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Project titleA novel acoustic/optical imaging modality for zebrafish cardiovascular system: from embryos to adult fish

AffiliationScientific and Technological Centre of Unique Instrumentation of RAS,

Research area 09 - ENGINEERING SCIENCES, 09-609 - Automated complexes for biology and medicine

KeywordsZebrafish, model organism Danio rerio, cardiovascular system, high frequency ultrasound imaging, high resolution optical imaging, non-invasive diagnostics, blood flow parameters, in vivo monitoring, photoplethysmography, capillaroscopy, pathogens, spectral imaging

Annotation
One of the most effective model object for testing the effects of drugs on the human cardiovascular system is the aquarium fish Danio rerio (Zebrafish), which is widely used to study the course of cardiac diseases, monitor the development of blood vessels, and solve other problems. Small size, ease of maintenance, high reproduction rate with multiple embryos (100-200), short period of embryonic development (3 days), and the possibility of research in a number of generations make Danio rerio a very convenient object for research and experiments and allow comparative experiments on offspring received from one pair. These advantages, as well as the possibility of continuous monitoring, allow to significantly increase the speed of obtaining data and their reliability in comparison with other model objects used for research and testing of drugs. The ultrasonic and optical methods, hardware and software developed by the PIs of this project, will allow visualizing tissues and a map of blood vessels, automatically calculating the heart rate and the blood flow rate in various vessels. The technology developed in the course of the project can be used for non-invasive study of the effects of various factors, such as pathogenic nature (viruses, bacteria, etc.), and therapeutic / protective (drugs, vaccines, etc.), as well as physical effects (radiation, temperature, etc.) on the functioning of the cardiovascular system. The project is dedicated to the development and debugging of the unique laboratory setup for non-invasive monitoring of the state of the cardiovascular system and tissues of Danio rerio at different stages of development (embryonic, larval, fry, juvenile, and adult), including in a number of generations. A combination of ultrasound echocardiography and optical methods such as high resolution ultrasound imaging, including vector Doppler and strain/stress imaging, photoplethysmography, video-capillaroscopy, and hyperspectral monitoring will be implemented. Using this setup it is planned to carry out a measurement cycle of the cardiovascular system`s parameters using medications to detect / exclude their cardiotoxicity and influence on the cardiovascular system and body tissues, including offspring. During the project, the functional capabilities of the setup (multimodality, high performance, automatic data integration) will be demonstrated, a methodology for holding researches of cardiotoxicity of drugs will be developed, and a database with the characteristics of the cardiovascular system and various tissues of Danio rerio at different stages of development will be formed, containing the research results of spectral imaging, sonography and video capillaroscopy in vivo with accompanying data of histological analyzes, including the introduction of various drugs. The effectiveness of the setup is planned to be confirmed by experimental studies of the influence of the influenza virus and anti-influenza vaccines on the cardiovascular activity of Danio rerio and by comparing the results with the known data.

