Introduction
Image-enhanced endoscopy is widely used for detection and diagnosis of diseases. The
principle of some diagnostic methods is recognition and classification of images,
which is based on an individual's experience. Filtered images are combined by using
image-processing techniques, which markedly improve visualization of lesions [1]. However, image-enhanced endoscopic detection and diagnosis of disease is still
based on each operator’s subjective judgment.
Recently, quantitative diffuse reflection spectroscopy for gastrointestinal examination
was developed for detection of noninvasive disease diagnosis [2]. The system uses hyperspectral imaging to successfully capture biological information
[3]. We previously reported that a large part of the diffuse reflection data of the
normal mucosa obtained by using this system were dominated by blood and scatter signals.
Furthermore, two-dimensional (2 D) mapping of relative hemoglobin (Hb) concentration
and Hb saturation mapping was also performed, which suggested its value for medical
use [4].
Relative Hb concentration and Hb saturation are useful for detection and diagnosis
of gastrointestinal disease. These data provide quantitative and objective information
relevant to biological activity. Associations of tumor hypoxia and vascularization
with malignant progression have been reported previously [5]. Despite reports of point and 2 D mapping of this information based on hyperspectral
data, there is no system as yet that provides real-time saturation video imaging for
practical use. Furthermore, the value of the information for clinical use, such as
for detection and diagnosis of lesions, is unclear.
Conventional hyperspectral data capture and analysis procedures are not always optimal
for use in a practical commercial system for assessing relative Hb concentration and
Hb saturation because of difficulty with the amount of data that can be captured,
difficulty ensuring light intensity, and complexity of postprocessing. In the current
study, we designed a simple process and algorithm to obtain Hb saturation and relative
Hb concentration. By capturing featured wavelength area data and reducing the calculation
load, we achieved a frame rate of 7.5 frames per second. We then designed and prototyped
an endoscopy system using this method that provided real-time relative Hb concentration
and Hb saturation mapping functionality. This simplified and practical system was
applied to relative Hb concentration and Hb saturation mapping of tumors in small
animal viscera in vivo.
Material and methods
Principle of the optical filter system
The Hb spectrum change during the oxy-Hb to deoxy-Hb transition is shown in [Fig. 1]. The basic spectra for saturation of 100 % oxygenated and 100 % deoxygenated Hb
are shown, and the spectral wavelength region from 524 to 582 nm is highlighted.
Fig. 1 Typical spectra of the oxy-hemoglobin (Hb; red) and deoxy-Hb (blue) spectrum 1.0 × 10−4 g/L.
Absorption data were derived from “Absorption of Hemoglobin by Scott Prahl, Oregon
Medical Laser Center.” https://omlc.org/spectra/hemoglobin/
An inverted change in transmittance between the isosbestic points, which were derived
from the oxy-deoxy transition, is observed, and a marked transmittance change between
524 and 582 nm was selected for this system. Measuring changes in the spectra corresponds
to change in the quantitative ratio of oxy-Hb and deoxy-Hb and can be analyzed by
using the spectral analysis method. The Hb saturation is defined by Eq. 1.
Single xenon light source and optical filter system were developed for our Hb concentration
and saturation mapping endoscope system, which is different from the previously reported
saturation mapping system in the concept of optical design [6]. The system has been certified by PMDA JAPAN (The Pharmaceuticals and Medical Devices
Agency; https://www.pmda.go.jp/english/symposia/0147.html) ([Fig. 2]). Red – green – blue image signals and narrow (544 to 570 nm), wide-filtered (524
to 582 nm) image signals were sequentially collected. By calculating the ratio of
the narrow-filtered signal to the wide-filtered signal of each pixel, we can obtain
the Hb saturation related value. The relationship of the ratio of the narrow and wide
filters with Hb saturation is shown in [Fig. 3a] and [Fig. 3b]. Using the values of the red, green, and blue signals and wide-filtered signals,
the Hb concentration was calculated without the influence of saturation. The Hb saturation
value was decided by using the ratio of the narrow-filtered signal to the wide-filtered
signal versus the Hb concentration curves.
