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Imaging in Renal
Raman Spectroscopy
R. De Crevoisier, K. Bensalah
Signal and Image Processing (U642)
Raman Spectroscopy
Definition of Raman
spectroscopy and rationale to
use it
Definition of Raman spectroscopy
= optic spectroscopy technique used to study
vibrational state of molecules, using a laser in
the visible or infrared range, and relying on
inelastic scattering
Interactions between light and molecules
light passing through a transparent substance
EXCITATION
Incident light/photons (monochromatic,
visible or infra-red wavelength)
Polarisation of the electron cloud
/impact on bounds of the
molecule
Î changes in vibrational state
molecules
specific vibrational energy
level of each molecule
Interactions between light and molecules
light passing through a transparent substance
EMISSION
releasing scattered photons in any direction
EXCITATION
2 types of
scattering
Incident light/photons
(monochromatic, visible or infra-red)
- Rayleigh
- Raman
relaxation
Polarisation of the electron cloud
/impact on bounds of the
molecule
Î changes in vibrational state
molecules
specific vibrational energy
level of each molecule
Interaction between light and molecules: Rayleigh scattering
= the more prominent scattering
light passing through a transparent substance
EXCITATION
Incident light/photons
(monochromatic, visible or infra-red)
EMISSION
Releasing scattered photons in any direction
relaxation
Polarisation of the electron
cloud /impact on bounds of
the molecule
specific vibrational energy
level of each molecule
Elastic effect:
- The light does not gain or lose energy during the scattering, stays at the same wavelength.
- The amount of scattering is strongly dependent on the wavelength.
Raman scattering: principle
light passing through a substance
EMISSION
EXCITATION
Inelastic effect:
Incident light/photons
The light photons lose or gain energy during the
scattering process, and therefore a shift in
wavelength (increase or decrease)
(monochromatic, visible or infra-red)
relaxation
specific vibrational energy
level of each molecule
Polarisation of the electron
cloud /impact on bounds of
the molecule
The scattered photon has less energy
than the incident photon, and therefore
a longer wavelength.
If the molecule is in a vibrational state to begin
with, and after scattering is in its ground state then
the scattered photon has more energy, and
therefore a shorter wavelength.
Raman scattering: principle
light passing through a substance
Inelastic effect:
The light photons lose or gain energy during
the scattering process, and therefore
increase or decrease in wavelength
EXCITATION
Incident light/photons
(monochromatic, visible or infra-red)
Polarisation of the electron cloud
Only about 1 in 107 photons undergo Stokes Raman scattering
(swamped by the far more prominent Rayleigh scattering).
low sensitivity
Î specific technology to amplify the Raman spectrum
Raman spectrum :
= intensity of the Raman scattering as a function of the difference of
wavelength between the incident light and the scattered light (in cm -1)
represents the vibrational states of the molecules constituting the tissue and
therefore the molecular composition of the tissue
Raman spectrum :
= intensity of the Raman scattering as a function of the difference of
wavelength between the incident light and the scattered light (in cm -1)
represents the vibrational states of the molecules constituting the tissue
and therefore the molecular composition of the tissue
In kidney cancer: objectives of Raman spectroscopy:
Î To discriminate: normal tissue / benign tumor / cancer ?
- rapid diagnosis: ‘‘optical biopsy tool’’ : indication of resection for small tumor ?
- status of margin (partial nephrectomy)
Î Prognostic/predictive factor ?
Î Physiopathology of tumor invasion (identification of the involved molecules) ?
Raman spectrum :
= intensity of the Raman scattering function of the difference of wavelength
between the incident light and the scattered light (in cm -1)
represents the vibrational states of the molecules constituting the tissue
In kidney cancer: objectives of Raman spectroscopy
Î To discriminate: normal tissue / benign tumor / cancer ?
- rapid diagnosis: ‘‘optical biopsy tool’’ (indication of resection for tumor ?)
- status of margin (partial nephrectomy)
Î Prognostic/predictive factor ?
Î Physiopathology of tumor invasion ?