Expected results
The main expected results of the Project include the following. 1. Development of a laboratory setup for non-invasive monitoring of the characteristics of the cardiovascular system, which also includes auxiliary subsystems for the introduction of drugs into various tissues of Danio rerio specimens at the embryonic, larval, fry, juvenile and adult stages of development, and for maintaining their vital activity. 2. Formation of a database with the characteristics of various Danio rerio tissues at different stages of development, containing the results of photoplethysmographic, videocapillaroscopic, acoustic and spectral imaging measurements with accompanying data of histological analyzes, including the introduction of various drugs. 3. The method of rapid testing of the cardiotoxicity of drugs based on the analysis of their effect on the cardiovascular activity of healthy or infected Danio rerio. The planned results correspond to the world level of research. Moreover, in terms of research methods (photoplethysmography, spectral imaging, sonography) of the cardiovascular system functioning at the embryonic stage, the planned results exceed the world level, which is confirmed by the priority of the authors from Russian and Taiwanese teams in this area [A. Machikhin, M. Volkov, A. Burlakov, D. Khokhlov, A. Potemkin. Blood Vessel Imaging at Pre-Larval Stages of Zebrafish Embryonic Development. // Diagnostics, 2020. V. 10 (886); Burlakov A.B., Khokhlov D.D., Machikhin A.S., Titov S.A., Lomonov V.A., Vinogradov V.A. Study of dynamic processes in biological systems by methods of acousto-optical video spectrometry. // Radio engineering and electronics, 2020.Vol. 65 (7). P. 717-724, Hsin Huang, Pei-Yu Chen, and Chih-Chung Huang “40-MHz High-Frequency Vector Doppler Imaging for Superficial Venous Valve Flow Estimation,” Medical Physics, V. 47, No. 9, pp.4020-4031, 2020, Chao-Chuan Chang, Pei-Yu Chen, Hsin Huang, and Chih-Chung Huang “In vivo visualization of vasculature in adult zebrafish by using high-frequency ultrafast ultrasound imaging”, IEEE Transactions on Biomedical Engineering. Vol. 66, No. 6, pp. 1742-1751, 2019 etc.]. This setup will make it possible to quickly explore the influence of drugs, vaccines, pathogens on the cardiovascular system of Danio rerio model object and on its tissues. Efficiency is ensured both by the properties of the model object and by the fact that most of the data will be processed automatically. In addition to studies on the safety of effects carried out on healthy fish, it is possible to study the protective properties of vaccines when vaccinated individuals are infected and to analyze the cardiotoxicity of drugs introduced to infected individuals. Therefore, the planned results have wide opportunities for practical use and high scientific and social significance.

Annotation of the results obtained in 2022
The main modules of the laboratory setup for non-contact monitoring of Danio rerio cardiovascular system characteristics using optical and acoustic methods have been developed. The spectral imaging module is implemented as a complete acousto-optical unit installed in the imaging system of the upright microscope. The videocapillaroscopy module is implemented as an imaging optical system installed in the video port of a trinocular microscope. Being equipped with a polarization-sensitive image sensor, this module also provides an ability of Mueller-matrix microscopy. The sonographic module consists of a two-coordinate motorized scanner, a driver unit, an electronic unit and an ultrasonic transducer. The sonographic module with a frequency range of 100 MHz is developed in two versions: as an attachment to an optical inverted microscope for observing transparent specimens and as an independent module with an additional portable optical microscope located on the side of the ultrasonic transducer and rigidly connected to it for studying opaque specimens. Original data processing algorithms providing contrast visualization of specific tissues and cardiovascular system characterization have been developed for all modules. These algorithms have been successfully tested in multiple in vivo experiments with Danio rerio at various developmental stages and have made it possible to obtain new scientific results. During the study of the spectral properties of Danio rerio tissues, it was shown for the first time that body pigmentation, which is opaque in the visible range and does not allow the use of optical methods for analysis in the transmission of organisms older than a few days, is transparent in the near infrared range of 1000-1500 nm. This makes it possible to significantly (up to several weeks) increase the age limit of organisms at which it is possible to analyze the characteristics of the functioning of the cardiovascular system (vascular morphology, blood flow velocity, heart rate, etc.) using videocapillaroscopy and photoplethysmography. A method for real-time Mueller-matrix microscopy for differentiation of Danio rerio tissues without scanning is proposed. The interaction of polarized light with tissues provides information about their structure and properties. This approach is effective for examining muscle tissue due to its birefringence. Measurements of the light intensity time dependences and polarization parameters in various tissues show that hatching is accompanied by a sharp change in the angle and degree of linearly polarized light after interaction with muscle tissue. It was also shown that the spatial distribution of depolarization parameters clearly indicates disorders of the embryonic skeleton. The obtained results demonstrate the high efficiency of the Mueller-matrix microscopy method for monitoring Danio rerio embryos without staining. It has been proposed and experimentally shown that the methods of Mueller-matrix microscopy and videocapillaroscopy can be realized simultaneously using a single polarization-sensitive image sensor. This approach makes it possible to acquire 4 polarization images at once and obtain both the temporal intensity dependencies and polarization properties of the light interacting with the sample. Experiments on Danio rerio embryos demonstrate the high efficiency of this approach for the simultaneous analysis of hemodynamics and muscle structure. For example, this can be useful to quantify the impact of various factors on the cardiovascular system and muscles simultaneously. Using the developed sonographic module, in vivo studies of Danio rerio heart activity at the age of 40 hours after fertilization to ten days were carried out. Algorithms for ultrasonic signals processing enabled spatio-temporal filtering, separation of the contributions from moving blood cells and heart walls, visualization of intense blood flow areas and measurement of blood flow velocity in selected areas. Analysis of the video acquired simultaneously with registration of ultrasonic signals made it possible to determine the heart rate, register the phases of the heart rhythm and observe heart performance anomalies. Comparison of the obtained heart contractions ultrasound phases made it possible to synchronize ultrasound signals at different points in the heart region. A sequence of two-dimensional ultrasonic frames with a frequency of up to 1 kHz were synthesized with the synchronization of spatiotemporal ultrasonic signals using video data. Correlation processing of such data made it possible to visualize the temporal evolution of blood flow distribution and measure its velocity in the selected section of the heart. The studies on the impact of various factors on the functioning of Danio rerio cardiovascular system were initiated from the investigation of a magnetic field influence in the early stages of development. The dynamics of changes in heart rate was of a circadian nature in all the studied groups, except for individuals exposed to a magnetic field under constant illumination. The results demonstrate the importance of the natural magnetic background for the early development of Danio rerio.

