CC BY
0DUALWAVE LASER ACTION ON BLOOD VESSELS: THEORY AND EXPERIMENT
VIKTOR CHUCHIN1,2, ANDREY BELIKOV1,3, ALEXANDRA MASHARSKAYA1,2 AND PAVEL PANCHENKO4
1Institute of Laser Technologies, ITMO University, Russia 2Sector of Medical Laser Technologies, "NPP VOLO" LLC, Russia 3Research Institute of Dentistry and Maxillofacial Surgery, Pavlov First St. Petersburg State Medical University, Russia 4Department of Upper Respiratory Tract Pathology, Saint-Petersburg Research Institute of Ear, Throat, Nose and Speech, Russia
ABSTRACT
Feedback in laser systems allows for real-time adjustment of laser parameters to achieve the required treatment outcomes more effectively [1]. One of the most widely implemented types of feedback in such systems is temperature-based feedback. This type of feedback monitors the thermal glow emitted from the heated tip of the optical fiber and ensures that the temperature remains constant [2, 3]. As biological tissue is subjected to increased heating, tissue proteins begin to denature, leading to irreversible structural changes. These alterations are critical, which is why it is important to identify markers indicating the exact temperature reached during laser exposure [4]. However, merely monitoring temperature does not fully capture the changes occurring in the tissue at the biochemical level.
Optical methods, in contrast, provide a more detailed insight into the chemical and structural transformations the tissue undergoes during and after thermal exposure. These methods could enable real-time tracking of the changes occurring in the tissues under laser irradiation. The foundation of an optical feedback mechanism could be based on monitoring the intensity of light reflected from the skin at specific wavelengths where the optical properties of the tissue show the most significant alterations due to laser heating.
During the heating process, reversible and irreversible biophysical processes occur in the blood that can have a pronounced effect on its optical properties. For instance, bound oxygen is released from hemoglobin, causing a transformation from oxyhemoglobin (HbO2) to deoxyhemoglobin (Hb), resulting in a reduction in blood oxygen saturation [5]. It is well known that this process begins at blood temperatures between 46°C and 48°C [6]. If the blood is further heated to temperatures between 65°C and 70°C, the iron within the hemoglobin molecules is oxidized from its ferrous (Fe2+) state to its ferric (Fe3+) state, thereby forming methemoglobin (MetHb). This thermal conversion of HbO2 to Hb and subsequently to MetHb occurs universally with any form of heating, including both continuous and pulsed laser irradiation [5]. At even higher temperatures, approximately 80°C, irreversible biochemical reactions lead to denaturation and coagulation of the hemoglobin [7].
In the visible and near-infrared regions of the spectrum, as the blood's oxygen saturation decreases, the light absorption by blood increases significantly in the wavelength ranges from 420 to 450 nm and 590 to 800 nm. However, absorption decreases in the wavelength range between 450 and 500 nm (Fig.1) [8]. Methemoglobin, a dysfunctional form of hemoglobin, is incapable of binding oxygen. Its absorption spectrum differs substantially from that of both oxyhemoglobin and deoxyhemoglobin [9]. Specifically, methemoglobin shows distinct absorption characteristics in the visible spectrum (415-630 nm) and in the infrared range (8001200 nm). This spectral shift can significantly impact the absorption profile of whole blood as hemoglobin is progressively replaced by methemoglobin [10]. Compared to oxyhemoglobin, methemoglobin exhibits its most significant reduction in the absorption coefficient at a wavelength of 577 nm, with a maximum increase at around 630 nm. In the case of deoxygenated hemoglobin, decreases and increases in absorption occur at 560 nm and 630 nm, respectively [11].
The changes in oxygen saturation levels and the replacement of hemoglobin by methemoglobin in the blood will clearly affect the intensity of light reflected from biological tissues. This phenomenon can be utilized to monitor changes in tissue optical properties and to optimize the parameters of laser treatments. For example, in transcutaneous laser sclerotherapy of telangiectasias, it is necessary to deliver the maximum amount of energy to the deeper layers of the skin without causing damage to the superficial layers. The ability to monitor reflected light intensity at wavelengths where the optical properties of biological tissue change most dramatically during laser heating could be a key element in the development of new laser systems and techniques with integrated feedback mechanisms.
The aim of the current study was to measure the reflection spectra of whole blood and rabbit skin before and immediately after laser exposure using wavelengths of 450 nm, 980 nm and their combination, includes evaluation and analyze the causes of changes in these reflection spectra.
An experimental setup was developed to perform laser heating of biological tissues using radiation at wavelengths of 450 nm, 980 nm, and their combination. The system also allowed for the measurement of reflection spectra within the range of 400-800 nm. Continuous wave diode lasers from the ALPH-01-"DIOLAN" series, manufactured by NPP VOLO LLC, were used as laser sources for both wavelengths. Considering the losses during light transmission, the maximum average laser power on the surface of the biological tissue reached 20 W at 450 nm and 90 W at 980 nm.
