rus
Issue #8/2024
М. Е. Stepanov, А. А. Vlasov, P. А. Demina, R. А. Akasov, G. Babaeva, V. I. Yusupov, Т. V. Egorova, К. R. Karimullin, А. N. Generalova, А. V. Naumov, Е. V. Khaydukov
Intravital Microscopy – a Window Into the World of Bioprocesses
Intravital Microscopy – a Window Into the World of Bioprocesses
DOI: 10.22184/1993-7296.FRos.2024.18.8.640.648
The article shows the potential of practical use of the dorsal skin fold optical microscopy
method as an effective diagnostic technology for biosystems. It has been experimentally
proved that even in the basic formulation, the presented method allows obtaining a large
amount of useful research data in conditions as close as possible to native ones.
The article shows the potential of practical use of the dorsal skin fold optical microscopy
method as an effective diagnostic technology for biosystems. It has been experimentally
proved that even in the basic formulation, the presented method allows obtaining a large
amount of useful research data in conditions as close as possible to native ones.
Теги: dark-field fluorescence microscopy life sciences light-field microscopy in white light optical clearing agents науки о жизни оптические просветляющие агенты светлопольная микроскопия в белом свете темнопольная флуоресцентная микроскопия
Intravital Microscopy – A Window Into The World Of Bioprocesses
М. Е. Stepanov 1, 2, А. А. Vlasov 1, P. А. Demina 1, 2, 3, 4, R. А. Akasov 1, 2, 3, G. Babaeva 5, V. I. Yusupov 3, 6, Т. V. Egorova 1, 6, К. R. Karimullin 6, А. N. Generalova 3, 4, А. V. Naumov 1, 6, Е. V. Khaydukov 1, 2, 3, 4, 7
Moscow State Pedagogical University, Moscow, Russia.
Petrovsky National Research Center of Surgery, Moscow, Russia.
Kurchatov Complex of Crystallography and Photonics, National Research Center “Kurchatov Institute”, Moscow, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia.
Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia, Moscow, Russia.
P. N. Lebedev Physical Institute of the Russian Academy of Sciences, Troitsk Branch, Moscow, Troitsk, Russia.
D. I. Mendeleev Russian University of Chemical Technology, Moscow, Russia.
The article shows the potential of practical use of the dorsal skin fold optical microscopy method as an effective diagnostic technology for biosystems. It has been experimentally proved that even in the basic formulation, the presented method allows obtaining a large amount of useful research data in conditions as close as possible to native ones.
Keywords: life sciences, optical clearing agents, light-field microscopy in white light, dark-field fluorescence microscopy
The article is received: 14.11.2024
The article is accepted:02.12.2024
Introduction
In life sciences, a large amount of important information can be obtained only by directly studying biosystems, considering all complex internal processes and interactions. Under in vivo conditions, while maintaining intercellular interactions, transport of substances and signals, one can hope to obtain a real picture of the processes occurring in biological tissues. A living organism consists of trillions of cells [1], where each individual cell is a chemical factory that synthesizes and disposes of some substances and stimuli to produce other ones and transfers them further down the chain to one of the trillions of recipients, or maybe all at once. And all this in absolute “silence”, using only the language of physico-chemical “gestures”. Yet, it is necessary and possible to study this intricate system, and photons turn out to be the main tool here, allowing information to be recorded in an intuitive way, on the one hand, and realizing sufficient resolution when analyzing a living system, on the other hand. Optical spectroscopy and microscopy methods are among the most effective ways to study the structure and dynamics of complex molecular systems, nanostructures and materials based on them [2–9] and are widely used for diagnostics and therapy in biology and medicine [10–14].
The standard in the field of observation is in vitro cell microscopy (from Latin “in glass”), when a cellular specimen is studied in real time under high magnification in artificially created conditions. However, it is not difficult to understand that most of the information associated with signals from other cells (endocrine – from distant cells, paracrine – from neighboring cells) is completely lost with this approach. A complete picture of the mutual influence of cells on each other can be preserved using ex vivo (lat. “from life”) histological approaches, when a fixed thin tissue sample is analyzed, but at the same time, there is obviously a loss of data on the development of processes over time.
From what has been said, in modern biology and medicine, approaches are extremely in demand that provide long-term monitoring of a living organism in real time at the level of its individual cells and/or cell systems, which is quite difficult to implement due to the limited penetration of radiation from the optical range of the spectrum in biological tissues. However, there are several methods to work around this problem. For example, clarification methods [15] can be used when the transparency of tissues (mainly integuments) for incident light increases due to modification of their structural and optical properties by immersion in biocompatible optical clearing agents that equalize the optical properties of diffusers. It is possible to do otherwise and shift radiation into spectral regions in which it weakly interacts with body tissues – into the so-called spectral “transparency windows” of tissues [15, 16] located in the near infrared or X-ray spectral range. Finally, you can get around the problem by choosing the geometry of the experiment to create a real window for observation. All these methods, combined under the general name “intravital microscopy” [17] (microscopy of biosystems), today make it possible to achieve significant progress in life sciences, since they do not disrupt the picture of complex intercellular interaction [18] and provide real-time observation at the level of individual cells and cellular structures [19].