Publications

1. A. Machikhin, C. Huang, D. Khokhlov, V. Galanova, A. Burlakov Single-shot Mueller-matrix imaging of zebrafish tissues: In vivo analysis of developmental and pathological features Journal of Biophotonics, Journal of Biophotonics, 2022, V. 15, Iss. 9, No. e202200088 (year - 2022) https://doi.org/10.1002/jbio.202200088

2. M. Volkov, A. Machikhin, V. Bukova, D. Khokhlov, A. Burlakov, V. Krylov Optical transparency and label-free vessel imaging of zebrafish larvae in shortwave infrared range as a tool for prolonged studying of cardiovascular system development Scientific Reports, Scientific Reports, 2022, V.12, No. 20884 (year - 2022) https://doi.org/10.1038/s41598-022-25386-w

3. S. Titov, A. Burlakov, L. Zykova, С. Huang Estimation of Blood Flow Velocity in the Heart of the Danio rerio Embryo Using Correlation of Ultrasonic Signals Proceedings of the 2022 International Conference on Information, Control, and Communication Technologies, IEEE Xplore, - (year - 2022)

4. S. Titov, L. Zykova, A. Burlakov, C. Huang, A. Bogachenkov High-Frequency Ultrasound Echocardiography of heart activity of Danio rerio Embryo IEEE Xplore, 2022 Wave Electronics and its Application in Information and Telecommunication Systems (WECONF), 2022, pp. 1-4 (year - 2022) https://doi.org/10.1109/WECONF55058.2022.9803424

5. V. Krylov, A. Machikhin, D. Sizov, A. Guryleva, A. Sizova, S. Zhdanova, V. Tchougounov, A. Burlakov Influence of hypomagnetic field on the heartbeat in zebrafish embryos Frontiers in Physiology, Frontiers in Physiology, V. 13, No. 1040083 (year - 2022) https://doi.org/10.3389/fphys.2022.1040083

6. D. Khokhlov, A. Machikhin Применение оптической когерентной томографии для исследования эмбрионов низших позвоночных in vivo Сборник тезисов докладов конгресса молодых ученых. Электронное издание. – СПб: Университет ИТМО, [2022]., Хохлов Д.Д., Мачихин А.С. Применение оптической когерентной томографии для исследования эмбрионов низших позвоночных in vivo // XI Конгресс Молодых Ученых Университета ИТМО (04.04.2022 – 08.04.2022). Электронное издание. – СПб: Университет ИТМО, [2022]. (year - 2022)

7. L. Zykova, A. Burlakov, S. Titov, A. Bogachenkov Оценка сердечно-сосудистой системы эмбрионов Danio rerio с помощью высокочастотного ультразвукового сканера Сборник трудов XXXIII Всероссийской школы-семинара «Волновые явления: физика и применения» имени А.П. Сухорукова 5-10 июня 2022 года («Волны-2022»), М.: Издательство Московского Университета., Сборник трудов XXXIII Всероссийской школы-семинара «Волновые явления: физика и применения» имени А.П. Сухорукова 5-10 июня 2022 года («Волны-2022»), Ч. 6 "Акустика неоднородных сред", С. 19-20. (year - 2022)