Fig. 2 Whole image of endoscopy system.
Fig. 3 a Simulated Hb saturation versus narrow-filter area spectral data of Hb for different
Hb concentrations. b Simulated Hb saturation versus wide-filter area spectral data of Hb for different
Hb concentrations. The wide-filter data are independent of Hb saturation and dependent
on Hb concentration.
Calibration of instruments for quantitative analysis
Calibration curves for Hb oxygen saturation of the “spectrometer” versus “endoscope”
were prepared by using the experimental arrangement in [Fig. 4a] and least-squares method data analysis shown in [Fig. 4b]. The Hb saturation level of the blood solvent was controlled by a reducing agent,
and each saturation condition was measured by the two instruments.
Fig. 4 a Experimental arrangement of Hb saturation calibration. b Calibration curve of Hb saturation captured by the developed endoscope system. Spectral
data and curve-fitting analysis used to obtain y-axis versus standard data (x-axis).
As an experimental specimen, a 3 × 10−3 g/L blood solution was prepared in a vessel. The path length of the solution was
10 mm. The Hb oxygenation level was controlled using Na2S2O4 [7]. A filtered image and a red-green-blue video image were captured by the endoscope.
Additionally, spectral data from 400 to 800 nm at 1-nm resolution were also collected
by using a single-fiber spectrometer HR2000 (Ocean Optics) nearly at the same time.
The Hb saturation value for each instrument was calculated. A single-fiber spectrometer
(HR2000) detected incident light through the fiber at the measuring point. The endoscope
detected 968 × 496 points as image data, and the average of the center 100 points
area was used for the plot ([Fig. 4b]).
Experimental observation of a tumor in vivo
A small animal model of direct tenuous implantation of HLaC-79 human squamous cancer
cells was prepared as an observation model. Tumor growth was investigated using an
in vivo imaging system (IVIS Spectrum PerkinElmer Inc., Waltham, Massachusetts, United
States) prior to dissection. Relative Hb concentration mapping and Hb saturation mapping
for the in vivo tumor were performed by using our system. The Hb concentration and
saturation combined image was also acquired. The image combination technique uses
a mask-based Hb concentration distribution. We can select the concentration scale
and range depending on the individual's difference.
All animal care and experimental protocols were approved by the Animal Ethics Committee
of the Kyushu University Faculty of Medicine and were performed according to the recommendations
of the Committee for Care and Use of Laboratory Animals, Kyushu University Faculty
of Medicine. Male C57BL/6 mice, aged 5 weeks, were purchased from Charles River Laboratories
Japan, Inc. (Yokohama, Japan) and maintained in temperature- and light-controlled
chambers (24 °C, 12 h/12 h light-dark cycle). Prior to the experiments, all animals
were fed a normal diet for 1 week for acclimation and had free access to tap water
and appropriate food (MF diet; Oriental Yeast Co., Tokyo, Japan).
Results
Calibration curve
For evaluation of the function of our system, we obtained a calibration curve and
performed a correlation with the actual saturation values by using the Hb saturation
phantom ([Fig. 4b]). The Hb saturation calibration curve was obtained by using stable oxygenation level
samples in transparent containers. The standard error of the predicted y-value for
each x in the regression was 4.8 %.
Experimental observation of in vivo tumor
Hb saturation mappings of the small animal ligation model are shown in [Fig. 5a] and [Fig. 5b]. A decrease in oxygen saturation due to vascular ligation has been indicated in
green.
Fig. 5 a Experimental vessel ligation model. Red, green, and blue video endoscope image of
the mouse viscera. The ligation point is indicated by an arrow. b Hb saturation map of the ligation model mouse by the endoscope system. Lower hemoglobin
saturation area is indicated in green.
