Combined Video Analysis of ICG and 5-ALA Induced

Protoporphyrin IX and Hemoglobin Oxygen Saturation in near

Infrared

T. A. Savelieva

1,2

, D. M. Kustov

, P. V. Grachev

1,2

, V. I. Makarov

, E. E. Osipova

and

V. B. Loschenov

1,2

Prokhorov General Physics Institute of the Russian Academy of Sciences, Vavilov st., Moscow, Russia

National Research Nuclear University MEPhI, Kashirskoe sh., Moscow, Russia

Keywords: Indocyanine Green, Protoporphyrin IX, Haemoglobin Oxygenation, Fluorescence, Diffuse Reflectance.

Abstract: Due to the high recurrence rate after the glial brain tumor removal, methods of intraoperative navigation

have a high relevance, providing the most complete removal of tumor tissues with maximum preservation of

healthy ones. In this work a combined visualization method is proposed with an assessment of fluorescence

and diffuse reflectance images. Fluorescence intensity of 5-ALA-induced protoporphyrin IX allows

visualization of tumor cells, distribution of indocyanine green fluorescence helps to visualize the vascular

system of the tumor, and parallel mapping of the degree of oxygenation demonstrate the hypoxic regions.

The images were obtained in the near infrared range of the optical spectrum in order to maximize the optical

probing depth in the window of biological transparency.

1 INTRODUCTION

This paper presents a video analysis technique,

which allows parallel-sequential implementation of:

- simultaneous registration of fluorescent

intraoperative images of tumors and surrounding

tissues in two wavelengths, corresponding to the

fluorescence of protoporphyrin IX, which

characterizes the activity of metabolic processes in

cells, and indocyanine green fluorescence,

characterizing the parameters of blood supply to the

tissue with the display in the overlay mode of two

fluorescent patterns;

- registration of intraoperative images of tumors and

surrounding tissues in diffusively reflected light at

wavelengths characterizing the ratio of oxy- and

deoxyhemoglobin to assess the degree of hypoxia of

different sections of tumor tissue.

Currently there are no systems for simultaneous

visualization of fluorescence of indocyanine green

and protoporphyrin IX in clinical practice, however,

these modes are available sequentially in the Zeiss

Opmi Pentero microscope, but there are no oxygen

saturation mapping regime. The possibility of a

multi-factorial intraoperative assessment of the

tumor state can improve the accuracy of resection.

There are a number of works on simultaneous

registration of the photosensitizer fluorescence

distribution and the image of the same area in

diffusively reflected broadband light to observe the

fluorescent tissues on the full color background

image and thereby improve navigation (Sato et al.,

2015; Loshchenov et al., 2018). In other side the

hemoglobin oxygenation visualization of brain

tissues by optical methods are performed in two

ways as a rule. Oximetric video systems in the

visible or near infrared spectral range are based on

mathematical processing of full-color images

(Mustari et al., 2018) or hyperspectral imaging

(Giannoni et al., 2018). But using the near infrared

region for this purpose is limited as a rule by the

mapping of spectroscopic signals obtained from

specific points of the cortex projection from a set of

fiber optic probes, as in Hitachi, fNIR Devices LLC,

Biopac, Artinis systems for functional near infrared

spectroscopy. These systems are designed for

functional transcranial spectroscopy in the near

infrared, while proposed system is intended for

intraoperative imaging of the tumor bed. And it has

such an important advantage over visualization

systems in the visible range as work in the biological

transparency window. As it is well known, in near

320

Savelieva, T., Kustov, D., Grachev, P., Makarov, V., Osipova, E. and Loschenov, V.

Combined Video Analysis of ICG and 5-ALA Induced Protopor phyrin IX and Hemoglobin Oxygen Saturation in near Infrared.

DOI: 10.5220/0007693703200324

In Proceedings of the 7th International Conference on Photonics, Optics and Laser Technology (PHOTOPTICS 2019), pages 320-324

ISBN: 978-989-758-364-3

infrared range of optical spectrum the absorption of

main tissue chromophores is minimal so the light

can penetrate deeper in the tissue. But in this optical

range we should consider the prevalence of

scattering under the absorption in tissue.