8. Machikhin A.S., Guryleva A.V., Burlakov A.B., Bukova V.I., Khokhlov D.D. Неинвазивное исследование мышечной ткани рыб в раннем онтогенезе Тезисы докладов Всероссийской научной конференции "Моря России: вызовы отечественной науки", Севастополь, 26-30 сентября 2022 г. - Севастополь: ФГБУН ФИЦ МГИ, 2022. - 392 с., Тезисы докладов Всероссийской научной конференции "Моря России: вызовы отечественной науки", Севастополь, 26-30 сентября 2022 г. - Севастополь: ФГБУН ФИЦ МГИ, 2022. - С. 325-327. (year - 2022)

9. Machikhin A.S., Guryleva A.V., Burlakov A.B., Selyukov A.G., Bukova V.I., Khokhlov D.D., Rudenko E.E. Метод неинвазивного исследования сердечно-сосудистой системы в раннем онтогенезе рыб Тезисы докладов Всероссийской научной конференции "Моря России: вызовы отечественной науки", Севастополь, 26-30 сентября 2022 г. - Севастополь: ФГБУН ФИЦ МГИ, 2022. - 392 с., Тезисы докладов Всероссийской научной конференции "Моря России: вызовы отечественной науки", Севастополь, 26-30 сентября 2022 г. - Севастополь: ФГБУН ФИЦ МГИ, 2022. - С. 323-325. (year - 2022)

10. Selyukov A., Burlakov A., Guryleva A., Bukova V., Khokhlov D., Efremova E. Неинвазивное исследование сердечно-сосудистой системы сиговых рыб в раннем онтогенезе Материалы VI Международной конференции «Информационные технологии и технические средства управления» (ICCT-2022), Астрахань, 3–7 октября 2022 года / Астраханский государственный технический университет. – Астрахань : Изд-во АГТУ, 2022., Материалы VI Международной конференции «Информационные технологии и технические средства управления» (ICCT-2022), Астрахань, 3–7 октября 2022 года / Астраханский государственный технический университет. – Астрахань : Изд-во АГТУ, 2022. С. 48-50 (year - 2022)

11. Volkov M.V., Margaryants N.B., Myalitsyn D.I., Potemkin A.B., Guryleva A.V. Вычисление траектории и скорости элементов кровотока личинки Zebrafish на основе анализа сигналов с применением метода фазовой корреляции Труды Российского научно-технического общества радиотехники, электроники и связи имени А.С. Попова: Материалы 15-й Международной научно-технической конференции «Акустооптические и радиолокационные методы измерений и обработки информации», ARMIMP-2022., Материалы 15-й Международной научно-технической конференции ARMIMP-2022, 2022. C. 60-62. (year - 2022) https://doi.org/10.25210/armimp-2022-16

12. - Программа для визуализации и количественного анализа параметров сердечно-сосудистой системы рыб -, 2022667840 (year - )

13. - Отсутствие геомагнитного поля привело к аритмии у эмбрионов рыб и нарушениям в их развитии Пресс-служба РНФ, - (year - )

