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Abstract
Laser speckle contrast imaging (LSCI) can be used to observe dynamic changes in the tissue microcirculation in vivo according to the dynamic interaction between red blood cells and coherent light. In this study, a dual-wavelength LSCI system based on a microscope was developed for in vivo observation of the microvascular pattern and measurement of the blood flow change in the animal model. Additionally, based on the dual-wavelength setup, including 635 and 855 nm wavelengths, the oxygenation of biological tissue was evaluated. Finally, the developed LSCI microscope was implemented for the studies of tissue microcirculation. The results indicate that the developed LSCI microscope could be a potential tool for in vivo observation of the tissue microcirculation and quantitative evaluation of hemodynamics in animal experiments.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Laser speckle contrast imaging (LSCI) is a wild-field and noninvasive method for the observation of dynamic motion in biological tissue with spatial and temporal resolutions [1,2]. LSCI is able to quantify blood flow in various biological applications such as cerebral blood flow [3,4], skin [5,6], diabetes [7,8], and burn wounds [9,10]. The cause of speckle variance is due to the intensity variation induced by the moving particles in biological tissue and coherent light [11,12]. The moving particles in biological tissue could be the blood flow or moving red blood cells. Such intensity variation causes spatial blurring when the intensity is averaged over an integration period of the camera. Moreover, a previous report indicated that the autocorrelation time is inversely related to the blood flow velocity [13]. Therefore, the microvasculature of biological tissue can be obtained and the blood flow can be measured by estimation of the autocorrelation time. Moreover, previous reports also proposed the use of multiple laser diodes with different center wavelengths for the evaluation of tissue oxygenation [14,15]. A. K. Dunn et al. proposed the utilization of multiple wavelengths to achieve high-resolution imaging of the total hemoglobin concentration, oxygenation, and blood flow [14]. W. Chen et al. combined LSCI and the imaging correlation technique to study the cerebral dynamic changes during cocaine administration [16]. Although LSCI has been widely used for in vivo imaging of various organs, the proposed system setups are difficult for widespread use in different applications in animal models.
In this study, we proposed a system setup to integrate LSCI with a commercial microscope based on a dual-wavelength configuration. Then, the developed LSCI was implemented for the in vivo observation of the dynamic microcirculation. To validate the abilities of the developed LSCI system, the animal experiments were performed by changing the external pressure on mouse skin. Based on the temporal analysis of speckle contrast, the dynamic change in the blood velocity was investigated. Additionally, oxygenation of the microcirculation was estimated for comparison before and after the application of external force on tissue.
2. System setup and experimental method
2.1. Setup of microscope-based LSCI
In this study, a microscope (SZ61, Olympus, Taipei, Taiwan) was used to develop a dual-wavelength LSCI system, as shown in Fig. 1. Two laser diodes with different wavelengths of 635 nm (QFLD- 635-40S, QPHOTONICS, Michigan, USA) and 855 nm (QFLD-850-100S, QPHOTONICS, Michigan, USA) were fixed on the same side of the microscope by a 3D printing mount. The output powers on the sample of both laser diodes were set to 20 mW, and the exposure areas of both beams were carefully adjusted to overlap on the sample surface. The reflected/scattering light from the sample was collected by the objective lens. Here, the magnification factor of the used microscope ranged from 6.7× to 45×, making the field of view and the imaging resolution adjustable. Then, the reflected light from the sample was composed of 635 nm and 855 nm components and then, was divided by a dichroic mirror (DMSP805, THORLABS, New Jersey, USA) for individual detection. Two identical cameras (acA1300-200um, Basler, Ahrensburg, German) were implemented to receive the light beams of two wavelengths, respectively. The mounts for the optical components and two cameras were made by a 3D printer. The exposure times of both cameras were set to 20 ms, corresponding to a frame rate of 50 Hz. Here, the field of view was 5 mm × 5 mm.