Figure 1: Absorption spectra of hemoglobin (Hb) [12], oxyhemoglobin (HbO) [12] and methemoglobin
(MetHb)
[9, 13]
For both laser sources, the diameter of the laser beam spot on the surface of the investigated objects was 6 ± 0.1 mm. To record the reflection spectrum of the objects, an Ocean Optics USB2000 fiber-optic spectrometer (Ocean Optics, Inc., USA) was used. A collimator, two diaphragms, and an electromechanical shutter were installed at the input end of the spectrometer's fiber. The diaphragms restricted the receiving aperture, ensuring that light from areas of the object not exposed to laser radiation did not reach the spectrometer. The electromechanical shutter, when closed, prevented the reflected laser radiation from entering the spectrometer's fiber. The spectrometer was operated in external trigger mode, with a fixed integration time of 50 ± 0.1 ms. The illumination source was a halogen lamp, and its radiation was directed through a liquid light guide with its output end aimed at the object under study. The electromechanical shutter was controlled using an Arduino DUE microcontroller, which had custom software developed specifically for this experimental setup.
For each sample of the studied object, the reflection spectrum before and immediately after laser exposure was normalized to the maximum intensity of the spectrum before laser exposure. To assess the changes in the reflection spectrum resulting from heating, the parameter AR was calculated according to equation 1:
AR=
(lafter'1 before) ,
'I (b
efore
(1)
where:
- IQjqfter intensity of light with wavelength reflected from biological tissue immediately after laser exposure,
-I(k)befo,-e intensity of light with wavelength □ reflected from biological tissue before laser exposure
Figure 2 presents a photograph of a rabbit's ear with the laser beam spot centered on the central auricular artery, along with the operating modes of the lasers. In the experiment, laser radiation was applied using either a single wavelength of 980 nm or 450 nm, as well as through a combined laser exposure using both wavelengths: first 980 nm followed by 450 nm, or vice versa. The duration of the laser pulse for both wavelengths, 980 nm and 450 nm, was constant at 150 ms. In the case of combined exposure, the time interval At between the start of the 980 nm and 450 nm pulses was varied, and At was set to 0, 150, or 300 ms.
To interpret the results, optical models of the mentioned biological tissue samples were used. These models were developed based on a previously created optical model of human skin [10] in the Trace Pro optical simulation software. This software allows the creation of three-dimensional models of optical systems, performs ray tracing using the Monte Carlo method, and analyzes the distribution of absorbed power within the system, taking into account scattering, reflection, absorption, and light anisotropy.
a)
0 50 100 150 200 250 300 350 400 450 t, ms
b)
Figure 2: Laser exposure site on a rabbit's pinna (a) and laser modes (b)
In the in vitro experiment, it was established that laser heating of a 300 ^m thick layer of rabbit venous blood using 980 nm radiation with a power of 90 W leads to a decrease in the reflectance of whole rabbit blood AR in the band with a maximum around 604 nm. According to the computer model, this can be associated with changes in blood oxygen saturation (Fig. 3).
'I(X)Before -I(VAfier -AR---AR=0
1 y
1 /
j r \ r
r_ \ .X
T r
g
1 _j f
f
— ^ JL * ~ £3
; V"
J /
g A f
* M
1 1 m
zL f_
ILf i1 r 1 1 f
/
Itak _ _ _ _ _ _ _ V _ _ _ __ _ ___ _ __ _
M — m --- r= — — — —4— - — — — - — - - --
400 500 600 700 800
Wavelength, nm
Figure 3: Reflection spectra of a rabbit venous blood sample in vitro before (blue line) and immediately after (red line) laser exposure (980nm, 150ms, 90W) and AR value for blood (green line)
11
In the in vivo experiment, it was found that immediately after laser exposure, the reflectance of rabbit ear skin increases across the entire measurable spectral range under all tested laser exposure modes (Fig. 4). For the single wavelength modes, 980 nm at 90 W (Fig. 4a) and 450 nm at 20 W (Fig. 4b), an increase in reflected light intensity was observed across the entire spectrum. However, in the combined modes (Fig. 4b-4g), a local minimum was observed around 604 nm. For the various laser exposure modes, the following changes in AR at 604 nm can be noted in the rabbit ear skin reflectance spectrum:
- for the 980 nm 90 W laser mode, an increase in reflectance intensity at 604 nm AR=0.011±0.001 is noted (Fig. 4a);