2 MATERIALS AND METHODS

2.1 Device

The four laser light sources were used. The source

with 635 nm wavelength was used for PpIX

fluorescence excitation. The source with 785 nm

wavelength was used for ICG fluorescence

excitation. The 687 nm laser light source was used

for getting diffuse reflectance image in the position

where deoxygenated hemoglobin absorption

significantly higher than oxygenated hemoglobin

absorption. The source with 805 nm wavelength was

used to obtain diffuse reflectance image in the

position close to isosbestic point of deoxygenated

and oxygenated hemoglobin absorption spectra. Two

video cameras were used for parallel-serial data

reception in two modes: fluorescence and diffuse

reflectance image registration.

In fluorescence mode in front of the cameras were

installed such optical band-pass filters as 710/40

Semrock for Pp IX fluorescence registration and

832/37 Semrock for ICG fluorescence registration.

In diffuse reflectance mode in front of the cameras

were installed 685/40 Semrock and 810/10 Semrock

band-pass filters to eliminate cross light from these

spectrum ranges.

The control of oxygenation level was performed

with fiber-optic spectroscopy technique with LESA-

01-BIOSPEC using algorithm described earlier

(Stratonnikov et al., 2006). The control of

fluorescence intensity was carried out with the same

spectroscopic device in fluorescence mode.

2.2 Diffuse Reflectance Image

Processing Algorithm

Schmitt and Kumar, (Schmitt and Kumar, 1996;

Kumar and Schmitt, 1997), demonstrated the

dependence of the diffuse reflectance spectrum in

the near infrared region for tissue with different

optical properties on the distance between the

illuminating and receiving fibers, using the diffusion

approximation to approximate the obtained

dependences. For the fluorescence spectra, it is usual

to assume that measuring the diffuse reflectance

spectrum of tissues in the same range as

fluorescence one is sufficient to take into account

the effect of the sample geometry. However, as it

was shown in Gebhart et al., 2007, the spectra of

diffuse reflectance and fluorescence show significant

differences when registering by the method of point

to point spectroscopy in contact with the sample and

at a certain distance, as well as by the method of

spectrally resolved visualization. An increase in the

contrast between the red and blue-green ranges of

the diffuse reflectance spectrum was found for the

illumination-reception geometry implemented in the

visualization system and point spectroscopy at a

certain distance from the sample. This effect is most

likely due to the low contribution of multiply

scattered photons in case of proximity of light

source and receiver. Differences in the diffuse

reflectance spectrum from samples containing

hemoglobin in various concentrations were greater

for the imaging system compared to the point

spectroscopy system, but they had similar trends. At

the same time, the dependence of the diffuse

reflectance spectra of samples with different

contents of scatterers differed for the considered

geometries both in nature and in magnitude. In this

connection, in the present work, in order to consider

the influence of the measurement geometry, a

mathematical simulation was carried out for the

cases of the diffusely reflected and fluorescent

radiation registration at different distances between

the illumination and receiving fibers for different

numerical apertures.

2.3 Objects for Validation

Validation of proposed method of combined video

analysis and image processing algorithms was

carried out on an array of optical phantoms that

simulate the optical properties of nerve tissues

containing the studied photosensitizers with a

concentration 0.625 mg/l, 1.25 mg/l, 2.5 mg/l and 5

mg/l of indocyanine green and 1.25 mg/l , 2.5 mg/l,

5 mg/l and 10 mg/l of Pp IX. Photosensitizers were

diluted with the 1% fat emulsion solution as a

scattering agent to simulate multiple scattering

occurring in investigated tissues.

Phantoms were performed in the cuvettes. To

analyze their images in environments similar to the

natural brain, they were immersed in a scattering

gelatin matrix (5% gelatin with a 1% intralipid

solution).

Optical phantoms simulating biological tissue with

blood were also performed. Whole blood was

diluted 16 times so that the hemoglobin

concentration was approximately equal to the

Combined Video Analysis of ICG and 5-ALA Induced Protoporphyrin IX and Hemoglobin Oxygen Saturation in near Infrared

321

average concentration in the white matter of the

brain. Fat emulsion solution was used as a scattering

medium. The samples were deoxygenated through

nitrogen ventilation.

