# A Ratiometric Fluorescence Imaging System for  Surgical Guidance

Authors: Eduardo H. Moriyama; Anthony Kim; Arjen Bogaards; Lothar Lilge; Brian C. Wilson
Journal: Advances in Optical Technologies
 (2008)

---

## Abstract

A 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometric algorithm to correct for the effects of tissue optical absorption and scattering, imaging  “geometry” and tissue autofluorescence background. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms for quantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX). The technical feasibility to use this system for fluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was induced exogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA).


---

## Body

## 1. Introduction

Intracranial brain tumors are the most common and aggressive primary tumors in the central
nervous system (CNS) and carry one of the worst prognosis of all types of
cancers. Advances in surgery, radiotherapy, and
chemotherapy have resulted in only modest improvement
in patient survival [1]. Radical surgical
resection is considered the standard procedure for treatment of high-grade
gliomas, and maximizing the degree of tumor resection has been related to
improvement of patient survival [2, 3]. However, due to the
infiltrative nature of most of the gliomas, complete resection is difficult to
achieve, resulting in high risk of tumor recurrence [2]. Hence, more sensitive and specific
techniques are needed to aid in the identification of
malignant tissue intraoperatively and, thereby, to
increase the completeness of tumor resection without damaging adjacent
critical normal brain structures and function [4]. Such techniques include magnetic
resonance imaging (MRI) [5, 6], ultrasound [7], and optical technologies [8–10].Fluorescence imaging is particularly promising to identify malignant tissuesin vivo, exploiting
either intrinsic fluorescence characteristics (autofluorescence) [11–13] or the preferential accumulation
or targeting of administered (exogenous) fluorophores [14–16]. Fluorescent agents such as fluorescein
[17] and porphyrins [8, 18, 19] are
suitable agents for detection of neoplastic cells during intraoperative tumor
removal, on account of their preferential accumulation and/or retention in
malignant tissues. Recently, the main effort has been on fluorescence-guided resection (FGR) of brain tumors using protoporphyrin IX (PpIX), which is preferentially synthesized in
brain tumor cells relative to normal brain following administration of
aminolevulinic acid (ALA). This approach has demonstrated more complete resection
of brain tumors compared to surgery under white light alone, in both
preclinical animal models [20, 21] and in patients [18, 22]. However, making FGR using
exogenous fluorophores, including ALA-PpIX, a quantitative and hence, objective
and reproducible technique is challenging, due the effects of
multiple scattering and absorption of the excitation and emission light and the
background of tissue autofluorescence. To date, only semiquantitative
information on the concentration and distribution of fluorescent probes in vivo [23–25] has been possible.Several approaches have been investigated
for quantitative fluorescence measurementsin vivo, including tomographic imaging [26, 27], fluorescence lifetime imaging [28], fiberoptic point fluorescence
spectroscopies [29, 30], and ratiometric correction
methods [31–33]. Here, we will focus on the
last of these, in which the objective is to make multiple spectral measurements
that are then combined through an empirical or biophysical model to correct for
the tissue attenuation and/or autofluorescence effects. As we will demonstrate,
the advantage is that this can be implemented in real-time imaging mode, which
is clinically desirable. In the simplest approach,
correction is performed using single excitation and emission wavelengths [34, 35],
but this does not correct for tissue autofluorescence [36]. As we recently showed in a modeling study [37], the use of multiple excitation and/or
emission wavelengths can virtually eliminate the tissue autofluorescence and
also enable quantitative measurements of fluorophores [36–40], such that
the fluorescent signal depends only on the concentration of the fluorophore. This
has been validated in phantoms for a wide range of fluorophore concentrations
(in the case of PpIX, e.g., to <0.1 μg mL−1) and tissue optical properties
[41].Here, we present a prototype FGR instrument that provides video-rate digital
fluorescence imaging incorporating a double-ratiometric correction method that
is optimized for intraoperative identification of brain tumors. Validation studies
in phantoms are presented to demonstrate the performance of the system, and
preliminary experiments are presented in resection of intracranial brain tumor
in a rat modelin vivo to illustrate its practicality and functionality.


