study of early spectral changes in cells transformation using advanced physical and mathematical...
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Study of early spectral changesStudy of early spectral changesin cells transformationin cells transformation
using advanced physical and mathematical using advanced physical and mathematical models.models.
By E. Bogomolny By E. Bogomolny
Supervisors:Supervisors: Prof. S.MordechaiProf. S.Mordechai Department of Physics (BGU)Department of Physics (BGU) Dr. M.Hulihel Institute of Applied Research (BGU)Institute of Applied Research (BGU)
IR radiation IR radiation UV radiationUV radiation FTIR MicrospectroscopyFTIR Microspectroscopy Light inducedLight induced FluorescenceFluorescence. .
1) Introduction.1) Introduction.
3) Results and Discussion.3) Results and Discussion.
2) Methodology.2) Methodology.
4) Conclusions.4) Conclusions.
Experimental descriptions. Spectra analysisExperimental descriptions. Spectra analysis.. Mathematical methods.Mathematical methods.
The Electromagnetic SpectrumThe Electromagnetic Spectrum
Gamma rayGamma ray SPECT, PET Imaging Nuclear transition
X-rayX-ray Mammography ,CAT. Inner shell
UVUV Potential electromagnetic range for diagnosis Valence electrons
VisibleVisible Pathology Valence electrons
InfraredInfrared Potential electromagnetic range for diagnosis Vibrational rotational level
MicrowaveMicrowave EPR imaging Rotational levels or fine structure
RadioRadio NMR imaging Hyperfine structure
IR radiationIR radiation
• Born-Oppenheimer ApproximationsBorn-Oppenheimer Approximations ::• 1) The electronic motion and the nuclear motion in molecules 1) The electronic motion and the nuclear motion in molecules
can be separatedcan be separated • 2) nuclear motion does not induce electronic transitions.2) nuclear motion does not induce electronic transitions.
ˆ ˆ ˆ ˆ ˆ ˆel el nuc el el nuc nuc nucH T V V T V
Multi-atomMulti-atom moleculemolecule
ˆn̂ucH T U R
el nuc nucU R E R V
2
1
ˆ2
elNj
elj e
pT
m
2
1
ˆ2
elNj
nucA A
pT
M
2
1
'ˆnuc nucN N
A Bnuc nuc
A B A A B
Z Z eV
R R
2
1
'ˆel elN N
el eli j i i j
eV
r r
2
,
'ˆ Ael nuc
j A j A
Z eV
r R
IR radiationIR radiation ˆ , , , ,nuc nuc nuc nucH R R E R
2
22 2 2 2 2 2 2
, , , , , ,1 1 1 2sin , , 0
sin sinnuc nuc nuc
nuc
R R Rr E U R R
r r r r r
2
22 2 2
( )1 2 ( 1)( ) 0
2nuc
nuc
d Rd J Jr E U R R
r dr dr r
Rewriting the Hamiltonian in spherical coordinates for diatomic systemRewriting the Hamiltonian in spherical coordinates for diatomic system
Equation similar to Hydrogen atomEquation similar to Hydrogen atom
Solution depends explicitly on U(R) . By Solution depends explicitly on U(R) . By expanding U(R) in Taylor series we obtain:expanding U(R) in Taylor series we obtain:
Equation can be separated to angular and radial partEquation can be separated to angular and radial part
2
,
1 1( ) ( )
2 2J eq eq
J JE U r h n
' * ''n n nR dx
The selection rules for vibrational and rotational The selection rules for vibrational and rotational transitions: transitions: ∆n = ±1 and ∆J= ± 1∆n = ±1 and ∆J= ± 1
The transition momentThe transition moment The dipole momentThe dipole moment
Only vibrational modes that have a transition dipole moment (or component of the transition dipole moment) can be observed in infrared spectroscopy
Different Types of Vibrational modesDifferent Types of Vibrational modes
Symmetric Stretch Asymmetric Stretch
Non linear molecules 3N-6Non linear molecules 3N-6
Rocking Twisting Scissoring Wagging
(Stretching)(Stretching)
(Bending)(Bending)
Different Types of Vibrational modesDifferent Types of Vibrational modesLinear molecules 3N-5Linear molecules 3N-5
