unfertilized (n=15) Averaged Raman spectra Phe. sym. ring breath 1003 Tyr PO 4 3 sym. Str. PO 2 str. amide III =C-H bend CH 2 def. CH 2 def. amide I C=C str pronuclear (n=9) 2-celled (n=26) 4-celled (n=21) 8-celled (n=34) Raman shift [cm -1 ] Applied Spectroscopy Reviews, 42: , 2007

11 Protein Lipid DNA/RNA Subtraction spectra Secondary structure of protein Protein DNA/RNA Lipid Pro- UF cell- UF 4 cell- UF cell- UF Raman shift [cm -1 ] DNA concentration increases after the fertilization.

12 Secondary structure of protein Secondary structure of protein Second derivative spectra UF Pro 2cell 4cell 8cell α-helix β-sheet UF Pro 2cel 4cel 8cel UF Pro 2cell 4cell 8cell ,001 0, α β ( cm -1 ) 0 UF 1 Pro 2 2-cell 3 4-cell 4 8-cell 5 6 α-helix dominant 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.

Relative intensity Tyrosine doublet (830 cm -1, 850 cm -1 ) UF Pro 2cell 4cell 8cell 2,5 2 1,5 Intensity ratio : R Tyr =I 850 / I 830 UF 4-cell 8-cell 2-cell Pro 1 0,5 0 0 UF 1 Pro 2 2-cell 3 4-cell

13 Relative intensity Tyrosine doublet (830 cm -1, 850 cm -1 ) UF Pro 2cell 4cell 8cell 2,5 2 1,5 Intensity ratio : R Tyr =I 850 / I 830 UF 4-cell 8-cell 2-cell Pro 1 0,5 0 0 UF 1 Pro 2 2-cell 3 4-cell 4 8-cell 5 6 R tyr : sensitive to UF R Tyr = 2.1 Pro R Tyr = cell R Tyr = cell R Tyr = cell R Tyr = 2.2 OH hydrogen bound OH ionic state strong hydrogen bound acceptor ionic state strong hydrogen bound acceptor Hydrogen bound behaves as drive force to form secondary structure of protein. Tyrosine The number variation of the ratio may relate with the change of the secondary stricture of proteins.

PC2(21.1%) PC2(2.6%) PC2(5.7%) Score plot -10 PCA: Each developmental stages Unfertilized Pronuclear 8-cell 15 10 5 0-10 0 10 20 30-5 PC1(69.

6%) Loading plot: PC1 lipid 1443 Unfertilized Pronuclear 8-cell Unfertilized 958 837 1304 1086 1272 1660 1741 good no good Pronuclear 8-cell 750 1000 1200

14 PC2(21.1%) PC2(2.6%) PC2(5.7%) Score plot -10 PCA: Each developmental stages Unfertilized Pronuclear 8-cell PC1(69.4%) PC1(97.0%) 2 1,5 1 0, , ,5-2 PC1(85.6%) Loading plot: PC1 lipid 1443 Unfertilized Pronuclear 8-cell Unfertilized good no good Pronuclear 8-cell Raman shift [cm -1 ] 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.

Principal Component Analysis (PCA): all stages 837 872 952 1076 1121 1366 Score plot Loading plot of PC1 1266 1301 1438 1653 1741 lipid Morphological feature good no good PCA classifies the dataset

15 Principal Component Analysis (PCA): all stages Score plot Loading plot of PC lipid Morphological feature good no good 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. BMC cell biology 11.1 (2010): 38 Hyperlipidaemic condition reduce embryonic quality. Human reproduction 25.3 (2010):

PC2(27.2%) 0-4 -2 0 2 4 6 2 1,5 1 0,5 0-0,5-1 -1,5-2 UF Principal Component Analysis (PCA) Score plot 3 2 1-1 -2-3 PC1(52.

16 PC2(27.2%) ,5 1 0,5 0-0,5-1 -1,5-2 UF Principal Component Analysis (PCA) Score plot PC1(52.4%) fertilized Average of PC1 score Pro 2-cell 4-cell 8-cell UF Pro 2-cell 4-cell 8-cell unfertilized PC1 PC α-helix C-C str β-sheet C-C str Raman shift [cm -1 ] 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. lipid

17 Summary 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

18 A promising tool for non-invasive, multiplexed measurement of blood constituents KGU-MIT Collaboration

20 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.

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

22 A Schematic Illustration of the Raman Spectroscopic Measurement Process for in vivo Continuous Glucose Monitoring

23 Representative Raman Spectrum Acquired from a Human Subject Undergoing OGTT

24 Plot of Prospective Prediction and Reference Glucose Concentration for a Representative Human Subject

25 Blood Glucose Predictions of the iconic Model for the Complete Human Subject Dataset

26 Acknowledgement Prof. Aritake Mizuno (JikeiUniversity) Hidetoshi Sato (Kwansei Gakuin Univ.) Mika Ishigaki (KGU) Kanet Wongravee (Chulalongkorn Univ. 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)

27 Thank You Very Much!!

Non-invasive methods of embryo selection

Non-invasive methods of embryo selection Liow Swee Lian O & G Partners Fertility Centre Gleneagles Hospital SINGAPORE Introduction More physiological laboratory procedures and culture systems have significantly