## 2. Materials and Methods

### 2.1. System Description

The fluorescence imaging system was designed primarily for surgical guidance and is shown in Figure1. The
light source comprises a 300 W xenon lamp (Cermax, Perkin Elmer, CA, USA) focused
through a filter wheel into a 5 mm diameter liquid light guide (Model 495 FR, Karl Storz, Tuttlingen,
Germany) that is coupled into a 10 mm diameter rigid clinical laparoscope (Model
871 1AA, Karl Storz, Tuttlingen
Germany). The collection component of the imaging system is
based on a 3-CCD compact camera (DXC-C33, Sony, ON, Canada) operating at 30 frames s−1(NTSC) with 796×494 pixels and 8-bit dynamic range. A long-pass 500 nm filter
(Custom made, Chroma Technologies, VT, USA)
is placed between the camera and the laparoscope. This filter deliberately
leaks a small fraction (∼10−4) of the UV/blue excitation light to allow
measurement of the diffuse reflectance signal from the tissue for the
ratiometric algorithm described below. Spectral response curves for the red,
green, and blue channels of the CCD were measured using a
standard color target (ColorChecker Chart: Gretag Macbeth, Grand
Rapids, MI, USA) illuminated by a tungsten-halogen light
source (LS-1: Ocean Optics, Dunedin, FL, USA). For this, the images of the
color target were taken through the laparoscope without the fluorescence
emission filter installed. Serial filters spanning from 420 to 750 nm were
placed in front of the laparoscope lens for each of the color target images.
The spectral response of the camera-laparoscope optical chain was determined
and averaged from 4 spectrally-neutral shades.(a) Photograph and (b) schematic of the system.
(a)(b)The fluorescence imaging system features a dual-excitation capability. The filter
wheel holds two different excitation filters, and its rotation rate is such
that consecutive camera frames are excited by alternating excitation
wavelengths. For the present study, the
excitation filters had central wavelengths,λex1, at the PpIX Soret maxima of 405 nm (full width at half maximum, FWHM = 96 nm) and at 440 nm (FWHM = 60 nm). The
latter was chosen as the shortest wavelength lying above the main PpIX Soret
band in order to allow for correction for the tissue autofluorescence. It is
assumed then that the tissue optical properties are comparable at these two
wavelengths, since they are not too far apart. Both wavelengths can induce high
levels of autofluorescence in tumor and normal tissues. The delivered power at
each wavelength was approximately 50 mW cm-2 at a typical working distance of 2 cm from the
tissue surface. The digital video output is captured by a laptop computer and
can be displayed for visualization of the processed fluorescence images in real
time. The image processing software (Hytek
Automation, Waterloo, ON,
Canada) was based on LabVIEW
(National Instruments Corp., Austin,
TX, USA) and could execute multiplication,
division, addition, or subtraction on each channel.


### 2.2. System Characterization

The spatial resolution of the
imaging system was determined using a standard 1951 USAF glass slide resolution
target (Edmund Optics, NJ, USA) placed 2 cm from the front end of the
laparoscope, resulting in a field of view of12.7×8.2 mm. To characterize the
system performance for fluorescence imaging, tissue-simulating liquid phantoms
were prepared, comprising PpIX (Sigma-Aldrich, ON,
Canada) dissolved in 1 mM dimethyl sulfoxide (DMSO) with methylene blue dye added
as the optical absorber and the lipoprotein emulsion Intralipid (Fresenius Kabi, Uppsala, Sweden)
to provide optical scattering [42].
The corresponding absorption and reduced scattering coefficients are given in Table 1, based on well-established literature values. The system sensitivity was
measured using varying PpIX concentrations: 2.5, 1.25, 0.62, 0.31, 0.15, 0.075, and 0.039 μg mL−1. At each concentration,
fluorescence images were taken at both excitation wavelengths at distances of
2, 3, 4, or 5 cm from the phantom surface, with the camera
focused at the 2 cm working distance. The signal in the red channel of the 3-CCD
was plotted as a function of PpIX concentration. The fluorescence signal was
collected at 0°, 15°, or 30° from the vertical axis to
determine the influence of the imaging geometry.Table 1
Optical properties of the liquid phantoms at 635 nm.
Reduced scatteringAbsorption coefficientcoefficientμs’ (cm−1)μa (cm−1)Phantom A71.6Phantom B73.2Phantom C141.6Phantom D143.2The ratiometric method applied here was developed
by our group previously and is based on using 2 excitation and 2 emission
wavelengths [41]. The first
excitation wavelength is in the absorption peak of PpIX (λex1=405 nm) and, for each image pixel,
the emitted red fluorescence (560–750 nm) is
divided by the signal from the diffusely reflected excitation light. Next, this
fluorescence/reflectance ratio is divided by the same ratio calculated using the
second-excitation wavelength (λex2=440 nm). Thus, the signal, Q,
in each pixel is given by(1)Q=F(λex1,λem1)R(λex1)×R(λex2)F(λex2,λem1).


