Observations des données:

recherche des régions spectrales

corrélées

6- Classification de spectres

Etant donné un nombre de spectres, comment

les classer par “ressemblance”

6- Classification de spectreshierarchical clustering

Step 1: the Euclidian distance between each pair of spectra is calculated.

Figure: 5 spectra represented in a 2-D space (say we recorded only the absorbance at two wavenumbers)

6- Classification de spectres

Step 2: grouping starts by linking the closest spectra.

Figure: grouping of spectra (#1 to 5) and clusters (beyond #5)

hierarchical clustering

6- Classification de spectres

Step 3: dendrogram representation.

Statistical significance of the distances

hierarchical clustering

0100200300400500600700

1517136812739111219263114241620274105304422253336341835402123293237414228383947434549504648

Dendrogram, distance: euclidean, linkage: ward, range: 1711-1485

hierarchical clustering

50 protéines

6- Classification de spectres

d1 r2d2 brasiliensis peruviana lainsoni0

1

2

3

4

5

6

7

8

donovani

hierarchical clustering

6- Classification de spectresK-means clustering

kmeans treats each spectrum as an object having a location in space. It finds a partition in which spectra within each cluster are as close to each other as possible, and as far from spectra in other clusters as possible.

kmeans uses an iterative algorithm that minimizes the sum of distances from each object to its cluster centroid, over all clusters. This algorithm moves objects between clusters until the sum cannot be decreased further. The result is a set of clusters that are as compact and well-separated as possible.

Identification de lignées cellulaires

Chemical differencesas(CH3)

10001200140016001800200022002400260028003000

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

cm-1

Absorb

ance

as(CH3) s(CH3)

as(CH2)

(C=O)ester (CH3)

(CH2)

as(PO2-)

(C-O)

s(PO2-)

as(N-(CH3)3+)

Amide I (C=O)amide

Amide II(N-H)amide

(C-OH)

Phospholipid (DMPC)

Glycoprotéine

Mucine

RNA

DNA

Correlated with growth, not with species

INFRARED MEASUREMENTS

X3

1000150020002500300035000

50

100

150

200

250

cm-1

Ab

sorb

anc

e

Infrared spectrum of a cell

• The conformation of the molecules, especially proteins

IR spectrum = fingerprint of:

• The chemical nature of the components (glycosylations, DNA, RNA, proteins, lipids,….)

Fingerprinting and cell classification

Dendrogram of a hierarchical cluster analysis performed on 240 spectra of different strains of Gram-positive andGram-negative bacteria, and of yeasts belonging to the genus Candida (a). Dendrogram obtained when cluster analysis is performed on the yeast spectra only (b).

FTIR of bacteria

FTIR of bacteria

Spectral typing of closely related microorganisms. (a) Clinical isolates of E. coli (numbers in right column) belonging to different serogroups: O 25, O 18, and O 114 according to their O-antigenic structure.

FTIR of bacteria

Application à des cellules eucaryotes

1. Identification de Leismania sp.

2. Cellules leucémiques K562 wt ou résistantes

3. Classification de cellules gliales

4. Mode d’action de molécules anticancéreuses

5. Etudes microscopiques de tissus

L. lainsoni versus L. brasiliensis

(87 spectra)

1000150020002500300035004000

0

100

200

300

400

500

600

700

800

cm-1

Abs

orba

nce

• recording: 2 cm-1, 256 scans

• noise evaluation

• Water vapor subtraction (when necessary), apodization at 4 cm-1 final resolution

• Baseline subtraction (typically 3620 3010 2700 2395 2247 1775 1718

1483 1434 1353 1196 948 845)

• Scaling for a same area under 1718-1483 cm-1

Spectra recording and processing

140014501500155016001650170017501800

0

200

400

600

800

1000

1200

1400

1600

cm-1

Abs

orb

anc

e

0 20 40 60 80 100 120 1400

0.05

0.1

0.15

0.2

0.25

150015201540156015801600162016401660168017000

0.1

0.2

0.3

0.4

0.5

0.6

0.7

cm-1

Ab

sorb

an

ce

1000150020002500300035004000

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0.18

cm-1

Ab

sorb

an

ce

100015002000250030000

0.02

0.04

0.06

0.08

0.1

0.12

cm-1

Abs

orba

nce

Identification de cellules eucaryotes

10001200140016001800200022002400260028003000

0

100

200

300

400

500

cm-1

Abs

orb

anc

e

Leishmania lainsoni

Leishmania

brasiliensis

Différences significatives (Student test)