Available studies:
Raman spectroscopy in prostate and bladder / Optical reflectance spectroscopy in kidney
…Very limited data for Raman spectroscopy in kidney
Raman Spectroscopy
Materials and methods
Bensalah, European Urology 2010
Patients and tumor characteristics
- Prospective study
- 36 patients having surgery for
renal tumor (imaging) between
June and November 2009
Bensalah, European Urology 2010
Instrumentation
Raman probe (to emit
and collect light)
Optic fiber
High-powered nearinfrared laser
Raman
spectrophotometer
Camera (to receive reflected
and scattered light)
Laptop computer
Bensalah, European Urology 2010
Instrumentation
Raman probe (to emit
and collect light)
Optic fiber
High-powered nearinfrared laser
Raman
spectrophotometer
Camera (to receive reflected
and scattered light)
Immediately after extraction, the specimens were stored on ice and
transferred to the pathology department.
Raman spectra were acquired within 15 min of extraction.
Bensalah, European Urology 2010
Raman measurements
After longitudinal section of the specimens, Raman spectra acquired by placing the
probe at several standardised locations (8 points): on the surface and on the section of
normal and tumoral tissue.
3–4 cm apart
avoiding necrotic sections
Kidney
2 spectra recorded on each spot
Raman measurements
Partial nephrectomy
Tumor
Healthy tissue
2 spectra recorded on each spot
Bensalah, European Urology 2010
Raman measurements
- laser beam : set at 50 mW and focused on a 500-microm spot on the
surface of the sample, duration of spectrum acquisition = 30 s
- wavelength of 785 nm (to avoid background fluorescence)
- each Raman spectrum comprised 11 769 points (with Raman shift wave
numbers ranging from 100 to 3444 cm-1)
- background fluorescence removed
- lower resolution signal computed using a wavelet decomposition
procedure (6th-level approximation)
Bensalah, European Urology 2010
Raman analysis
Clear cell carcinoma
Original spectrum
Final spectrum: background fluorescence removed
Statistical analysis
Objective: to generate a Raman classification of:
- normal and tumoral renal tissue
- benign and malignant renal tumors
- high-grade (Fuhrman III–IV) and low-grade (Fuhrman I–II) tumor
- clear-cell, papillary, and chromophobe tumors
Î support vector machine (SVM) with a linear kernel and a sequential minimal
optimisation solver (+ leave-one-out cross-validation technique)
Bensalah, European Urology 2010
Raman Spectroscopy
Results
Bensalah, European Urology 2010
Distribution of Raman spectra
Bensalah, European Urology 2010
Discrimination between normal tissue and tumor
Sensitivity: 82%
Specificity: 87%
Accuracy: 84%
Bensalah, European Urology 2010
Discrimination between low-grade (Fuhrman I–II) and high
grade (Fuhrman III–IV) tumors
Sensitivity: 84%
Specificity: 80%
Accuracy: 82%
Bensalah, European Urology 2010
Discrimination between clear cell carcinoma and non clear
cell tumors (papillary or chromophobe)
Sensitivity: 96%
Specificity: 87%
Accuracy: 93%
Bensalah, European Urology 2010
Discrimination between benign and malign tumors
No conclusion: number of spectra too small
Bensalah, European Urology 2010
Raman Spectroscopy
Conclusions
Bensalah, European Urology 2010
Conclusions
Raman spectroscopy can accurately (Se.
and Sp.> 80%) differentiate:
- normal and tumoral renal tissue
- high- and low-grade RCC
- histologic subtypes of RCC
Conclusions
Advantages:
•
immediate results : acquisition time = 30 s, optical graph in real time
•
objective method using robust algorithms (not need human interpretation)
•
reproducibility of the spectra measurement: in the same area: synchronized
robot (100 spectra from 5 pts, tumor/healthy tissue)
•
non-invasive: probe just placed on the tissue (to emit and collect light)
•
easily inserted through a catheter, an endoscope, or a trocar (‘‘optical
biopsy tool’’ )
•
non toxic and easily transferred to human care: probe made of nontoxic
material and the laser already widely used in various clinical situations
Conclusions
Remaining Issues and limitations:
•
Very limited number of data Î need for confirmation of the results from
other studies +++
•
Comparison with other optic spectroscopy modalities (reflectance and
fluoscence) discriminating between normal/tumoral renal tissue and between
benign/malignant renal tumors at surgery
•
No clear specific molecular “Raman signature”: variations of the overall
spectrum signal Î complexity of the molecular meaning of the spectra?
•
Ex vivo specimen analysis (impact of blood flow underestimated,
haemoglobin = chromophore) Î need for in vivo studies
Thank you !
Belle Ile, Bretagne, France