### 2.3.In Vivo Tests

To test the feasibility and functionality of this imaging system and
the double-ratio algorithmin vivo,
PpIX fluorescence and diffuse reflectance images were acquired in an
established rat brain tumor model [39]
undergoing fluorescence-guided tumor resection. The rat glioma tumor
model, CNS-1, was chosen as being highly infiltrative, a common characteristic
of high-grade human gliomas that should present a valid test of the ability to detect residual tumor at the
surgical margins. FGR was performed at 21 days following implantation of
luciferase-transfected CNS-1luc cells (3×105 cells, 2 mm below the dura, n=2 per ALA dose) in the left brain hemisphere of Lewis rats (Charles River, MA,
USA). PpIX was induced by administering aminolevulinic acid (ALA) in
hydrochloride form (Sigma, Oakville, ON, Canada).
This was dissolved in phosphate-buffered saline (20 mg mL−1) with the pH adjusted to ~5.5 by 1N NaOH and then injected intraperitoneally (i.p.) at 20, 50, or 100 mg kg−1 at 2–4 hours before the surgical
procedure/imaging. The animals were subdued by an i.p.
mixture of ketamine and xylazine (80 and 13 mg kg−1, resp.).
A 2 cm incision was made in the scalp along the midline and held open by a
retractor. A 1 cm craniotomy was performed using a burr drill,
and the dura was carefully removed. To measure the uptake of PpIX in
tumors, PpIX fluorescence spectra (450–750 nm) were
acquired by means of a 400 μm fiberoptic probe, coupled to a spectrometer
(Model S2000, Ocean Optics, FL,
USA), placed in gentle contact with the tissue surface,
and compared with PpIX fluorescence spectra from CNS-1luc cell lysates measured using a spectrofluorimeter (SpectraMax
M5; Molecular Devices, CA, USA) at 405 nm excitation after incubation
with 1 mM ALA for 4 hours. FGR was performed by positioning the
laparoscope tip at 2 cm above the surgical site. Tumor was identified by areas
of evident red fluorescence and resected by means of suction through a glass
tip with 0.5 mm inner diameter. Spectral images were acquired during tumor
resection, which was terminated when red fluorescence was no longer detectable
on the video monitor. The resected tissue was fixed in 10% formalin for sectioning
H&E staining. Following resection, the presence of residual tumor cells was
assessed semiquantitatively by applying 100 μL of 1 mM luciferin (Xenogen, Alameda, CA,
USA) in PBS as the substrate for bioluminescence, which was observed in vivo using a commercial
bioluminescence imaging system (IVIS, Xenogen).  Immediately afterwards, the animals were
sacrificed by intracardiac injection of 1 mg kg−1 of sodium
pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, ON, Canada),
and the whole brain was removed intact for sectioning and H&E staining
for histopathological assessment.