Fig. 1. Schematic of the LSCI system setup based on a commercial microscope. (a) Scheme of the microscope-based LSCI system; (b) setup of optical components for dual-wavelength detection; (c) 3D-printed detection end for dual-wavelength detection. DM: dichroic mirror and LM: lens mount
2.2. Theory
The interaction of coherent light with a scattering medium probably causes constructive and destructive interference, further resulting in variation of the scattering intensity. When a camera is used for intensity detection, the detected intensity randomly varies in space and forms a varying pattern called speckle. Moreover, if the scattering particles are moving, this causes the speckle intensity to change with time. Thus, the temporal and spatial analyses of speckle images enable the motion of particles to be investigated. From previous studies, it can be noted that blood flow, consisting of the moving red blood cells, results in the spatial blurring of speckle patterns. The speckle pattern changes rapidly when the blood flow velocity is higher. Therefore, the relative velocity of blow flow can be obtained by quantifying the blurring of the speckle pattern. To evaluate the blurring of the speckle pattern, the speckle contrast, κ, is used and it can be defined as
where σt is the temporal standard deviation of speckle intensity and ‹I› is the mean intensity [17]. Then, the relationship between the speckle contrast and the correlation time, τc, can be expressed asAdditionally, the previous report indicated that the τc is inversely proportional to the blood flow velocity, V [15]. Therefore, the relationship between the speckle contrast and the blood flow velocity can be rewritten as
Therefore, the relative change in the blood flow velocity can be estimated from Eq. (4). Moreover, to further evaluate the changes in oxyhemoglobin (ΔHBO) and de-oxyhemoglobin (ΔHB) as a function of time, t, the speckle intensities from 633-nm and 855-nm lasers can be substituted into the following equation:2.3. Image processing algorithm
In the setup of the microscope-based LSCI system, two cameras were used for individual detection of the scattering signals from two wavelengths. Figure 2 describes the flow chart of the image processing algorithm for the estimation of speckle contrast, blood flow, and oxygenation. Here, the raw speckle images of both wavelengths were acquired from two cameras, and time-series images for each camera were recorded individually. Then, temporal analyses between ten sequential images were performed by estimation of the time-dependent speckle variance at each pixel. Subsequently, the estimated speckle variance results were normalized and smoothed. Finally, the speckle noise was removed. According to Eqs. (4) and (5), the changes in the blood flow velocity and the hemoglobin and de-oxyhemoglobin concentrations were calculated.
2.4. Animal preparation and experimental method
In this study, C57BL/6 mice aged 7–8 weeks were utilized for the animal experiments. The mouse ears were fixed on a platform and the mice were anesthetized during LSCI imaging. Two experiments were performed: release and compression. For the release experiment, a 120-gram metal rod was placed on the mouse ear skin in the beginning, and then the rod was removed. To investigate the difference in the vessels, blood flow velocity, and oxygenation, the ear skin was imaged with the LSCI system before and after removing the metal rod from the skin surface. In contrast, the compression experiment was performed by placing the metal rod on the mouse ear, and the skin was continuously imaged with LSCI before and after placing the metal rod.
3. Results
In this study, a microscope-type LSCI system with dual-wavelength detection was developed for animal studies in vivo. To investigate the dynamical changes in tissue microcirculation due to the application of the external force, two experiments were performed including the release and compression experiments and the experimental results are shown in Sections 3.1 and 3.2, respectively.
3.1 Release experiment
In this experiment, a metal rod with a weight of 120 grams was placed on the mouse ear skin, and then the metal rod was removed to investigate the dynamic difference in microcirculation. Additionally, the same skin area was imaged when the rod was replaced and was continuously imaged after removing the rod. Using Eq. (1), the speckle contrast images at various time points after removing the metal rod on mouse skin were obtained. Figures 3(a)–(d) show the 635-nm laser speckle contrast images of mouse ear skin obtained at 0, 5, 10, and 15 s after the metal rod was removed. In contrast, Figs. 3(e)–(h) represent the 855-nm laser speckle contrast images of the same skin area obtained at 0, 5, 10, and 15 s after removing the metal rod. The red areas in Figs. 3(a) and 3(e) denote the location of the metal rod. From Fig. 3, the vessels indicated by the yellow arrows can be gradually identified with time, illustrating that the blood flow gradually recovered after removal of the compression source. Aside from the speckle contrast results, the relative change in the blood flow velocity was also calculated from Eq. (4). Figure 4 shows the distribution of the corresponding blood flow velocity change estimated from Fig. 3. From Fig. 4, the change in the blood flow velocity can be identified. Since the external force caused the blood flow to become slow and weak, the velocity distribution was relatively weak and not significant. After the rod was removed, the blood flow velocity increased, and the blood flow was recovered for some vessels which cannot be identified in Figs. 4(a) and 4(e). Compared with Figs. 4(a) and 4(e), the flow velocity increased with time after removing the metal rod, illustrating that the blow flow significantly increased after removing the rod. Here, since the blood flow velocity was inversely proportional to the square of the speckle contrast, as shown in Eq. (4), it was difficult to measure the absolute blood flow velocity. However, it was possible to calculate the relative change in the blood flow velocity from Eq. (4). To clearly differentiate the change in the relative blood flow velocity, the estimated 1/κ2 values were normalized, as shown in Fig. 4.
