SHAPE Society

1. Identifying Vulnerable Plaque
with Spectroscopy
Strategic Plan: Determine pH and
Lactate by Fiber Optic Catheter and
Near Infrared (NIR) Spectroscopy
By Tania Khan
UMass Medical, THI, UTH, WPI, LMNOP….ubiquitous!

2. Spectroscopy Basics
In general, spectroscopy is the use of the electromagnetic
spectrum to perform physical or chemical analysis

3. Spectroscopy Basics
1) Non-ionizing radiation (light) is used to interrogate
sample E=hc/λ. Example wavelengths:
♦ Visible 0.4 – 0.7 microns
♦ Near-Infrared 0.7 – 2.5 microns
♦ Infrared 2.5 – 10 microns
2) Wavelengths are separated for detection
3) Detector converts intensity to voltage signal as a
function of wavelength
Energy is either absorbed, transmitted, or reflected
by molecules present in sample

4. Other Investigators of Vulnerable
Plaque
Focus on lipid accumulation to predict
vulnerability
♦ Feld et.al. (MIT)
– Raman spectroscopy: currently working on building
Raman fiber optical catheter; TC, logistic regression
– FTIR spectroscopy: only for ex-vivo pathological
identification (microscopy)
♦ Lodder et.al. (Univ. of Kentucky)
– NIR Catheter to study in-vivo rabbits cholesterol
contents; source-detector separation not identified or
optimized
– Lasers and super computers calculations does not take
into account inflammatory responses; not full

6. Fig. 1. (a) NIR spectra of several pure compounds of an arterial intima; (b) NIR
spectra of the model mixtures without water; (c) NIR spectra of the model mixtures
with water; (d) NIR spectra of aortic specimens (1 Micron=Wavelength 1000 nm).
♦Jaross et.al. (Germany)
– FT-NIR spectroscopy and
Partial Least Squares for total
cholesterol
Atherosclerosis 147 (1999): 327-
337.

7. Dr. Soller’s Lab
♦ Optical measurement of tissue pH: Patent #5,813,403
♦ Uses Partial Least Squares to calibrate the
spectrometer to pH (multivariate; several wavelengths
contribute to pH determination)
♦ Based on full spectrum analysis of pH-induced change
of proteins
♦ Lessons Learned: volume of optical measurement >>
volume of electrode measurement
♦ Tissue pH heterogeneity can be solved with smaller
optical probe (my research)
♦ Optimize source-detector separations with knowledge
of optical properties as a function of wavelength
(Monte Carlo Modeling)

8. Hypotheses
Focuses of inflammatory response that leads to vulnerability.
Activated macrophages make environment acidic.
Lactate predominates due to anaerobic metabolism.
♦ Smaller source-detector separations may improve
multivariate calibration to reference pH or lactate
measurements.
♦ Lactate concentration and/or tissue pH may be better
predictors of plaque vulnerability.
♦ Near infrared spectroscopy, combined with multivariate
calibration, will assess the lactate concentration and pH;
distinguish between different stages of atherosclerotic
plaques.

9. NIR Spectrum
0.15
0.2
0.25
0.3
0.35
0.4
0.45
1700 1800 1900 2000 2100 2200 2300 2400 2500
wavelength (nm)
arbitraryunits(absorbance)
Near infrared absorbance of lactic acid

10. Diffuse Reflectance
NIR Spectroscopy
♦ Absorption is due to
combinations and overtones
of fundamental vibrations
♦ Reflectance Mode: path
length varies for different
tissues and wavelengths
♦ Catheter geometry and
optical coupling important
♦ Small source-detector
separations: light penetrates
tissue while restricting
volume interrogated
plaque
interface
to
spectrometer
~3 mm

11. Tissue Penetration Study
♦ NIR reflectance off mirror 100% signal
♦ Tissue stacks placed on probe end
♦ Incremental increase in signal with mirror
~50 um slices
Aortic tissue
Mirror-Enhanced
Reflectance
Tissue Absorption
& Scattering
Mirror
Fiber
Probe

12. Plaque Measurements
♦ Full spectrum absorbance data (400-2500 nm, FOSS NIRSystems)
♦ 24 gauge needle thermistors (Cole-Parmer model 8402-20)
♦ 750 µm diameter pH electrodes (Microelectrodes, MA)
♦ Punch needle biopsy 1 – 5 mg pieces for lactate assay
♦ Measurements on plaque in 37° incubator
♦ Histology on the rest of the plaque
°C
pH
spectrometer
10-20
m
m
~2 mm

13. Small Diameter Probe
♦ Preliminary visible/NIR
spectra (UMass Worcester)
Visible Spectra
0.1
0.15
0.2
0.25
0.3
0.35
0.4
400 500 600 700 800 900 1000 1100
Wavelengt h
Fibrofatty Ulcerated FTNIR Plaque
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1000 1100 1200 1300 1400 1500 1600 1700 1800 1900
w avelength
A.U.
fibrofatty ulcerated area

14. Large Diameter Probe: Full Spectrum of
Rabbit Aortic Arch
Aortic Arch
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
400 550 700 850 1000 1150 1300 1450 1600 1750 1900 2050 2200 2350 2500
wavelength
absorbance
aortic arch WHHL 0.9 mm NZ aortic arch
WHHL
New
Zealand

15. Long-Term Goals
♦ Determine a mathematical model to train the
spectrometer to determine pH and lactate
♦ Correlate spectroscopically measured pH and lactate to
histology
♦ Determine optimal penetration depth and the minimum
number of wavelengths needed for accurate, repeatable
plaque measurements
♦ Incorporate linear scanning or detector arrays for a
“spectral image” of vessel wall
♦ Locate and identify vulnerable plaque with Near Infrared
Spectroscopy

16. Plan for Next 4 Weeks:
♦ Identify and assess lactate assay (Dr. Dasgupta’s lab)
♦ Identify appropriate culture medium and environment
(DMEM, pO2, pCO2, other parameters)
♦ Calibrate pH electrodes, temperature probes, etc.
♦ Test on normal and atherosclerotic rabbit aorta in culture
♦ Conduct depth penetration study with current probe (~1 cm)
♦ Receive parts for small dimension probe (~3 mm)
♦ Plan pH, lactate, fresh human specimens and spectroscopic
analysis study
♦ Get THI lab up and running!!
NIR Spectroscopy: the right key to open the lock!