## 2.1. System Description

The fluorescence imaging system was designed primarily for surgical guidance and is shown in Figure1. The
light source comprises a 300 W xenon lamp (Cermax, Perkin Elmer, CA, USA) focused
through a filter wheel into a 5 mm diameter liquid light guide (Model 495 FR, Karl Storz, Tuttlingen,
Germany) that is coupled into a 10 mm diameter rigid clinical laparoscope (Model
871 1AA, Karl Storz, Tuttlingen
Germany). The collection component of the imaging system is
based on a 3-CCD compact camera (DXC-C33, Sony, ON, Canada) operating at 30 frames s−1(NTSC) with 796×494 pixels and 8-bit dynamic range. A long-pass 500 nm filter
(Custom made, Chroma Technologies, VT, USA)
is placed between the camera and the laparoscope. This filter deliberately
leaks a small fraction (∼10−4) of the UV/blue excitation light to allow
measurement of the diffuse reflectance signal from the tissue for the
ratiometric algorithm described below. Spectral response curves for the red,
green, and blue channels of the CCD were measured using a
standard color target (ColorChecker Chart: Gretag Macbeth, Grand
Rapids, MI, USA) illuminated by a tungsten-halogen light
source (LS-1: Ocean Optics, Dunedin, FL, USA). For this, the images of the
color target were taken through the laparoscope without the fluorescence
emission filter installed. Serial filters spanning from 420 to 750 nm were
placed in front of the laparoscope lens for each of the color target images.
The spectral response of the camera-laparoscope optical chain was determined
and averaged from 4 spectrally-neutral shades.(a) Photograph and (b) schematic of the system.
(a)(b)The fluorescence imaging system features a dual-excitation capability. The filter
wheel holds two different excitation filters, and its rotation rate is such
that consecutive camera frames are excited by alternating excitation
wavelengths. For the present study, the
excitation filters had central wavelengths,λex1, at the PpIX Soret maxima of 405 nm (full width at half maximum, FWHM = 96 nm) and at 440 nm (FWHM = 60 nm). The
latter was chosen as the shortest wavelength lying above the main PpIX Soret
band in order to allow for correction for the tissue autofluorescence. It is
assumed then that the tissue optical properties are comparable at these two
wavelengths, since they are not too far apart. Both wavelengths can induce high
levels of autofluorescence in tumor and normal tissues. The delivered power at
each wavelength was approximately 50 mW cm-2 at a typical working distance of 2 cm from the
tissue surface. The digital video output is captured by a laptop computer and
can be displayed for visualization of the processed fluorescence images in real
time. The image processing software (Hytek
Automation, Waterloo, ON,
Canada) was based on LabVIEW
(National Instruments Corp., Austin,
TX, USA) and could execute multiplication,
division, addition, or subtraction on each channel.


## 2.2. System Characterization

The spatial resolution of the
imaging system was determined using a standard 1951 USAF glass slide resolution
target (Edmund Optics, NJ, USA) placed 2 cm from the front end of the
laparoscope, resulting in a field of view of12.7×8.2 mm. To characterize the
system performance for fluorescence imaging, tissue-simulating liquid phantoms
were prepared, comprising PpIX (Sigma-Aldrich, ON,
Canada) dissolved in 1 mM dimethyl sulfoxide (DMSO) with methylene blue dye added
as the optical absorber and the lipoprotein emulsion Intralipid (Fresenius Kabi, Uppsala, Sweden)
to provide optical scattering [42].
The corresponding absorption and reduced scattering coefficients are given in Table 1, based on well-established literature values. The system sensitivity was
measured using varying PpIX concentrations: 2.5, 1.25, 0.62, 0.31, 0.15, 0.075, and 0.039 μg mL−1. At each concentration,
fluorescence images were taken at both excitation wavelengths at distances of
2, 3, 4, or 5 cm from the phantom surface, with the camera
focused at the 2 cm working distance. The signal in the red channel of the 3-CCD
was plotted as a function of PpIX concentration. The fluorescence signal was
collected at 0°, 15°, or 30° from the vertical axis to
determine the influence of the imaging geometry.Table 1
Optical properties of the liquid phantoms at 635 nm.
Reduced scatteringAbsorption coefficientcoefficientμs’ (cm−1)μa (cm−1)Phantom A71.6Phantom B73.2Phantom C141.6Phantom D143.2The ratiometric method applied here was developed
by our group previously and is based on using 2 excitation and 2 emission
wavelengths [41]. The first
excitation wavelength is in the absorption peak of PpIX (λex1=405 nm) and, for each image pixel,
the emitted red fluorescence (560–750 nm) is
divided by the signal from the diffusely reflected excitation light. Next, this
fluorescence/reflectance ratio is divided by the same ratio calculated using the
second-excitation wavelength (λex2=440 nm). Thus, the signal, Q,
in each pixel is given by(1)Q=F(λex1,λem1)R(λex1)×R(λex2)F(λex2,λem1).


