Quantitative Reflectance and Fluorescence Spectroscopy
Dominic Robinson, Arjen Amelink, Riette de Bruijn, Christopher Hoy,
Ute Gamm, Floor van Zaane, Rob Baatenburg de Jong
The Center for Optical Diagnostics and Therapy
Department Otolaryngology - Head and Neck Surgery
Erasmus Medical Center
Rotterdam, The Netherlands
TNO Delft
Quantitative spectroscopy compliments imaging
Wide-field Reflectance Imaging
Quantitative Reflectance Spectroscopy
Is contrast due to more light getting absorbed
or less light getting reflected?
We see blood vessels, but how oxygenated
is the blood?
Where would a biopsy be most useful?
If the intrinsic absorption ‘artifacts’
could be removed
are there differences in scattering properties
that are diagnostically useful?
Ovarian Mouse Tumors, © BioOptics World 2011
Fill in your clinically relevant question
Quantitative spectroscopy compliments imaging
Wide-field Fluorescence Imaging
Quantitative Fluorescence Spectroscopy
Is contrast due to the concentration of the
fluorophore or the depth or
differences in tissue optical properties?
The signal changed during therapy. Did the
drug/dye decrease, or “change”, or did the
optical properties change?
For a well characterized drug/dye, what is
the true concentration at a given location?
Ovarian Mouse Tumors, © BioOptics World 2011
Raw fluorescence: how bad is the problem?
Wide-field Fluorescence Imaging
Increasing absorption
Increasing scattering
Quantitative Fluorescence Spectroscopy
Is contrast due to the concentration of the
fluorophore or the depth or
differences in tissue optical properties?
The signal changed during therapy. Did the
drug/dye decrease, or “change”, or did the
optical properties change?
For a well characterized drug/dye, what is
the true concentration at a given location?
Kim et al, JBO 067006 (2010)
Reflectance and Fluorescence Spectroscopy
Raman Spectroscopy: Vibrational energy levels
Absorption and fluorescence spectroscopy:
Electronic energy levels
Reflectance
Scattering
Light absorption in tissue
Absorption coefficient ma: average number of absorption events per unit distance
[mm-1]
ma depends on concentration of chromophores and their wavelength dependent
extinction coefficients e(l).
Light attenuation in the absence
of scattering:
ma z
0
(Beer-Lambert Law)
II e

oxyhemoglobin
deoxyhemoglobin
(vessel diameter)
cytochrome c
bilirubin
beta-carotene
lipids
Water
melanin
Light scattering in tissue
Scattering coefficient ms: average number of scattering events / unit distance [mm-1]
Phase function p(q): angular scattering probability distribution
ms depends on scattering efficiency (n, feature size) and concentration of scattering
centers
Average distance between scattering events
(mean free path) = 1/ ms
Light scattering in tissue
Scattering coefficient ms: average number of scattering events / unit distance [mm-1]
Phase function p(q): angular scattering probability distribution
ms depends on scattering efficiency (n, feature size) and concentration of scattering
centers
Average distance between scattering events
(mean free path) = 1/ ms
Light scattering in tissue
Scattering coefficient ms: average number of scattering events / unit distance [mm-1]
Phase function p(q): angular scattering probability distribution
logarithmic polar plot of
angular scattering probability
(phase function)
90
120
Scatterer Size Distribution
60
Frequency
150
30
180
0
210
0.01
0.1
1
Particle diameter (mm)
10
330
240
300
270
Light scattering in tissue
Phase function can be characterized by its Legendre moments, g1, g2, …
Legendre functions
Phase function
p(cos(q))
0.1
0.01
0.001
0.0001
0
40
80
120
160
q(degrees)
Forward scattered photons are similar to un-scattered photons:
μs  (1 g1 )μs
The relative contribution of the backscattering peak can also be quantified: γ 
(1  g )
(1  g )
2
1
Another problem
We don’t know the pathlength of light
Wide-field imaging
Red and NIR photons long pathlength
Green and Blue photons short pathlength
If we don’t know the pathlength – can’t do calculations
Understand how Reflectance relates to the fiber diameter
Scattering
5 years work!
Single Fiber Spectroscopy: Empirical Models
Reflectance
0
RSF  RSF
e
LSFR
df
0
SF
R
Single
Diameter μ a
Multi-diameter

0.18
f
  lim 1  2.31 e

2
[%]
1.45
0.64
0.64  (μa d f )
( μ d ) (
s
 μa LSFR
0.63 2 μ's d f
)
[-]
0.57

(
)
μ'
d
s
f
 
  0.63 2  (μ's d f

μ γ
Simultaneous
of multiple diameters
Single
Fibersolution
Spectroscopy
(MDSFR)
s
)
0.57



Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Scattering and
Absorption
NIR
Fiber-optic
spectroscopy:
measuring
Fluorescence
NIR
Multi-diameter single fiber reflectance (MDSFR)
1.5
Data
Model Fit
R0
1.0
df = 1.0 mm
0.5
df = 0.6 mm
df = 0.2 mm
0
Single Diameter
Single Diameter
μ a μ s
μa
Simultaneous solution of multiple diameters
Raw fluorescence  Intrinsic fluorescence
μ s
γ
Pre-clinical data
OSC-gfp-luc tumours in the mouse tongue
Sequential placement of 0.4 & 0.8 mm fibers
van Leeuwen-van Zaane et al In vivo quantification of the scattering properties of tissue using multi-diameter single fiber
reflectance spectroscopy.Biomed Opt Express. 2013 Apr 9;4(5):696-708.
Scattering parameters m’s and 
Scattering parameters m’s and 
Diagnostically relevant in the clinic ? – under investigation
Technology: Probes
Co-localized single fiber measurements: fiber bundels
200 μm
Halogen
Lamp
Spectrometers
365 nm LED
Technology: Devices
3 diameters (concentric)
2 diameters (adjacent)
Reflectance ma, m’s and 
Fluorescence lex = 365/405/690/785 nm
Image Guided Surgery, Reflectance Spectroscopy
Optical Properties
• Absorption
– Physiology
Blood volume
Saturation
Vessel diameter
cytochrome c
Bilirubin
beta-carotene
Lipids
Water
• Scattering
– Architecture
Scattering coefficient
Tissue Ultrastructure
Oral Cavity: StO2
Normal oral mucosa
Large SCC
10
StO2 92 (4)  0.5 (0.2)
StO2 4 (2)  3 (0.2)
Reflectance (arb. units)
8
StO2 2 (3)  10 (0.2)
6
4
2
0
400
500
600
700
800
wavelength (nm)
900
1000
Superficial lesions - smaller differences – tumour margins under investigation
Single Fiber Fluorescence: Models
FSF  FSF0 e
LSFF
df
(
 0.71 μ s d f
Qm
(
S  0.0935 μs d f
f
a ,x
)
)

0.31
 0.36
 μa LSFF
[-]
1  1.81 μ s d f
(
1  μa d f
FSF0
[-]
[mm-1]
d f n S
e
)


1
1.61



 0.31 ms , xd f 1 0.31 μs,m d f 1 


(
)
(
)
[-]
f
Single Diameter + μ a + μ s  Qm a, x
[Kanick et al., Biomed. Opt. Exp., 3 137, (2012)]
Validation: Fluorescein phantoms
Optical Phantoms:  and m’s easy to control F2- (with and without absorption)
Hoy CL et al, et al Title: Method for rapid multi-diameter single fiber reflectance and fluorescence spectroscopy
through a fiber bundle. JBO 2013;18(10):107005.
Data with fitted Qµa
Scattering Model
μs d f
μs d f
Solved for Qm af,x by regression. Qm af,x = 7.0 ± 0.3 (10)-3 mm-1
QSFF = 0.92 ± 0.04, compared with published Q  0.88 for pH ~7.4
Image Guided Surgery, Reflectance Spectroscopy
Optical Properties
Absorption & Scattering
Quantitative Fluorescence
•
•
Auto-fluorescence
Targeted fluorophores
Aid Fluorescence Image Guided Surgery
For a well characterized dye
what is the true concentration (Q.uaf)
given location
Tumor margin
CW800-Cetuximab
Quantitative Spectroscopy Application Areas
Clinical Relevance
Measurable
Quantities
Absorption
μa
Scattering
μs  (1 g1 )μs
γ
(1  g )
(1  g )
Fluorescence
Qm a,f x
2
1
Microvascular
Properties
(StO2, BVF, Dvessel)
Tissue Ultrastructure
(Δn correlation function)
Concentration of
targeted fluorophores
Tumor vs Normal
tissue
Resection Margins
Pre-malignant disease
Fluorescence
Image Guided
Surgery
The field effect
Challenges
•
•
Point measurement(s)
• Complimentary to imaging and (micro-) endoscopy
• Really want to do imaging
• Coherent fiber bundles
• Structured light illumination
Measurement time
• Depends on the number of fiber diameters a few seconds
• Goal < 1 sec