Testing of the developed method and system on

laboratory animals was also carried out in the

fluorescence mode (with simultaneous

administration of indocyanine green and

protoporphyrin IX) and in the diffuse reflectance

mode (to control the degree of oxygenation) before,

during and after deoxygenation of the animal’s

blood by nitrogen ventilation.

3 RESULTS AND DISCUSSION

3.1 Analysis of Co-distribution of ICG

and PP IX on Optical Phantoms

The results of the video system validation on optical

phantoms are shown in Figure 1 in the fluorescence

mode for fluorescence phantoms. In the left column

of images in black-white channel the laser light

source with 785nm wavelength for fluorescence

excitation and optical filter 832/37 nm for

fluorescence registration were used. In the right

column of images in color channel the laser light

source with 633 nm wavelength for fluorescence

excitation and optical filter 710/40 nm for

fluorescence registration were used. These two

pictures can be combined in one in special mode to

navigate during surgery.

Validation showed that the sensitivity of the

system in the fluorescence mode is no worse than

1.25 mg/l for indocyanine green detection and 0.125

mg/l for Pp IX detection without hardware signal

amplification in real time for fluorescent inclusions

in the scattering medium, whose transverse

dimensions do not exceed 1 mm.

3.2 Algorithm of Diffuse Reflectance

Image Processing with Separation

of Absorption and Scattering

Component

The diffuse reflectance spectra depends on scattering

as well as absorption of light by the tissue. If the

purpose of registering a diffuse reflectance signal is

to analyze the content of chromophores in a tissue

by their absorption, these two processes should be

separated. At the same time, information about

tissue scattering can be an independent diagnostic

criterion.

In a highly scattering medium, the radiation

corresponding to one pixel of the image is

characterized by a wide distribution of photon path

lengths in the tissue. Thus, the greater the scattering

compared to the absorption, the greater the influence

of the neighboring pixels on each other. In general

case, the solution for one pixel of the image is a

linear combination of solutions for a point source

and a receiver for the sum of possible photon paths

in the tissue.

As a result of numerical simulation of the

propagation of light in a high-scattering medium

with optical properties, which varied in a series of

physiologically relevant values, the following

approximating dependence of the diffuse reflection

signal (Rd) on absorption coefficient (µ

) and

scattering coefficient (µ’

) was proposed:





























































(1)

Here, A

and B

are adjustable parameters which

depend on the illumination and signal detection

geometry, μ'S, reduced scattering coefficient defined

as μ'

= μ

·(1 – g), g, scattering anisotropy factor,

and μа, absorption coefficient. Detailed description

of this approximation is given in earlier publication

(Savelieva et al., 2015).

is the signal that we receive from each camera

(on two wavelengths) in the diffuse reflection mode

of registration, taking into account the normalization

to the image of standard white illuminated with the

same broadband light source. Solving equation (1)

we get the values of the absorption coefficients on

chosen wavelengths.

The matrix for converting the concentrations of

tissue chromophores studied in this work to

absorption coefficients is as follows:





















































(2)

where µ

λi

– absorption coefficient on

wavelength λ

, ε

λi

– molar extinction coefficient of j

chromophore on wavelength λ

, C

– the

concentration of j chromophore.

Solving system of equations (2) we get the

values of deoxygenated hemoglobin concentration

(



) and oxygenated haemoglobin concentration

(



). From these parameters we can calculate the

deoxygenation parameter as:















(3)

This parameter was introduced in this work for

more convenient observation and it is additional to

PHOTOPTICS 2019 - 7th International Conference on Photonics, Optics and Laser Technology

322

the level of hemoglobin oxygen saturation; in total,

they give 1.

On Figure 2 we can see two pictures in diffuse

reflectance mode as an example of registered

images.

Figure 1: Imaging of distribution of ICG and Pp IX in

optical phantoms in fluorescence mode.

3.3 Analysis of Hemoglobin Oxygen

Saturation with Diffuse Reflectance

Visualization in NIR Region

The time dependences of the deoxygenation

parameter (the ratio of the deoxygenated

hemoglobin concentration to the total concentration

of hemoglobin) were obtained during nitrogen

ventilation of an optical phantom containing a

solution of whole blood in the scattering medium,

which served as calibration curves for analyzing

similar dependencies recorded in experiments with

laboratory animals. On Figure 2 we can see the

example of pictures in two wavelengths obtained

during in vivo experiment. Laboratory animals were

ventilated with nitrogen to achieve significant

changes in the deoxygenation parameter without

harming the animal's health.