## 2.3.In Vivo Tests

To test the feasibility and functionality of this imaging system and
the double-ratio algorithmin vivo,
PpIX fluorescence and diffuse reflectance images were acquired in an
established rat brain tumor model [39]
undergoing fluorescence-guided tumor resection. The rat glioma tumor
model, CNS-1, was chosen as being highly infiltrative, a common characteristic
of high-grade human gliomas that should present a valid test of the ability to detect residual tumor at the
surgical margins. FGR was performed at 21 days following implantation of
luciferase-transfected CNS-1luc cells (3×105 cells, 2 mm below the dura, n=2 per ALA dose) in the left brain hemisphere of Lewis rats (Charles River, MA,
USA). PpIX was induced by administering aminolevulinic acid (ALA) in
hydrochloride form (Sigma, Oakville, ON, Canada).
This was dissolved in phosphate-buffered saline (20 mg mL−1) with the pH adjusted to ~5.5 by 1N NaOH and then injected intraperitoneally (i.p.) at 20, 50, or 100 mg kg−1 at 2–4 hours before the surgical
procedure/imaging. The animals were subdued by an i.p.
mixture of ketamine and xylazine (80 and 13 mg kg−1, resp.).
A 2 cm incision was made in the scalp along the midline and held open by a
retractor. A 1 cm craniotomy was performed using a burr drill,
and the dura was carefully removed. To measure the uptake of PpIX in
tumors, PpIX fluorescence spectra (450–750 nm) were
acquired by means of a 400 μm fiberoptic probe, coupled to a spectrometer
(Model S2000, Ocean Optics, FL,
USA), placed in gentle contact with the tissue surface,
and compared with PpIX fluorescence spectra from CNS-1luc cell lysates measured using a spectrofluorimeter (SpectraMax
M5; Molecular Devices, CA, USA) at 405 nm excitation after incubation
with 1 mM ALA for 4 hours. FGR was performed by positioning the
laparoscope tip at 2 cm above the surgical site. Tumor was identified by areas
of evident red fluorescence and resected by means of suction through a glass
tip with 0.5 mm inner diameter. Spectral images were acquired during tumor
resection, which was terminated when red fluorescence was no longer detectable
on the video monitor. The resected tissue was fixed in 10% formalin for sectioning
H&E staining. Following resection, the presence of residual tumor cells was
assessed semiquantitatively by applying 100 μL of 1 mM luciferin (Xenogen, Alameda, CA,
USA) in PBS as the substrate for bioluminescence, which was observed in vivo using a commercial
bioluminescence imaging system (IVIS, Xenogen).  Immediately afterwards, the animals were
sacrificed by intracardiac injection of 1 mg kg−1 of sodium
pentobarbital (Euthanyl, MTC Pharmaceuticals, Cambridge, ON, Canada),
and the whole brain was removed intact for sectioning and H&E staining
for histopathological assessment.


## 3. Results

### 3.1. System Performance

Figure2(a) shows the spectral response
of the CCD camera in each of the 3 channels. The spatial modulation transfer
function (MTF), measured using the resolution pattern at a working distance of
2 cm from the laparoscope tip, is shown in Figure 2(b).
The spatial resolution, defined at an MTF value of 50%, is 0.12 mm (8.5 line
pairs per mm).(a) Spectral
response curves for the red, green, and blue channels of the 3-chip CCD, (b) fluorescence
modulation transfer function measured at 2 cm working distance. The
solid line is a logarithmic fit to the experimental data points.
(a)(b)


### 3.2. Efficacy of the Ratiometric Algorithm

The red-channel signal, corresponding to PpIX fluorescence, is plotted as a function of PpIX
concentration for phantoms of different optical properties in Figure3(a)
and for different laparoscope-tissue distances in Figure 3(c). These demonstrate the strong dependence
of the “raw” fluorescence signal on the tissue characteristics (due to attenuation of the excitation
and fluorescent light) and imaging geometry, confirming the need to apply corrections to the data.
Figures 3(b) and 3(d) show that the double-ratio method markedly reduces these differences,
for example, at 2.5 μg mL−1 (i.e., the highest concentration used), the relative standard
deviations of the fluorescence signal (i.e., stdev/mean * 100%) are 26.1%, 13.1%, 28.2%, and 10.1%
for Figures 3(a)–3(d), respectively. Figure 4 shows the corresponding data for varying angle between
the laparoscope and the tissue surface.(a), (c) Relative
PpIX fluorescence intensities as a function of PpIX concentration before and (b), (d) after
application of the double-ratio algorithm, measured in the phantoms under
different conditions. Linear regression fits to the combined data are shown in
the corrected plots.
(a)(b)(c)(d)Relative PpIX fluorescence
intensities as a function on position on the CCD image (centered at the 400
pixel value), showing how this varies with the angle between the optical axis
(i.e., laparoscope direction) and the tissue surface, for the different optical
properties: (a) 0°, (b) 15°, (c) 30°. Graphs on the
left are the uncorrected data; those on the right are after applying the double-ratio
algorithm (with linear fits to the combined data). In each plot, the signals
are summed along the axis perpendicular to the x-axis of the graphs on the 3D
CCD array. All measurements were made at 2 cm working distance.
(a)(b)(c)