Annotation of the results obtained in 2023
50 MHz and 200 MHz sonographic modules have been developed, manufactured and experimentally tested. A technique has been developed for the initial positioning of an ultrasound transducer in the heart area using optical images of the surface of an opaque individual at the juvenile stage. A technique has been developed for synchronizing ultrasonic signals measured at individual points in the studied region of the heart, without using video data, which is inaccessible to opaque individuals. The technique is based on determining the period of heart contractions and comparing the moments of the ultrasonic signal measurement initiation at different points with a synthesized rhythm. An effective algorithm has been developed for calculating the spatiotemporal distribution of blood flow velocity, based on two-dimensional correlation of the high-frequency component of spatiotemporal ultrasound signals. An analysis of the error in synchronization of ultrasonic signals with the heart rhythm using a photoplethysmography signal was performed. Experiments aimed at testing acoustic methods for ultra-fast visualization of cardiac activity, vector Doppler measurement of blood flow velocity, assessment of shear stress of the vessel wall and mapping the deformation of cardiac tissues of Danio rerio at juvenile stages were carried out in cooperation with a foreign scientific team. The developed modules were integrated into a single experimental setup designed for in vivo research of Danio rerio cardiovascular system using simultaneously acoustic and optical methods. It allows one to simultaneously record ultrasound and optical data from opposite sides of the body, ensuring their spatial and temporal synchronization. A method for calibrating the setup has been developed to ensure the possibility of joint processing of optical and acoustic data; in particular, a technique has been proposed for aligning the axis of the optical microscope with the origin of sonographic module scanner coordinates. It is shown that joint processing of acoustic and optical data makes it possible to determine the spatiotemporal distribution of blood flow velocity in the heart area and measure the dimensions of its chambers in various directions. Algorithms for joint processing of optical and acoustic data were debugged in order to simultaneously determine the longitudinal and transverse dimensions of the heart chambers depending on the phase of the cardiac cycle, and the features of the formation of photoplethysmography and Doppler velocimetry signals in the area of atrium and ventricle were studied. A series of acoustic and optical measurements of healthy individuals was carried out, aimed at determining the optimal operating modes of the setup. Using the developed experimental setup and algorithms for processing optical and acoustic data, non-invasive studies of the influence of various factors on the cardiovascular system of Danio rerio were carried out. Toxicological experiments were conducted to assess the chronic toxicity of pesticides widely used in agriculture: imidacloprid and glyphosate. The influence of a hypomagnetic field on the characteristics of the functioning of Danio rerio heart in early ontogenesis was studied. The pathophysiological changes in the cardiovascular system of Danio rerio under the influence of toxicants of inorganic origin (cadmium) were analyzed.

Publications

1. Machikhin A.S., Guryleva A.V., Bukova V.I., Khokhlov D.D., Burlakov A.B. Simultaneous blood vessel and Mueller-matrix imaging of zebrafish embryos Proceedings of SPIE, Proc. SPIE 12320, Optics in Health Care and Biomedical Optics XII, 123200E (19 December 2022) (year - 2023) https://doi.org/10.1117/12.2642955

2. Titov S.A., Bogachenkov A.N., Zykova L.A., Guryleva A.V., Burlakov A.B., Machikhin A.S. Стенд для исследования сердечно-сосудистой системы Danio rerio методами акустической и оптической микроскопии Физические основы приборостроения, Физические основы приборостроения. 2023. Т. 12. № 2 (48). С. 86-93. (year - 2023) https://doi.org/10.25210/jfop‑2302-DYCSOQ

3. Volkov M.V., Margaryants N.B., Myalitsin D.I., Potemkin A.V., Guryleva A.V. Вычисление параметров кровотока личинки zebrafish с применением метода фазовой корреляции Оптический журнал, Оптический журнал, 2023, Т. 90, № 12, С. 61-72. (year - 2023) https://doi.org/10.17586/1023-5086-2023-90-12-61-72

4. Bukova V.I., Guryleva A.V. Application software for cardiovascular and muscular studies of zebrafish larvae 2023 Wave Electronics and its Application in Information and Telecommunication Systems (WECONF), 2023 Wave Electronics and its Application in Information and Telecommunication Systems (WECONF), 2023, pp. 1-4 (year - 2023) https://doi.org/10.1109/WECONF57201.2023.10147984

5. Bukova V.I., Guryleva A.V., Machikhin A.S. Приложение для оценки параметров сердечно-сосудистой и мышечной систем Danio rerio Материалы XXVI Международной научной конференции "Волновая электроника и инфокоммуникационные системы", Материалы XXVI Международной научной конференции "Волновая электроника и инфокоммуникационные системы". В 3-х частях. Часть 2. Санкт-Петербург, 2023. С. 74-79. (year - 2023)