Fig. 3. Laser speckle contrast images of mouse ear skin obtained after releasing the compression for (a)(e) 0 s, (b)(f) 5 s, (c)(g) 10 s, and (d)(h) 15 s. (a)–(d) and (e)–(h) are the laser speckle contrast images obtained from the 635-nm and 855-nm laser beams, respectively. Each image area covers 4 mm × 4 mm.
Fig. 4. Distribution of the corresponding blood flow velocity change of Fig. 3 obtained after removing the external force for (a), (e) 0 s, (b), (f) 5 s, (c), (g) 10 s, and (d), (h) 15 s. (a)–(d) and (e)–(h) are the results obtained from the 635-nm and 855-nm laser beams, respectively. Each image area covers 4 mm × 4 mm.
3.2 Compression experiment
In contrast to the release experiment, the compression effect on microcirculation dynamics was also investigated. Compared with the release experiment, the experimental procedure was reversed for the compression experiment. In the beginning of this experiment, no extra force was applied for mouse skin, and then, the metal rod was placed on mouse skin. Before placing the metal rod, the mouse was continuously imaged with LSCI until placing the rod for 30 s. Figures 5(a)–(d) show the 635-nm laser speckle contrast images of mouse skin obtained at various time points of 0, 10, 20, and 30 s after placing the metal rod on the skin. In contrast, Figs. 5(e)–(h) are the 855-nm laser speckle contrast images of mouse skin obtained at time points of 0, 10, 20, and 30 s after the metal rod was placed. It can be noted that the vessels gradually disappeared due to the application of the external force to reduce or stop the blood flow. The yellow arrows in Figs. 5(a) and 5(e) indicate the vessels those disappeared after the rod was placed.
Fig. 5. Laser speckle contrast images of mouse ear skin obtained after applying the external force for (a)(e) 0 s, (b)(f) 10 s, (c)(g) 20 s, and (d)(h) 30 s. (a)–(d) and (e)–(h) are the laser speckle contrast images obtained from the 635-nm and 855-nm laser beams, respectively. Each image area covers 4 mm × 4 mm.
Similarly, Fig. 6 represents the distribution of the corresponding blood flow velocity change of Fig. 5. The red areas denote the region of the placed metal rod. The decrease in the blood flow velocity can be found when Fig. 6(a) is compared with Fig. 6(d). The results also illustrate that the external force affected the blood flow, reducing the flow and the flow velocity. Additionally, the results indicate that the influence induced by the external force in the upper skin region (corresponding to the upper regions of Fig. 6(a)-(d)) was significantly more severe than that in the lower skin region. The difference is probably due to the unbalanced application of the extra force on the skin.
Fig. 6. Distribution of the corresponding blood flow velocity change of Fig. 5 obtained after applying the external force for (a), (e) 0 s, (b), (f) 10 s, (c), (g) 20 s, and (d), (h) 30 s. (a)–(d) and (e)–(h) are the results obtained from the 635-nm and 855-nm laser beams, respectively. Each image area covers 4 mm × 4 mm.
To further understand the dynamic and quantitative change in the relative blood velocity, the time-dependent velocity distribution was estimated at specific vessel locations. Three regions (Regions A, B, and C) were chosen for estimation, as indicated by the black rectangles in Fig. 4(h). Additionally, two regions in Fig. 6 were analyzed, as indicated by the black rectangles in Fig. 6(e). Figure 7 plots the change in the blood flow velocity of Figs. 4 and 6. To investigate the change in the blood flow velocity, the blood flow velocity at t=0 s was set as the baseline. As shown in the Fig. 7(a), it can be noted that the velocity changes significantly increased after removing the external force. In contrast, the velocity changes rapidly decreased after the application of the external force and became stable after 20 s as shown in Fig. 7(b).