### 3.3. Biological Studies

In vitro and in vivo spectroscopic analysis of CNS-1 cells and tumors after
administration of ALA
for 4 hours (1 mM and 100 mg kg−1, resp.) confirmed
the presence of the PpIX fluorescence peak at 635 nm (see Figure 5(a)).
Figures 5(b)–5(d) show examples that demonstrate the quality of the fluorescence images
obtained with the system. Evident red fluorescence was observed from tumor areas at all ALA doses,
demonstrating the capability to discriminate between tumor and normal brain tissues. The
tumor-associated fluorescence increased from the lowest to the highest ALA dose, roughly
proportionally. The normal brain showed no such trend. However, there was high
variability in the fluorescence signals that was likely due in part to the
small number of animals used in this feasibility study and in part
to the intrinsic heterogeneity of the tumor model. The imaging system also
detected diffuse and specular reflectance in the blue component, which aids in overall
tissue orientation during surgery. No significant differences in
PpIX fluorescence were found in normal brain tissues between the tumor-bearing
and contralateral hemispheres (data not shown).(a) Normalized PpIX fluorescence
spectra of tumor and normal contralateral brainin vivo, 4 hours after i.p. administration
of 100 mg kg−1 of ALA, and corresponding spectra in CNS-1 cells in vitro
compared to controls (no ALA).  The insert shows the excitation and emission
spectra of PpIX in solution. (b)–(d) In vivo
fluorescence images of PpIX in tumor after i.p. injection of 20, 50, and 100 mg kg−1 ALA, respectively. (e) PpIX fluorescence from tumor-bearing animals 4 hours after
injection of different ALA doses in tumor and contralateral normal brain (means ±1
standard deviation: N=2).
(a)(b)(c)(d)(e)We also tested the system’s ability to monitor fluorescence-guided resection of tumor. As
illustrated by the example in Figure6, it was possible in all animals to
completely resect the visible fluorescing (tumor) tissue, as confirmed by
histopathology (see Figure 7(c)).
Despite this, bioluminescence imaging (see Figures 7(a)-7(b)) indicated
the presence of small amounts of residual tumor, corresponding to ~100 cells at or close to the resection surface (as
estimated from the bioluminescence signal).(a), (c) Example ofin vivo PpIX fluorescence (λex: 405 nm) and (b), (d) white
light images in the tumor resection cavity pre (a), (b)
and post fluorescence-guided resection (c), (d). Surgery was performed 4 hours
after ALA
injection (100 mg kg−1, i.p.). The blue areas represent specular
reflection from the tissue surface.
(a)(b)(c)(d)Example ofin vivo bioluminescence imaging pre (a) and post (b) fluorescence-guided resection. The bioluminescence imaging of residual
CNS-1luc cells immediately after FGR corresponds to approximately 100 cells detected at the surface of
the resection cavity. (c) H&E stained tissue section
from the red-positive region in Figure 6(a). The arrows
show nests of tumor cells.
(a)(b)(c)


## 3.1. System Performance

Figure2(a) shows the spectral response
of the CCD camera in each of the 3 channels. The spatial modulation transfer
function (MTF), measured using the resolution pattern at a working distance of
2 cm from the laparoscope tip, is shown in Figure 2(b).
The spatial resolution, defined at an MTF value of 50%, is 0.12 mm (8.5 line
pairs per mm).(a) Spectral
response curves for the red, green, and blue channels of the 3-chip CCD, (b) fluorescence
modulation transfer function measured at 2 cm working distance. The
solid line is a logarithmic fit to the experimental data points.
(a)(b)


## 3.2. Efficacy of the Ratiometric Algorithm

The red-channel signal, corresponding to PpIX fluorescence, is plotted as a function of PpIX
concentration for phantoms of different optical properties in Figure3(a)
and for different laparoscope-tissue distances in Figure 3(c). These demonstrate the strong dependence
of the “raw” fluorescence signal on the tissue characteristics (due to attenuation of the excitation
and fluorescent light) and imaging geometry, confirming the need to apply corrections to the data.
Figures 3(b) and 3(d) show that the double-ratio method markedly reduces these differences,
for example, at 2.5 μg mL−1 (i.e., the highest concentration used), the relative standard
deviations of the fluorescence signal (i.e., stdev/mean * 100%) are 26.1%, 13.1%, 28.2%, and 10.1%
for Figures 3(a)–3(d), respectively. Figure 4 shows the corresponding data for varying angle between
the laparoscope and the tissue surface.(a), (c) Relative
PpIX fluorescence intensities as a function of PpIX concentration before and (b), (d) after
application of the double-ratio algorithm, measured in the phantoms under
different conditions. Linear regression fits to the combined data are shown in
the corrected plots.
(a)(b)(c)(d)Relative PpIX fluorescence
intensities as a function on position on the CCD image (centered at the 400
pixel value), showing how this varies with the angle between the optical axis
(i.e., laparoscope direction) and the tissue surface, for the different optical
properties: (a) 0°, (b) 15°, (c) 30°. Graphs on the
left are the uncorrected data; those on the right are after applying the double-ratio
algorithm (with linear fits to the combined data). In each plot, the signals
are summed along the axis perpendicular to the x-axis of the graphs on the 3D
CCD array. All measurements were made at 2 cm working distance.
(a)(b)(c)