100012001400160018002000220024002600280030000

50

100

150

200

250

300

cm-1

Abs

orb

anc

e

Différence des moyennes

Moyenne pour L. lainsoni

Moyenne pour L. brasiliensis

* Student positive, alpha=0.01

Classification supervisée / non supervisée

0 100 200 300-50

-45

-40

-35

-30

-25

-20

-15

1

2

3

4

5

67

8

9

10

11

12

13

14

15

16

17

18

19

20 21

22

23

24

25

26

2728

2930

31

32

3334

3536

37

38

39

40

41

42

43

44

45

CP 1

CP

2

Score plot, selected wavenumbers: 3025 2996; 2945 2835; 1760 1725; , unknown in magenta

-20 0 20 40-28

-26

-24

-22

-20

-18

-16

-14

-12

-10

12

34 5

6

7

8

9

10

11

12

13

1415

16

17

18

19

2021

22

23

24

25

26

27

28

2930

31

32

33

3435

36

37

3839

40

41

42

43

44

45

CP 3

CP

4

-5 0 5-3.5

-3

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

1.5

1

2

3

4

5

67

8

9

10

11

12 13

1415

16

17

1819

20

21

22

23

24

25 26 2728

29

30

31

32

3334

3536

37

38

39

40

41

42

43

44

45

CP 5

CP

6

0 10 20 30 40 500.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

1

2

3

4

5

6

7

8

9

10

1112131415

16

17

181920

2122

23

24

25

26

2728

29

30

31

3233

34

35

36

3738

39

40

41

4243

4445

Spectrum number

Gro

up

Lin reg of scores on groups, selected wavnb: 3025 2996; 2945 2835; 1760 1725;

Analyse non supervisée: decomposition en composants principaux

(cross validation)

Analyse supervisée: régression linéaire

(cross validation)

Distance between spectraModel built after variable selection and principal component analysis

10 14 23 15 29 39 1 9 5 3 2 22 25 28 35 32 34 44 4 8 38 6 13 19 45 46 7 49 16 55 50 31 18 36 26 20 48 27 11 30 37 47 24 40 56 12 42 41 17 59 60 43 21 33 84 79 85 51 53 71 77 57 67 58 83 69 75 65 72 86 54 64 66 80 81 82 63 74 70 68 78 87 52 61 62 73 760

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Leishmania lainsoniLeishmani

a brasiliensis

gr1 gr3 gr2 gr4 gr5 gr6 gr9 gr7 gr8

0.5

1

1.5

2

2.5

3

3.5

4

L. lainsoni versus L. brasiliensis

MANOVA using 1753, 1724, 3008 and 1430 cm-1

L. brasiliensis

L. lainsoni

Classification de quatre espèces

Leishmania peruviana

Leishmania lainsoni

Leishmania donovani

Leishmania brasiliensis

1400145015001550160016501700175018000

100

200

300

400

500

cm-1

Abs

orba

nce

Distance analysis between species

d1 r2d2 brasiliensis peruviana lainsoni0

1

2

3

4

5

6

7

8

donovani

Chemical differences

10001200140016001800200022002400260028003000

-4

-3

-2

-1

0

1

2

3

cm-1

Abs

orba

nce

* Student positive, alpha=0.01

Mean L. brasiliensis – Mean L. lainsoni

as(CH3)

10001200140016001800200022002400260028003000

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

cm-1

Absorb

ance

as(CH3) s(CH3)

as(CH2)

(C=O)ester (CH3)

(CH2)

as(PO2-)

(C-O)

s(PO2-)

as(N-(CH3)3+)

Amide I (C=O)amide

Amide II(N-H)amide

(C-OH)

Phospholipid (DMPC)

Glycoprotéine

Mucine

RNA

DNA

Correlated with growth, not with species

100012001400160018002000220024002600280030000

50

100

150

200

250

300

cm-1

Absorb

ance

Difference of the means

Strain 1, mean

Strain 2, mean

Effect of culture growth. Comparing two strains.