The small animal cancer model is shown in [Fig. 6a], [Fig.6b], [Fig. 6c], and [Fig. 6 d]. The red, green, and blue video endoscope image and saturation map and Hb concentration-masked
Hb saturation map are shown in [Fig. 6d]. The malignant disease area of the high Hb concentration and the low Hb saturation
is enhanced by this procedure.
Fig. 6 a Experimental cancer model. Red-green-blue video endoscope image of the mouse viscera.
The tumor area is indicated by an arrow. b Hb saturation map of the tumor generated by the endoscope system. c Hb concentration map of the tumor generated by the endoscope system. d Hb concentration-masked Hb saturation map. Higher Hb concentration area was enhanced
by an Hb concentration mask. Higher Hb concentration and lower Hb saturation area,
influenced by the tumor, were observed as a blue area.
Discussion
As suggested in previous studies, quantitative assessment based on spectroscopy of
Hb concentration and Hb saturation is one of the latest developments in real-time
endoscopy field [5]. We developed a concise and practical method based on this concept and tested its
functionality for current endoscope systems in clinical use
Optical design and analysis
Spectroscopy approaches for measurement of typical Hb concentration and Hb saturation
have been previously reported [5]
[6]
[8]. In the current study, we developed and validated an Hb concentration and saturation
mapping system for endoscopy. The system provides red-green-blue video movie imaging
and time-sequential Hb concentration and saturation map.
We selected an optical filter to integrate the intensity and simplify the postprocessing
procedure. The cut-on and cutoff wavelengths of the two optical filters were designed
to match the isosbestic points of Hb because the influence of cut-on and cutoff wavelength
errors in the filters are smaller than those of the center area of the peak ([Fig. 1]).
It is also important for spectral band selection to consider the influence of scatter.
Scatter in the defuse reflection data of the mucosa was previously reported in numerous
studies [9].
The scatter influence of mucosa is serious in the wavelength area of < 500 nm, and
the Hb saturation signal weakens as scatter level increases. For this reason, a > 500 nm
area spectral peak was selected. Furthermore, for calculation of the saturation related
value, the spectral baseline of the narrow-filtered signal (544 – 570 nm) and the
wide-filtered signal (524 – 580nm) were overlapped, and the influence of scatter in
the reflection spectra was minimized. The overlapped optical baseline helped generate
rational and reliable calculation results.
We developed a simplified calculation procedure based on (Equation 1, [Fig. 3]) and a combination technique for obtaining an Hb concentration-masked Hb saturation
map. This technique helped us to recognize an Hb concentration and saturation area
in one figure ([Fig. 6d]). In addition, the vascular pattern and its saturation value can be recognized effectively
with this method. This practical use of the imaging method loading in this system
should be indispensable for clinical research and applications. The mapping frame
rate of this system is comparable to those of white-light image systems; however,
to prevent artifacts, which are caused by image shift and deformation of the subject
itself, a higher frame rate is preferable (> 15 frames/sec), and improvement in capture
rate is necessary.
Future clinical applications
Measurement of relative Hb concentration and Hb saturation mapping is not limited
to endoscopy and can be used to monitor blood flow recovery and tissue oxygen supply
after surgery ([Fig. 5b]). In addition to providing quantitative assessment of blood status, these values
can be used to detect spatial abnormalities in tissues. Fusion analysis using scientific
instruments is also effective for increasing accuracy of diagnosis. Overall, we suggest
that future development of medical equipment use spectral imaging and analysis. For
example, 2 D Hb saturation surgical system, which can indicate tissue activity, can
be used. We hope that the results of this study will provide new criteria for detection
and diagnosis using endoscopy of various diseases and to improve gastroenterology
clinical outcomes [10].
Conclusion
In this study, we designed and validated an optical filter-equipped endoscope system
that enables 2 D visualization of Hb concentration and Hb saturation maps. Finally,
differences in oxygenation levels between normal mucosa and those of in vivo tumors
in a small animal model were determined by using our new endoscope system.