- for the 450 nm 20 W laser mode, an increase in reflectance intensity at 604 nm AR=0.007±0.001 is noted (Fig. 4b);
- for the simultaneous 980 nm and 450 nm laser exposure (At=0), an increase in reflectance intensity at 604 nm AR=0.029±0.002 is noted (Fig. 4c);
- for the mode where 980 nm is applied first, followed by 450 nm after At=150 ms, an increase in reflectance intensity at 604 nm AR=0.011±0.001 is noted (Fig. 4d);
- for the mode where 980 nm is applied first, followed by 450 nm after At=300 ms, an increase in reflectance intensity at 604 nm AR=0.012±0.001 is noted (Fig. 4e);
- for the mode where 450 nm is applied first, followed by 980 nm after At=150 ms, an increase in reflectance intensity at 604 nmzfi?=0.007±0.001 is noted (Fig. 4f);
■ I(X)before •
■I(l)afrer
•AR '
AR=0
• 1(1) before •
•I(X)after
•AR <
AR=0
0.1 1
0.05 0.9
s s
T. os
0 ps
0.7
-0.05 0.6
-0.1 0.5
400 500 600 700 Wavelength, nm
800
400 500 600 700 Wavelength, nm
-0.1
800
ps <i
a)
b)
• I(A)before •
- KDqfter
•AR •
AR=0
• I(X)before •
■I(l)after
•AR •
AR=0
0.05
-0.05
400 500 600 700 Wavelength, nm
800
ps
<1
0.05
400 500 600 700 Wavelength, nm
0.05
800
«
<1
C)
d)
■ I(X)before •
■I(X)after
•AR <
AR=0
• I(X)before •
■I(l)after
•AR •
AR=0
400 500 600 700 Wavelength, nm
e)
800
1 ■
0.05 0.9 ■
s 3 o.s -
B rt
0
<1 0.7 ■
-0.05 0.6 ■
-0.1 0.5 J
400 500 600 700 Wavelength, nm
f)
«
<1
800
■ I(l)before •
■ I(X)after
•AR
AR=0
£
400 500 600 700
Wavelength, nm
soo
Pi <1
Figure 4: Reflection spectra of a rabbit pinna skin in vivo before (blue line) and immediately after (red line) laser exposure and AR value for pinna skin (green line) (a-g) for the studied laser exposure modes 1-7,
respectively
- for the mode where 450 nm is applied first, followed by 980 nm after At=300 ms, an increase in reflectance intensity at 604 nm AR=0.008±0.001 is noted (Fig. 4g).
The changes mentioned above in the rabbit ear skin reflectance spectrum can be associated with the conversion of oxyhemoglobin into deoxyhemoglobin under the influence of laser radiation.
REFERENCES
[1] A.Zaj^c et al., "Real-time control procedures for laser welding of biological tissues," Bulletin of the Polish Academy of Sciences: Technical Sciences. 139-146, 2008.
[2] Belikov, A. V., Feldchtein, F. I. and Altshuler, G. B., "Dental surgical laser with feedback mechanisms, pat. US 2012/0123399 A1/ № 13/379,916; appl. 31.12.2010; pub. 17.05.2012," 1-34 (2012).
[3] G.E. Romanos, et al., "Uncovering dental implants using a new thermo-optically powered (TOP) technology with tissue air-cooling," Lasers in Surgery and Medicine. 411-420, 2015.
[4] Spliethoff, J. W., Tanis, E., Evers, D. J., Hendriks, B. H. W., Prevoo, W., & Ruers, T. J. M.. "Monitoring of tumor radio frequency ablation using derivative spectroscopy," Journal of Biomedical Optics. 097004, 2014.
[5] Kimel S. et al., "Influence of laser wavelength and pulse duration on gas bubble formation in blood filled glass capillaries," Lasers in Surgery and Medicine. 281-288, 2005.
[6] Verkruysse W. et al., "Optical absorption of blood depends on temperature during a 0.5 ms laser pulse at 586 nm," Photochemistry and photobiology. 276-281, 1998.
[7] Jia H., Chen B., Li D., "Dynamic optical absorption characteristics of blood after slow and fast heating," Lasers in medical science. 513-525, 2017.
[8] Bosschaart N. et al. "A literature review and novel theoretical approach on the optical properties of whole blood," Lasers in medical science. 453-479, 2014.
[9] Khatun F., Aizu Y., Nishidate I., "In Vivo Transcutaneous Monitoring of Hemoglobin Derivatives Using a Red-Green-Blue Camera-Based Spectral Imaging Technique," International Journal of Molecular Sciences. 1528-1545, 2021.
[10] Belikov A.V., Chuchin V.Yu., "Numerical study of the effect of methemoglobin concentration in the blood on the absorption of light by human skin," Scientific and Technical Journal of Information Technologies, Mechanics and Optics. 685-695, 2023 (in Russian).
[11] Jia H., Chen B., Li D., "Dynamic optical absorption characteristics of blood after slow and fast heating," Lasers in medical science. 513-525, 2017.
[12] Nachabe R., Evers D.J., Hendriks B.H.W., Lucassen G.W., Van der Voort M., Wesseling J., Ruers T.J.M. "Effect of bile absorption coefficients on the estimation of liver tissue optical properties and related implications in discriminating healthy and tumorous samples," Biomedical optics express. 600-614, 2011.
[13] Kuenstner J.T., Norris K.H. "Spectrophotometry of human hemoglobin in the near infrared region from 1000 to 2500 nm," Journal of Near Infrared Spectroscopy. 59-65, 1994.