Figure 2: Imaging of optical scattering phantoms with

haemoglobin in diffuse reflectance mode.

The curve of change in the deoxygenation

parameter of the animal brain depending on the

ventilation time is shown in Figure 3.

Figure 3: Analysis of deoxygenation in NIR in vivo.

Combined Video Analysis of ICG and 5-ALA Induced Protoporphyrin IX and Hemoglobin Oxygen Saturation in near Infrared

323

4 CONCLUSIONS

The video analysis technique for intraoperative

navigation during neurosurgery is proposed that

allows simultaneous registration of fluorescent

intraoperative images of tumors and surrounding

tissues at two wavelengths corresponding to the

fluorescence of protoporphyrin IX, which

characterizes the activity of metabolic processes in

cells, and fluorescence of indocyanine green, which

characterizes the parameters of tissue blood supply

with display in mode overlaying two fluorescent

pictures, as well as recording of intraoperative

images of tumors in diffusevely reflected broadband

light to measure the degree of tissue hypoxia.

ACKNOWLEDGEMENTS

The reported study was funded by RFBR according

to the research project № 17-00-00162 (K) (17-00-

00159) and partially supported by the

Competitiveness Program of MEPhI.

REFERENCES

Sato, T., Suzuki, K., Sakuma, J., Takatsu, N., Kojima, Y.,

Sugano, T., Saito, K., 2015, Development of a new

high-resolution intraoperative imaging system (dual-

image videoangiography, DIVA) to simultaneously

visualize light and near-infrared fluorescence images

of indocyanine green angiography.

ActaNeurochirurgica, Volume 157, Issue 8, pp 1295–

1301.

Loshchenov, M., Blondel, W., Amouroux, M., Steiner, R.,

Potapov, A., Golbin, D., Loran, O., Babaev, A.,

Borodkin, A., Daul, C., Trinh, D.-H., Loschenov, V.,

2018, Multimodal fluorescence imaging navigation for

surgical guidance of malignant tumors in

photosensitized tissues of neural system and other

organs, Proc. SPIE 10677, Unconventional Optical

Imaging, 106773N.

Mustari, A., Nakamura, N., Kawauchi, S., Sato, S., Sato,

M., Nishidate, I., 2018, RGB camera-based imaging of

cerebral tissue oxygen saturation, hemoglobin

concentration, and hemodynamic spontaneous low-

frequency oscillations in rat brain following induction

of cortical spreading depression, Biomed. Opt.

Express 9, 933-951.

Giannoni, L., Lange, F., Tachtsidis I., 2018, Hyperspectral

imaging solutions for brain tissue metabolic and

hemodynamic monitoring: past, current and future

developments. J. Opt. 20 044009

Stratonnikov, A.A., Meerovich, G.A., Ryabova, A.V.,

Savel'eva, T.A., Loshchenov, V.B., , 2006,

Application of backward diffuse reflection

spectroscopy for monitoring the state of tissues in

photodynamic therapy, Quantum Electronics, vol. 36,

no. 12, pp. 1103–1110.

Schmitt, J. M., and Kumar, G., 1996, Spectral distortions

in near-infrared spectroscopy of turbid materials.

//Appl. Spectrosc. 50, 1066–1073.

Kumar, G., Schmitt, J. M., 1997, Optimal probe geometry

for near-infrared spectroscopy of biological tissue.

//Appl. Opt., 36, 2286–2293.

Gebhart, S. C., Majumder, S. K., Mahadevan-Jansen, A.,

2007, Comparison of spectral variation from

spectroscopy to spectral imaging. //Applied Optics,

46(8).

Savelieva, T. A., Loshchenov, V. B., Goryainov, S. A.,

Shishkina, L. V., and Potapov. A. A., 2015, A

spectroscopic method for simultaneous determination

of protoporphyrin IX and hemoglobin in the nerve

tissues at intraoperative diagnosis, Russian Journal of

General Chemistry, vol. 85, no. 6, pp. 1549–1557.

PHOTOPTICS 2019 - 7th International Conference on Photonics, Optics and Laser Technology

324