Symmetric Stretch Asymmetric StretchSymmetric Stretch Asymmetric Stretch
Bending (two types)Bending (two types)
Introduction FTIR-MSPIntroduction FTIR-MSP((Fourier Transform Infrared Micro-Spectroscopy)Fourier Transform Infrared Micro-Spectroscopy)
Introduction FTIR-MSPIntroduction FTIR-MSP
MichelsonMichelsonInterferometerInterferometer
IR Source (Globars)
Sample
Microscope
Lens
Detector MCT
Michelson InterferometerMichelson Interferometer
IR spectra of bio-moleculesIR spectra of bio-molecules
Cell parameters Values
Radius [µm] 100
Volume [µm3] >10000
Generation time [h] 20-24
proteins % [w/w] 60
RNA % [w/w] 3-4
DNA % [w/w] ~1
Lipids % [w/w] 15-20
Polysaccharides %[w/w]
6-84000 3600 3200 2800 2400 2000 1600 1200 800
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
carb
oh
ydra
tes
nu
cle
ic a
cid
s
proteins
2958 C
H 3 str
asym
m
Lipids vib modesWater vib modes
2364
(C
O 2)
2917
CH 2, C
H3 s
tr. a
sym
m
3219
OH
str
. ban
d o
f w
ater
2849
CH 2, C
H3 s
tr. s
ymm
1740
CO
str
. (li
pid
s)
1652
am
ide
I
1542
am
ide
II14
52 C
H 3 asy
mm
. ben
din
g
1080
PO 2 s
ymm
. str
.
A
bso
rpti
on (
A.U
.)
Wavenumber (cm-1)
S0
S1
T1
transition involving emission/absorption of photons
radiationless transition
fl
uo
resc
ence
flu
ore
scen
ce101
0-6-6 t
o 1
0 t
o 1
0-9-9 s
ec s
ec
--hνhν
in
tern
al
inte
rnal
co
nve
rsio
nco
nve
rsio
n
inte
rsys
tem
inte
rsys
tem
cr
oss
ing
cro
ssin
g
FluorescenceFluorescenceThe Jablonski diagramThe Jablonski diagram
Phos
phor
esce
nc
Phos
phor
esce
nc
ee1010
-4-4 to 1
0 se
c
to 1
0 se
c+h+h
Ab
sorp
tio
nA
bso
rpti
on
1010 -1
5-1
5 s
ec
se
c
Fluorescence properties Fluorescence properties
(Quantum yield )
Number of fluorescent (emitted ) photonsNumber of absorbed photons
Q
Compounds with multiple conjugated double bonds, Compounds with multiple conjugated double bonds, i.e. extended i.e. extended electronic systems, has high quantum electronic systems, has high quantum yield and therefore detectable fluorescenceyield and therefore detectable fluorescence
1) Scattering of light due to particulates in the sample. 2) Phosphorescence of the sample . 3) Shifts in chemical equilibrium as a function of concentration. 4) Non-monochromatic radiation, 5) Stray light.
)(max nm )(max nm
Endogenous fluoropores
Excitationmax
Emissionmax
Q (Quantum yield)
Tryptophan 279.8 348 0.20
Tyrosine 274.6 303 0.14
Phenylalanine 257.4 282 0.04
NADH 340 440 0.019
Intrinsic fluorophoresIntrinsic fluorophores
Schematic diagram of Schematic diagram of spectrometerspectrometer
Emission spectra Excitation spectraEmission spectra Excitation spectra
250 260 270 280 290 3000
2
4
6
8
10
12
14
16
Emission monochromator set on 340
Aromatic amino acids fluorescence
Inte
nsi
ty
wavelength nm300 320 340 360 380 4000
2
4
6
8
10
12
14
16
Excitation monohromator set on 285 nm
Aromatic amino acids fluorescence
Inte
ns
ity
wavelength nm
• Main goals: Main goals: Study cancerous transformation using FTIR and LIF . Study cancerous transformation using FTIR and LIF .
Special emphasis was made to identify spectral changes before cell cultures Special emphasis was made to identify spectral changes before cell cultures
passed complete morphological transformation.passed complete morphological transformation.
• Experimental stepsExperimental steps
• Growing the cells Growing the cells
• Infection of the cell cultures by murine sarcoma virus (MuSV) (known to induce Infection of the cell cultures by murine sarcoma virus (MuSV) (known to induce
cancerous transformation)cancerous transformation)
• Measuring samples using FTIR and LIF as function of post infection time.Measuring samples using FTIR and LIF as function of post infection time.