## 3.3. Biological Studies

In vitro and in vivo spectroscopic analysis of CNS-1 cells and tumors after
administration of ALA
for 4 hours (1 mM and 100 mg kg−1, resp.) confirmed
the presence of the PpIX fluorescence peak at 635 nm (see Figure 5(a)).
Figures 5(b)–5(d) show examples that demonstrate the quality of the fluorescence images
obtained with the system. Evident red fluorescence was observed from tumor areas at all ALA doses,
demonstrating the capability to discriminate between tumor and normal brain tissues. The
tumor-associated fluorescence increased from the lowest to the highest ALA dose, roughly
proportionally. The normal brain showed no such trend. However, there was high
variability in the fluorescence signals that was likely due in part to the
small number of animals used in this feasibility study and in part
to the intrinsic heterogeneity of the tumor model. The imaging system also
detected diffuse and specular reflectance in the blue component, which aids in overall
tissue orientation during surgery. No significant differences in
PpIX fluorescence were found in normal brain tissues between the tumor-bearing
and contralateral hemispheres (data not shown).(a) Normalized PpIX fluorescence
spectra of tumor and normal contralateral brainin vivo, 4 hours after i.p. administration
of 100 mg kg−1 of ALA, and corresponding spectra in CNS-1 cells in vitro
compared to controls (no ALA).  The insert shows the excitation and emission
spectra of PpIX in solution. (b)–(d) In vivo
fluorescence images of PpIX in tumor after i.p. injection of 20, 50, and 100 mg kg−1 ALA, respectively. (e) PpIX fluorescence from tumor-bearing animals 4 hours after
injection of different ALA doses in tumor and contralateral normal brain (means ±1
standard deviation: N=2).
(a)(b)(c)(d)(e)We also tested the system’s ability to monitor fluorescence-guided resection of tumor. As
illustrated by the example in Figure6, it was possible in all animals to
completely resect the visible fluorescing (tumor) tissue, as confirmed by
histopathology (see Figure 7(c)).
Despite this, bioluminescence imaging (see Figures 7(a)-7(b)) indicated
the presence of small amounts of residual tumor, corresponding to ~100 cells at or close to the resection surface (as
estimated from the bioluminescence signal).(a), (c) Example ofin vivo PpIX fluorescence (λex: 405 nm) and (b), (d) white
light images in the tumor resection cavity pre (a), (b)
and post fluorescence-guided resection (c), (d). Surgery was performed 4 hours
after ALA
injection (100 mg kg−1, i.p.). The blue areas represent specular
reflection from the tissue surface.
(a)(b)(c)(d)Example ofin vivo bioluminescence imaging pre (a) and post (b) fluorescence-guided resection. The bioluminescence imaging of residual
CNS-1luc cells immediately after FGR corresponds to approximately 100 cells detected at the surface of
the resection cavity. (c) H&E stained tissue section
from the red-positive region in Figure 6(a). The arrows
show nests of tumor cells.
(a)(b)(c)


