Title: Study of early spectral changes in cells transformation using advanced physical and mathematical models.
1Study of early spectral changesin cells
transformationusing advanced physical and
mathematical models.
By E. Bogomolny
Supervisors
Prof. S.Mordechai
Department of Physics (BGU)
Dr. M.Hulihel Institute of Applied
Research (BGU)
21) Introduction.
IR radiation
UV radiation FTIR Microspectroscopy Light
induced Fluorescence.
2) Methodology.
Experimental descriptions. Spectra analysis.
Mathematical methods.
3) Results and Discussion.
4) Conclusions.
3The Electromagnetic Spectrum
Gamma ray SPECT, PET Imaging Nuclear transition
X-ray Mammography ,CAT. Inner shell
UV Potential electromagnetic range for diagnosis Valence electrons
Visible Pathology Valence electrons
Infrared Potential electromagnetic range for diagnosis Vibrational rotational level
Microwave EPR imaging Rotational levels or fine structure
Radio NMR imaging Hyperfine structure
4IR radiation
Multi-atom molecule
- Born-Oppenheimer Approximations
- 1) The electronic motion and the nuclear motion
in molecules can be separated - 2) nuclear motion does not induce electronic
transitions.
5IR radiation
Rewriting the Hamiltonian in spherical
coordinates for diatomic system
Equation similar to Hydrogen atom
Equation can be separated to angular and radial
part
Solution depends explicitly on U(R) . By
expanding U(R) in Taylor series we obtain
6 The selection rules for vibrational and
rotational transitions ?n 1 and ?J
1
The transition moment
The dipole moment
Only vibrational modes that have a transition
dipole moment (or component of the transition
dipole moment) can be observed in infrared
spectroscopy
7Different Types of Vibrational modes
(Stretching)
Non linear molecules 3N-6
Symmetric Stretch
Asymmetric Stretch
(Bending)
Rocking Twisting
Scissoring Wagging
8Different Types of Vibrational modes
Linear molecules 3N-5
Symmetric Stretch
Asymmetric Stretch
Bending (two types)
9Introduction FTIR-MSP(Fourier Transform Infrared
Micro-Spectroscopy)
10Introduction FTIR-MSP
11Michelson Interferometer
12The measurements sequence of polychromatic IR
source
A -log10T
13IR spectra of bio-molecules
Cell parameters Values
Radius µm 100
Volume µm3 gt10000
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 6-8
14Fluorescence The Jablonski diagram
Absorption 10-15 sec
hn
Phosphorescence 10-4 to 10 sec
15Fluorescence properties
Compounds with multiple conjugated double bonds,
i.e. extended p electronic systems, has high
quantum yield 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.
16 Intrinsic fluorophores
Q (Quantum yield) Emission l max Excitation l max Endogenous fluoropores
0.20 348 279.8 Tryptophan
0.14 303 274.6 Tyrosine
0.04 282 257.4 Phenylalanine
0.019 440 340 NADH
17Schematic diagram of spectrometer
- Emission spectra Excitation spectra
18Methodology
- Main goals Study cancerous transformation using
FTIR and LIF . Special emphasis
was made to identify spectral changes before cell
cultures passed complete morphological
transformation. - Experimental steps
- Growing the cells
- Infection of the cell cultures by murine sarcoma
virus (MuSV) (known to induce cancerous
transformation) - Measuring samples using FTIR and LIF as function
of post infection time. - Spectra analysis by software algorithms and
obtaining spectral biomarkers - Results of analysis by mathematical methods
19Methodology
normal
transformed
normal
Murine fibroblast cell lines (NIH/3T3)
Mouse embryonic fibroblast (MEF)
Properties NIH/3T3 MEF MEF
Source long term cycle in vitro stem cells from embryos during mouse pregnancy stem cells from embryos during mouse pregnancy
Susceptible to murine leukemia virus, murine sarcoma virus. murine leukemia virus, murine sarcoma virus. murine leukemia virus, murine sarcoma virus.