• Spectra analysis by software algorithms and obtaining spectral biomarkersSpectra analysis by software algorithms and obtaining spectral biomarkers
• Results of analysis by mathematical methodsResults of analysis by mathematical methods
MethodologyMethodology
MethodologyMethodology
Murine fibroblast cell lines (NIH/3T3)Murine fibroblast cell lines (NIH/3T3)normal normal transformedtransformed
Mouse embryonic fibroblast (MEFMouse embryonic fibroblast (MEF)normal normal
PropertiesProperties NIH/3T3NIH/3T3 MEFMEF
Source long term cycle in vitro
stem cells from embryos during mouse pregnancy
Susceptible to murine leukemia virus, murine leukemia virus, murine sarcoma virus murine sarcoma virus ..
Cell cultures use advantages
High utilization, less sensitive to environment condition than MEF cells
Primary cells
High gene expression
MethodologyMethodology
MuSVMuSV
ControlControl
14 d14 d
1 d1 d3 h3 h
1 d1 d
7 d7 d
7 d7 d
1)1) The cell cultures were grown in RPMI medium supplemented with 10% or The cell cultures were grown in RPMI medium supplemented with 10% or 2% (or mixture of them depends on cell condition) new born calf serum 2% (or mixture of them depends on cell condition) new born calf serum (NBCS) and the antibiotics such as penicillin, streptomycin and neomycin.(NBCS) and the antibiotics such as penicillin, streptomycin and neomycin.
2) MuSV was obtained from centrifuged highly concentrated transformed NIH ) MuSV was obtained from centrifuged highly concentrated transformed NIH cell lines. cell lines.
3) Measurements as a function of time were taken3) Measurements as a function of time were taken
a)a) FTIR measurements. FTIR measurements. Drop which contain 2 Drop which contain 2 l of PBS with concentration of l of PBS with concentration of 1 million cells/ml seeded directly on 2x2 cm2 ZnSe crystals slide. After ensuring 1 million cells/ml seeded directly on 2x2 cm2 ZnSe crystals slide. After ensuring complete dryness, the FTIR measurements were made.complete dryness, the FTIR measurements were made.
b)b) Fluorescence measurementsFluorescence measurements were taken in cuvette or microplate into PBS were taken in cuvette or microplate into PBS
3 h3 h
FTIR spectra analysisFTIR spectra analysis
100015002000250030003500
Wavenumber cm-1
0.0
50.1
00.1
50.2
00.2
5
Sin
gle
cha
nne
l
Background spectraBackground spectra
Baseline correctionBaseline correction NormalizationNormalization
Fluorescence Spectra analysisFluorescence Spectra analysis
Mathematical methodsMathematical methods Cluster analysisCluster analysis
Cluster Analysis ( Ward`s method )
0 2 4 6 8 10
Heterogenieity
5 days
3 days
1 day
4 hours
NIH/3T3
13 days
7 days
11 days
9 days
NIH/MuSV
Normal state classification
Cancerous state classification
2
1
Ward method ( minimum variance)kn
kk iki
E z z
Euclidean distance between two points p and qEuclidean distance between two points p and q 21
N
i ii
p q
2958 2852
1120 1015
1059 1099
1028 1085
2
A /A
A /A
A /A
A /A
symPO Shift
Mathematical methodsMathematical methodsDiscriminant classification functionDiscriminant classification function
D = w0+w1Z1 + w2Z2+ w3Z3 + .... wiZi
discriminant score
weighting coefficient
variable
MusvMusv NormalNormal 4h4h 1d1d 3d3d 5d5d 7d7d 9d9d 11d11d 13d13d
Z1 4.2 1.6 1,7 1,8 2,2 2,9 3,7 3,9 4,04 4,29
D 100 0 4.5 6.5 24.2 50.7 80.4 83.4 93.8 102.3
D = w0+w1Z1
W0=-61.7 W1=38.5
FTIR data analysisFTIR data analysis
I arealipids
II areaNucleic acids
FTIRFTIR data analysis data analysis
AA29582958/A/A28522852
FTIR spectral indicators FTIR spectral indicators (lipids absorbance)(lipids absorbance)
A2958/A2852 NIH/3T3 NIH/Musv
Mean 1.07 1.27
t-value 10.25
Max value 1.12 1.4
Min value 1.02 1.2
17%17%