6. Korzhevina V.I., Luk'yanov T.F., Machikhin A.S., Guryleva A.V., Burlakov A.B., Krylov V.V. Влияние глифосата и имидаклоприда на сердечный ритм Danio rerio и Daphnia magna Сборник трудов Всероссийской научно-практической конференции «Современные проблемы биологии, химии, экологии» / сост. С. Н. Леднев. – Ярославль: ЯрГУ, 2023. – 152 с., Сборник трудов Всероссийской научно-практической конференции «Современные проблемы биологии, химии, экологии» / сост. С. Н. Леднев. – Ярославль: ЯрГУ, 2023. – 152 с. – (17–21 октября 2023 г., Ярославль). С. 54-56. (year - 2023)

7. Krylov V.V., Machikhin A.S., Sizov D.A., Guryleva A.V., Sizova A.A., Zhdanova C.M., Chugunov V.K., Burlakov A.B. Влияние гипомагнитных условий на сердцебиение эмбрионов Danio rerio Сборник тезисов XXIV съезда физиологического общества им. И.П. Павлова. Санкт-Петербург, 11–15 сентября 2023 г., Сборник тезисов XXIV съезда физиологического общества им. И.П. Павлова. Санкт-Петербург, 11–15 сентября 2023 г. / СПб.: Изд-во ВВМ, 2023. – 612 с. С. 405-406. (year - 2023)

8. Machikhin A.S., Chakraborty A., Zykova L.A., Titov S.A., Guryleva A.V., Bukova V.I., Burlakov A.B Мультимодальный стенд для визуализации и измерения параметров сердечно-сосудистой и мышечной систем Danio rerio Оптические методы исследования потоков: Труды XVII Международной научно-технической конференции, 26– 30 июня 2023. – М.: Научно-технологический центр уникального приборостроения РАН, 2023. – 795 с., Оптические методы исследования потоков: Труды XVII Международной научно-технической конференции, 26– 30 июня 2023. – М.: Научно-технологический центр уникального приборостроения РАН, 2023. – 795 с. С. 610 - 616. (year - 2023)

9. Slavin A.E., Shuman L.A., Guryleva A.V., Bukova V.I., Burlakov A.B., Burlakova O.V., Machikhin A.S. Применение фотоплетизмографии для оценки состояния сердечно-сосудистой системы рыб Danio rerio в условиях загрязнения водной среды кадмием Акустооптические и радиолокационные методы измерений и обработки информации: Труды XVI Международной научно-технической конференции, Суздаль, 9-12 октября 2023. ISBN 978-5-6051133-3-1. М.: НТЦ УП РАН, 2023., Акустооптические и радиолокационные методы измерений и обработки информации: Труды XVI Международной научно-технической конференции, Суздаль, 9-12 октября 2023. ISBN 978-5-6051133-3-1. М.: НТЦ УП РАН, 2023. С. 204-208. (year - 2023) https://doi.org/10.2510/armimp-2023-FQFNOM

10. Volkov M.V., Margaryants N.B., Myalitsin D.I. Применение систем высокоскоростной видеокапилляроскопии при исследовании параметров кровотока в коже человека и модельных организмов Акустооптические и радиолокационные методы измерений и обработки информации: Труды XVI Международной научно-технической конференции, Суздаль, 9-12 октября 2023. ISBN 978-5-6051133-3-1. М.: НТЦ УП РАН, 2023., Акустооптические и радиолокационные методы измерений и обработки информации: Труды XVI Международной научно-технической конференции, Суздаль, 9-12 октября 2023. ISBN 978-5-6051133-3-1. М.: НТЦ УП РАН, 2023. С. 190-192. (year - 2023) https://doi.org/10.2510/armimp-2023-IODQVQ

11. Zykova L.A., Titov S.A. Алгоритм обработки пространственно-временных сигналов в сонографических исследованиях низших позвоночных на ранних стадиях развития Современные технологии научного приборостроения и информационно-измерительных систем: Труды Международной научно-технической конференции, Москва, 23.06.2023. М.: НТЦ УП РАН, 2023., Современные технологии научного приборостроения и информационно-измерительных систем: Труды Международной научно-технической конференции, Москва, 23.06.2023. М.: НТЦ УП РАН, 2023. С. 119-120. (year - 2023)

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