Fig. 7. Time-dependent velocity changes of (a) the release experiment and (b) the compression experiment. Regions A, B, and C in (a) are indicated by the black rectangles in Fig. 4(h). Regions A and B are indicated by the black rectangles in Fig. 6(e).
Furthermore, the oxygenation of biological tissue including the changes in oxyhemoglobin and de-oxyhemoglobin was measured. Figures 8(a) and 8(b) represent the ΔHBO and ΔHB of the release experiment at 15 s after the metal rod was removed, and Figs. 8(c) and 8(d) show the ΔHBO and ΔHB of the compression experiment at 30 s after placing the metal rod on the skin. Here, one region in each experiment was selected as indicated by the black rectangles, and Figs. 8(e) and 8(f) plot the time-dependent ΔHBO and ΔHB values for the release and compression experiments, respectively. In Fig. 8(e), since the blood flow increased after the rod was removed, the ΔHBO and ΔHB values increased after removing the rod and reached a saturation level after 4 s. In Fig. 8(f), the blood flow was blocked when the rod was placed on the skin, and thus, the ΔHBO and ΔHB values decreased. It can be found that the ΔHBO and ΔHB values were unchanged after 20 s. The results demonstrate that the developed LSCI system can be used to study the tissue microcirculation in vivo, including the microvasculature, relative flow velocity, and oxygenation.
Fig. 8. (a), (b) ΔHBO and ΔHB of the release experiment at 15 s after the metal rod was removed. (c), (d) ΔHBO and ΔHB of the compression experiment at 30 s after placing the metal rod on the skin. (e), (f) Time-dependent ΔHBO and ΔHB values for the release and compression experiments, respectively. The imaging areas of (a)-(d) are 4 mm × 4 mm.
4. Discussion
In this study, a dual-wavelength LSCI system integrated with a commercial microscope was demonstrated and the developed system was successfully used for in vivo animal experiments. Moreover, two experiments were conducted to observe the dynamic changes of microcirculation with/without the application of the external force including the release and compression experiments. With the developed system, the vessels of mouse skin can be visualized, and the change in the blood flow velocity can be estimated as well as the concentration change of ΔHBO and ΔHB. Although the results have demonstrated that the dynamics of tissue microcirculation can be in vivo investigated, the imaging abilities can be further improved including the imaging speed, the imaging quality, and the magnification ratio. Currently, the exposure time of two cameras was set to 20 ms, corresponding to a frame rate of 50 Hz. Additionally, temporal analyses between ten sequential images were performed by estimation of the time-dependent speckle variance at each pixel. However, it is difficult to investigate the faster change in tissue microcirculation. The imaging speed can be further improved by using the high-speed cameras. Moreover, as shown in Figs. 4 and 6, the 635-nm results are much blurrier than the 855-nm results, probably resulting from the weaker reflection or backscattering from tissue when tissue was illuminated by a 635-nm laser beam. Alternatively, increasing the illumination power on the tissue surface is able to further improve the image quality and remove the image blurring. Additionally, the microscope with a magnification ratio ranging from 6.7× to 45× was used in this study. However, due to the limited magnification ratio, it is difficult to observe the changes in capillaries. Thus, a microscope with a higher magnification ratio enables to observe dynamic microcirculation of smaller vessels.
5. Conclusions
In this study, a microscope-based LSCI system was developed and a dual-wavelength configuration was proposed for studying the tissue microcirculation in vivo. Two laser diodes with wavelengths of 635 and 855 nm were implemented to acquire the dynamic change in speckle variance, which can be used to measure the relative change in the blood flow velocity. Additionally, since the absorption coefficient of hemoglobin is a function of the wavelengths, the dual-wavelength configuration can be used for the estimation of tissue oxygenation including the changes in the oxyhemoglobin and de-oxyhemoglobin concentrations. Therefore, two experimental protocols were designed including the release and compression experiments. The results depict that the developed dual-wavelength LSCI system enables in vivo observation of the dynamic changes in the microcirculation such as the changes in the blood flow velocity and the changes in the oxyhemoglobin and de-oxyhemoglobin concentrations. It can be noted that the developed system in this study could be a potential platform for real-time investigation of the microcirculation in animal models.
Funding
Chang Gung Memorial Hospital, Linkou (CMRPD2J0131); Ministry of Science and Technology, Taiwan (MOST 107-2628-E-182-001-MY3).
Disclosures
The authors declare no conflicts of interest.
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