FTIR spectral indicators FTIR spectral indicators (nucleic acids area)(nucleic acids area)
AA10991099/A/A1058 1058 (nucleic acids (nucleic acids
vibrational modes)vibrational modes)
AA11211121/A/A1015 1015 (RNA/DNA)(RNA/DNA)
Wavenumber shift of POWavenumber shift of PO22--
(conformation structure (conformation structure of nucleic acids)of nucleic acids)
AA10281028/A/A1085 1085
(Gluocose /phospate)(Gluocose /phospate)
FTIR spectral indicators (nucleic absorbance)FTIR spectral indicators (nucleic absorbance)T-value 16.1T-value 16.1 147%147%
T-value 13.1T-value 13.1 66%66%
T-value 8.9T-value 8.9 96%96%
T-value 5.7T-value 5.7 43%43%
TransformationTransformation
Morphological changes Morphological changes can be observed by can be observed by microscopemicroscope
Transformation of ATransformation of A11211121/A/A10151015 RNA/DNA RNA/DNA
After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change After 9-10 days we can observe morphological changeAfter 9-10 days we can observe morphological change
After 8-9 days we can observe morphological changeAfter 8-9 days we can observe morphological change
Finding fluorescence differenceFinding fluorescence difference
Aromatic amino acids Aromatic amino acids fluorescence transformationfluorescence transformation
After 5-6 days we can observe After 5-6 days we can observe morphological changemorphological change
After 8-9 days we can observeAfter 8-9 days we can observe morphological changemorphological change
Cluster Analysis of NIH/3T3 fast Cluster Analysis of NIH/3T3 fast transformationtransformation
Cluster Analysis (Ward`s method). According five indicators . Fast transformation
0 1 2 3 4 5 6 7 8 9
Heterogenieity
2 days1 day
3 hoursNIH/3T3
5 days14 days
3 days9 days7 days
NIH/MuSV
Cancerous state classification
Normal state classification
2958 2852
1120 1015
1059 1099
1028 1085
2
A /A
A /A
A /A
A /A
symPO Shift
After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change
0 5 10 15 20 25
Heterogenieity
3 days9 days7 days5 days
14 daysNIH/MuSV
2 days3 hours
1 dayNIH/3T3
Cancerous state classification
Normal state classification
Cluster Analysis ( Ward`s method )Fluorescence classification . Fast transformation
Fluorescence Intensity of Aromatic amino acids
Fluorescence Intensity of NADH
Cluster Analysis of NIH/3T3 fast Cluster Analysis of NIH/3T3 fast transformationtransformation
After 5-6 days we can observe morphological changeAfter 5-6 days we can observe morphological change
2958 2852
1121 1015
1059 1099
1028 1085
A /A
A /A
A /A
A /A
1
2
7
sym
AAA
NADH
W
PO Shif
S
I
I W
Discriminant classification functionDiscriminant classification function
ConclusionsConclusions
1)1) Both spectroscopic technique (FTIR-MSP and LIF) allow to detect Both spectroscopic technique (FTIR-MSP and LIF) allow to detect spectral changes before cell cultures pass complete morphological spectral changes before cell cultures pass complete morphological transformation.transformation.
2) The spectroscopic technique shed light on 2) The spectroscopic technique shed light on carcinogenesiscarcinogenesis metabolic processes:metabolic processes:a) Nucleic acids activitya) Nucleic acids activityb) Glucose / phosphateb) Glucose / phosphatec) Lipids c) Lipids d) Aromatic amino acids d) Aromatic amino acids e) NADHe) NADH
3)3) The transformation tendency can be described by sigmoid functionThe transformation tendency can be described by sigmoid function
AcknowledgementsAcknowledgements
Thanks to my supervisors Thanks to my supervisors Prof. S.Mordechai and Dr Prof. S.Mordechai and Dr M.Hulihel.M.Hulihel.
And all members of our lab And all members of our lab Dr.A.Salaman, Dr.R.Sahu and Ph.D Dr.A.Salaman, Dr.R.Sahu and Ph.D students U.Zeligstudents U.Zelig and Z.Hamodi.and Z.Hamodi.
ThankThank youyou