Cell cultures use advantages High utilization, less sensitive to environment condition than MEF cells High utilization, less sensitive to environment condition than MEF cells Primary cells High gene expression
20Methodology
- 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
(NBCS) and the antibiotics such as penicillin,
streptomycin and neomycin. - 2) MuSV was obtained from centrifuged highly
concentrated transformed NIH cell lines. - 3) Measurements as a function of time were taken
- FTIR measurements. Drop which contain 2 ml of
PBS with concentration of 1 million
cells/ml seeded directly on 2x2 cm2 ZnSe crystals
slide. After ensuring complete dryness, the FTIR
measurements were made. - Fluorescence measurements were taken in cuvette
or microplate into PBS
MuSV
1 d
3 h
14 d
7 d
1 d
3 h
Control
7 d
21FTIR spectra analysis
Background spectra
Baseline correction
Normalization
22Fluorescence Spectra analysis
23Mathematical methods
Euclidean distance between two points p and q
24Mathematical methods
- Discriminant classification function
D w0w1Z1 w2Z2 w3Z3 .... wiZi
D w0w1Z1 W0-61.7 W138.5
Musv Normal 4h 1d 3d 5d 7d 9d 11d 13d
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
25FTIR data analysis
I area lipids
II area Nucleic acids
26FTIR data analysis
A2958/A2852
27FTIR spectral indicators
(lipids absorbance)
A2958/A2852 NIH/3T3 NIH/3T3 NIH/Musv
Mean 1.07 1.07 1.27
t-value 10.25 10.25 10.25
Max value 1.12 1.4 1.4
Min value 1.02 1.2 1.2
17
28FTIR spectral indicators
(nucleic acids area)
A1121/A1015 (RNA/DNA)
Wavenumber shift of PO2- (conformation structure
of nucleic acids)
A1099/A1058 (nucleic acids vibrational modes)
A1028/A1085 (Gluocose /phospate)
29FTIR spectral indicators (nucleic absorbance)
T-value 16.1 147
T-value 8.9 96
T-value 13.1 66
T-value 5.7 43
30Transformation
Morphological changes can be observed by
microscope
31Transformation of A2958/A2852
32Transformation of A1121/A1015 RNA/DNA
After 5-6 days we can observe morphological change
After 9-10 days we can observe morphological
change
After 8-9 days we can observe morphological change
33Finding fluorescence difference
34Aromatic amino acids fluorescence as a function
of cell concentration
Intensity BC concentration
Ex. Wavelength 285.00(nm)
Em. Wavelength 340.00 (nm)
Excitation filter Auto
Emission filter Open
PMT Voltage (V) Medium (600 V)
Replicates 5
Sample averaging ON
Cells type Value of B Error t-Value
NIH/3T3 38.825 0.373 103.944
NIH/MuSV 30.675 0.381 80.608
MEF 50.975 1.475 34.564
MEF/MuSV 34.786 0.650 53.542
35NADH fluorescence as function of cell
concentration
IIntensityBCconcentration
Ex. Wavelength 340.00 (nm)
Em. Wavelength 425.00 (nm)
Excitation filter Auto
Emission filter Open
PMT Voltage (V) High (800 V)
Replicates 5
Sample averaging ON
Cells type Value of B Error t-value
NIH/3T3 59.595 0.459 130.125
NIH/MuSV 45.006 0.746 60.331
MEF 48.716 0.504 96.594
MEF/MuSV 30.675 0.381 80.608
36Aromatic amino acids fluorescence
transformation
After 5-6 days we can observe morphological
change
After 8-9 days we can observe morphological
change
37NADH fluorescence transformation
After 5-6 days we can observe morphological
change
After 8-9 days we can observe morphological
change
38Cluster Analysis of NIH/3T3 fast transformation
After 5-6 days we can observe morphological change
39Cluster Analysis of NIH/3T3 fast transformation
After 5-6 days we can observe morphological change
40Cluster Analysis of NIH transformation
(Slow transformation)
41Discriminant classification function
42Conclusions
1) Both spectroscopic technique (FTIR-MSP and
LIF) allow to detect spectral changes before cell
cultures pass complete morphological
transformation.
2) The spectroscopic technique shed light on
carcinogenesis metabolic processesa) Nucleic
acids activityb) Glucose / phosphatec) Lipids
d) Aromatic amino acids e) NADH
3) The transformation tendency can be described
by sigmoid function
43- Acknowledgements
-
Thanks to my supervisors
Prof. S.Mordechai and Dr
M.Hulihel. - And all members of our lab Dr.A.Salaman,
Dr.R.Sahu and Ph.D students U.Zelig and Z.Hamodi.
44