02

46

810

12

9001000

11001200

13001400

1500

-50

0

50

100

150

200

Culture day

0 2 4 6 8 10-150

-100

-50

0

50

1240

1085

CONCLUSIONS

1. Certaines régions spectrales décrivent la croissance de la culture indépendamment de l’espèce

2. D’autres régions décrivent l’espèce indépendamment de l’état de la culture

3. La spectroscopie FTIR peut devenir un outil rapide et économique pour la détermination de Leishmania sp.

90011001300150017001900210023002500270029003100-1

1

3

5

7

9

frequency, cm-1

inte

nsity

A.B.C.D.

trace A: Representative infrared spectrum of resistant K562 cells. trace B: Representative infrared spectrum of sensitive K562 cells. trace C: Difference infrared spectrum between resistant and sensitive K562 cells, this spectrum is magnified 4 times.trace D: Result of the Student test performed at alpha level = 5% , the wavelengths in blue are significantly different between the two cell lines.

Example of information retrieved from the data: the intensity ratio between 2958cm -1 (CH3 stretching) and 2923cm-1 (CH2 stretching) is increased by 20% in resistant cells, suggesting a qualitative modification of the lipids in the cell membranes.

2) Resistant / sensitive K562

-2 -1.5 -1 -0.5 0 0.5 1 1.5 2

x 10-3

-10

-8

-6

-4

-2

0

2

4

6

8x 10

PC3 = 0.041%

PC

6 =

0.0

103

%

2D plot of K562 sensitive (22 spectra, blue points) and resistant (26 spectra, red stars) cells spectra reduced by PCA.

Unsupervised classification

Belot et al., Glia 2001, 36, 375-390

3) Identification de phénotypes: le cas des gliomes

In vitro parameters :

Motility : Maximum Relative Distance from the Origin

Motility : Average Speed

Growth : Anchorage-dependent growth

Growth : Anchorage-independent growth (in semi-solid agar)

Invasion : percentage of cells invading a collagen matrix

In vivo parameter :

Aggressiveness : Median Survival Time

2852 cm-1

2983 cm-1

2945 cm-1

Identification de phénotypes: le cas des gliomes

Sample preparation

FTIR de cellulesIdentification de phénotypes: le cas des gliomes

FREQUENCY (CM-1)

100011501300145016001750285029253000

INF

RA

RE

D A

BS

OR

BA

NC

E (

A.U

.) 16 cell lines used

Identification de phénotypes: le cas des gliomes

Calculated values

Actu

al v

alue

s

A172

H4

HS683

SW1088

SW1783

T98U118

U87

U373

5

6

7

8

9

10

11

12

5 6 7 8 9 10 11 12

Calculated values

Actu

al v

alue

sA172

H4

HS683

SW1088

T98U118

U87

U373

-2

2

6

10

14

18

22

-2 2 6 10 14 18 22 26

Multiple regression explaining the average speed

R = 0.96 (P = 0.003).

Multiple regression explaining the median survival periods of the nude mice grafted with glioma cells.

R = 0.97 (P = 5 10-3).

Average speed Median survival time

Identification de phénotypes: le cas des gliomes

Mode d’action de molécules

anticancéreuses

Studies on cells

- PC-3 prostate cancer cells in culture

- Washed in 0.9% NaCl

- Deposited on a BaF2 window

Daunorubicine

Doxorubicine

Irinotecan

Mercaptopurine

Méthotrexate

Paclitaxel

Vinblastine

Vincristine

Non traitées100011001200130014001500160017001800

0

200

400

600

800

1000

1200

cm-1

Abs

orba

nce

406080100120140160180

taxol.ir

vcri.ir

vbla.ir

dauno.ir

doxo.ir

merco.ir

metox.ir

Dendrogram, distance: euclidean, linkage: ward, range: 1724-1300

Daunorubicin

Doxorubicin

Mercaptopurine

Methotrexate

Paclitaxel

Vinblastine

Vincristine

Distance

Hierarchichal classification of “ difference spectra”

FTIR of drug signature on cancer cells

FTIR of drug signature on cancer cells