## 4. Discussion

The ability to accurately quantify fluorescence signals in optically-turbid media such
as tissue is challenging, due to its complex dependence on many factors in
addition to the fluorophore concentration, and in particular the tissue
absorption and scattering properties, the measurement geometry and the background
tissue autofluorescence. These issues have been discussed in many papers, as
summarized by Bradley and Thorniley [36]
and Bogaards et al. [41].
Several different fluorescence imaging systems have also been developed for in vivo applications, including the purpose of guiding tumor surgery and particularly
in the brain. The intent in developing the present instrument was two-fold.
Firstly, we wished to make a device that is compact and independent of any
other surgical instrumentation. The approach implemented by Stummer and
colleagues [18, 22, 43], for
example, is to integrate fluorescence capabilities into an operating
microscope. This certainly is a valid approach and has advantages in terms of
ease of use, but limits its general surgical applicability. We intend in the
near future to carry out a direct comparison of the performance of the present
open-field device with the through-microscope technique. The second intent was
to implement the double-wavelength ratiometric algorithm. While single-ratio
correction has been used in other studies as a method to improve the
information content in
fluorescence-based diagnostics (e.g., R1=F(λex1,λem1)/R(λex1),
or R2=F(λex2,λem2)/R(λex2)) [32, 39, 44], the 2-excitation/2-detection wavelengths
algorithm developed previously by our group [41] and implemented here appears to be particularly effective in
minimizing the effects of tissue autofluorescence, geometric effects,
and optical attenuation. The phantom studies presented above on the latter two
factors confirm our previous modeling study of the efficacy of different
correction algorithms [41]. In
particular, including the reflectance signal (by allowing a
small fraction of the excitation light to leak through the excitation filter)
minimizes the dependence on variations in autofluorescence. As shown by the
phantom studies (see Figures 3 and 4),
the algorithm also largely removes the dependence on the tissue optical properties
and imaging geometry, since the effects of these factors are similar at the 2
excitation wavelengths, so that they are largely cancelled by taking the ratio
of the signals at these wavelengths, which aids in applying an objective and quantitative
criterion (e.g., a threshold concentration) to differentiate between diseased
and non-diseased tissues. This overcomes a significant weakness
of previous studies, including clinical trials, in which only subjective and
qualitative criteria were applied [45].The principal advantage of using a 3-chip CCD camera in this device is that the
red, green, and blue components can be independently augmented or
attenuated in the resulting image, depending on the spectral band that exhibits
the highest contrast between tumor and normal tissue. Custom software
integrates the dual excitation and RGB components into a real-time (video rate)
composite that can be tailored to enhance a large number of different fluorophores.The spectral images of PpIX in phantoms andin
vivo demonstrated excellent spatial resolution and contrast for visualization
of residual tumors and the margins between normal and tumor tissues (see Figure 5). Hence, the technology should “extend the surgeon's eye” in the identification
and localization of tumor tissue. In particular, the system should work better
than simply imaging the uncorrected fluorescence signal, especially in areas of
low fluorescence intensity, due to reduction of the dependence on tissue autofluorescence.
Again, this will be evaluated by head-to-head comparisons in phantoms, animal
models, and patients.One of the most exciting possibilities for this method of ALA-PpIX fluorescence
imaging for guiding brain tumor resection is to combine it with photodynamic therapy
(PDT), and several groups, including our own, are pursuing
this approach [20, 39]. The
advantage of ALA-PpIX is that the same agent can serve both purposes, since
PpIX is also well established as a PDT sensitizer. Further, since the PpIX is
endogenously synthesized, we and others have shown its high selectivity for
brain tumor relative to normal brain tissues, especially white matter [46–48].As indicated in the Introduction, ALA-PpIX-based FGR has been shown to
improve the completeness of glioma resection compared to standard white-light
visualization [18–21]. However, the present study (see Figure
7), and earlier work in a different brain
tumor model [49] using bioluminescence post
resection, show the likelihood of there being small amounts of residual tumor
tissue that is below the fluorescence detection threshold. Hence, PDT applied
intraoperatively immediately after resection should offer an additional level
of tumor destruction and such combination studies are in progress, both
preclinically [20, 42] and in
human trials [50].In conclusion, to date this instrument has met our objectives of a free-standing, easy-to-use,
and highly sensitive intraoperative
fluorescence imaging system for improved tumor resection. The phantom and
previous modeling studies [41] have demonstrated the
effectiveness of the double-ratio algorithm. Although limited in extent, the
initial in vivo studies
presented here have demonstrated the technical feasibility of the system to be
used for fluorescence image-guided resection. Work is in progress in the CNS-1luc brain tumor model using the system to optimize the ALA
dose and the time interval between its
administration and the resection to give the highest sensitivity and
specificity. We have also confirmed the technical feasibility of the instrument
in preliminary clinical tests in patients carried out during prostatectomy and
in assessment of recurrent vulvar malignancy (data not shown). Systematic
clinical tests are in progress or planned for a number of different tumor sites.



---

*Source: 532368-2008-10-16.xml*