Kurochkin M.A. , Stiukhina E. S., Fedosov I.V., Postnov D.E.

1. Image processing for vascular pulsation measurements in response to laser light irradiation using micro-PIV approach Kurochkin M.A. , Stiukhina E. S., Fedosov I.V., Postnov D.E.

2. Motivation Biological tissue activity is critically dependent on the timely delivery of nutrients and oxygen through the blood. It is known that endothelium provide dynamic regulation of blood vessel elasticity in response to different environmental conditions change like endothelial shear stress and circumferential wall stress, respectively. Vascular wall shear stress plays a key role in the development of atherosclerosis and cardiogenesis pathology. Abnormal blood flow and blood hemorheological characteristics modification are critical in the diagnosis of vascular disease. Thus, the blood flow velocity quantitative data may provide important information for early vascular diseases detection. In chicken embryos it is possible to directly visualize blood flow microcirculation and RBC velocity measurements. We employed a micro-PIV technique to assess blood flow in chicken embryo blood vessels, using red blood cells (RBCs) as tracers. Blood vessel diameter and RBC velocity fluctuation in response to highly localized laser light irradiation are presented.

3. Particle Image Velocimetry (PIV) Recorded series of mesenteric vascular network Euclidean skeleton Background thresholding PIV algorithm Velocity map Velocity map (colored)

4. Experimental arrangement The self-made optical setup build around Lomo microscope base equipped bright-field condenser with white LED light source. Laser light (532 nm, 20mW, Lascompany) enters coaxial via the back entrance and is reflected by a dichroic mirror to a 10x/0.27 infinity corrected long work distance objective to irradiate vascular network segment.Vascular pulsation is imaged by f=200 tube lens on a CMOS detector (acA2040-180km, Basler Inc) before and after laser light irradiation. Schematic view of the optical system used forfluorescent microcapsules imaging

5. Optical probe Experimental data were collected using chicken embryos of 12-th – 14-th day of incubation. Blood flow direction is marked with a white arrows. To produce the occlusion probe, laser beam was focused at the location indicated by a X-mark. Green laser module power was attenuated to 14,5 mW with a pair of rotating polarizing filters. Under these conditions, the irradiation during 3 sec was found to be enough to cause local vascular dilatation. Blood flow velocity and vascular diameter pulsation was measured in area that is marked by the numbers I (upstream), II (laser light irradiation ) and III (downstream) . (A) (B) Chicken embryo chorioallantoic membrane (CAM) A) Before and B) after laser light irradiation. X-mark indicates the irradiation zone

6. vascular pulsation measurements algorithm • After optimization of brightness and contrast of obtained images series, an automated search of the axial line of the vessel was performed. For this purpose, the selection of the segment made by the operator within the field of view was used as an initial approximation. From the ends of the segment a wererestored perpendiculars k and m, along which the was calculated brightness profiles P1 and P2.Thus, the coordinates of the axial length of the line a' were the calculated. Per-frame profiles were averaged within each series, in order to minimize fluctuations of optical density contribution at the vessel walls. Within the found boundaries of the vessel was placed a set of rectangular interrogation regions, needed for the calculation of the velocity field by PIV and oriented parallel to a'. The typical width of each of the calculated area was 20 or 14,5 μm pixels in the object space, which roughly corresponds to twice the size of a red blood cell. The typical length of the calculated area is 50 pixels or 36 μm in the object space

7. Results (B) (A) Fig. Bshows the results obtained in the experiment above the impact region diameter measurement vessel (I), in the area (II), and downstream (III) before and after laser-assisted vascular irradiation. As it can be seen, laser irradiation caused an increase in vessel diameter is fixed in the impact area. Some reduction in the diameter of the upstream zone of influence is also a significant result of the measurement, however, discussion of the effect mechanisms beyond the scope of this article. Fig. A illustrates an attained temporal and spatial resolution in the determination of the average flow rate values. With regard to the necessary averaging over a sequence of frames, measuring the speed attained sampling frequency is about 168 frames per second that can adequately capture the pulse oscillation speeds for most laboratory animals. Fig. A also allows assessing the variation of maximum and minimum blood flow rate at various locations in the test vessel compared with its initial state (horizontal dot-dash lines). Therefore for 1 second 10 velocity fields is calculated for the selected stream portion. As can be seen, when the blood flows through the vessel its velocity varies from 0.7-0.8 mm / s in the input (the neck) portion, up to 0.1-0.3 mm / s in the central (extended) portion, and then rises again to values of 0.3-0.6 mm / s. Clearly visible dark spot in the region II corresponds to a higher flow rate, however, it represents measurement artifact caused instability clot forms erythrocytes, which is especially likely in a sharp change in the vessel diameter, as decay (loss of brightness) of the clot and the formation of the new in the distance, PIV method can potentially be interpreted as a rapid movement of the same optical in homogeneity.

8. Summary • We propose a non-distructive laser based approach for assessment of a microvessel wall local status. It is implemented on measurements using µPIV of local vessel diameter variation and blood flow velocity in response to focused laser irradiation. This date potentially enable estimation of vessel wall elasticity and muscle tone smooth. • We designed a self-made optical system for in-vivo visualization and digital processing of CAM blood flow dynamics, Including the stand-alone unit for local laser-assisted vascular irradiation • We have developed and tested the adaptive μPIV-based technique for measurement of blood flow pulsation. The method allows to locate the interrogation regions within the analyzed vessel segment.

9. Acknowledgments • Research and Development Project (RF Governmental contract №2014/203), Research Project №1490 "Development of optical methods and instrumentation for measurements and control of structure and dynamics of biological media“ • Project number 3.1340.2014/K ”Development of methods for diagnosis of functional state of microvasculature cell layers on the basis of optical imaging techniques” • Grant of Foundation for Assistance to Small Innovative Enterprises in science and technology UMNIK №8959GU/2015 • VVT is thankful for support by Tomsk State University Foundation named after D.I. Mendeleev

10. Thank you! maxim_optics@mail.ru