In this paper, we demonstrate an example of an implementation of an in vivo method for observing laboratory mice using an original dorsal skinfold design. In this design, the fold of skin on the animal’s back is lifted above the main surface of the body and fixed by a special camera with an optically transparent window, which makes it possible to conduct observations with a total duration of up to several weeks [20]. It is shown that even in the most basic configuration, using an inverted optical microscope, it is possible to observe subcutaneous layers of biological tissues and blood vessels (including capillaries), as well as register the processes occurring in them at the level of individual cells in real time.
1. The method of experiment
The experimental scheme is shown on Fig. 1. Microscopic studies were performed using an inverted fluorescence microscope Motic AE31E with an incandescent lamp as a white light source and a mercury lamp for fluorescence imaging. For additional labeling of biological tissues, a fluorescent dye Cy‑5‑amine with absorption (emission) maxima at wavelengths of 646 (662) nm was used. An appropriate set of spectral filters was used to excite and detect fluorescence. A Raptor Photonics camera (digital FALCON EMCCD FA285-CL) was used to register optical signals.
The dorsal chamber and the table compatible with a standard microscope were designed by us independently and made of biocompatible polycarbonate on a 3D printer [21]. The key difference of the dorsal chamber we used is the minimized weight of the device. It is well known that commercially available titanium chambers reach up to 30–40% of the animal’s weight, which causes not only excessive injury to animals [22], but also leads to significant physiological changes in the observation process, for example, due to the production of the stress hormone corticosterone [23].
To prepare for the experiment, the dorsal chamber was surgically mounted on the mouse’s back with fixation using a 5–0 monofil thread. All manipulations with animals were performed using general injection anesthesia by administering a combination of Zoletil-Xylazine drugs (20–40 mg/kg of Xylazine intramuscularly and 5–10 mg/kg of Zoletil intraperitoneally 10 minutes after Xylazine). To reduce light scattering, a thin layer of skin was resected on one side of the skinfold up to the muscular fascia. Microscopy was performed using a set of standard 4–40× micro lenses. The resulting images and videos were processed in the ImageJ program.
2. Light-field microscopy
in white light
The results obtained using the brightfield technique are illustrated in Fig. 2. The technique makes it possible to observe various components of subcutaneous tissues: the vascular bed, as well as rounded subcutaneous fat cells (adipocytes). At the same time, the morphology of the vascular network can be traced almost to the scale of capillaries: the red marks show the sequential branching of a large vessel up to terminal arterioles (~15 μm). The detailed picture makes it possible to study many pathophysiological processes. For example, vascular growth and development (angiogenesis) can be investigated in response to damage or chemical stimulus, or pathological formation in the dorsal window area. In addition, the model makes it possible to directly observe vasodilation and vasoconstriction (respectively, dilation or narrowing of the lumen of blood vessels) under chemical or thermal influences or inflammation, which is extremely valuable, since usually such data are studied only indirectly [24]. Clear distinctness of vasa vasorum vessels which are thin vessels branching directly from large ones and feeding their walls and nearby tissues (branches are marked in Fig. 2a in green), – can be used to study some common vascular pathologies. For example, violations in their work hypothetically can cause inflammation in the walls of large vessels and lead to the development of atherosclerosis [25].
As can be seen from Fig. 2b, adipose (fat) tissue is also available for research. This allows us to study physiology, as well as diseases associated with metabolic disorders, including diabetes mellitus [26]. We calculated the statistics of maximum adipocyte diameters (Fig. 2b) and found that it corresponds to the normal distribution law, while the average size of the adipose tissue element is 42 ± 12 μm.
3. Dark-field fluorescence microscopy
Widefield fluorescence microscopy makes it possible to significantly expand the range of bioprocesses under study and to obtain valuable data in real time. Within a few seconds after the introduction of fluorescent dye into the bloodstream of a small animal, large vessels begin to noticeably fluoresce around the dorsal skinfold with a maximum intensity at times of 3 to 5 minutes. The coloring details can be evaluated on Fig. 3, where the same area of the dorsal window is shown in two modes at low magnification (4× lens).
Fig. 3b shows that at a characteristic scale of 20–30 μm, approximately corresponding to the size of adipocytes, a thin mesh structure of the colored capillary network appears in the fluorescent mode. At a higher magnification (magnification of the 40× lens), it becomes possible to observe the trajectories of individual blood components (Fig. 4) continuously circulating through arterioles and capillaries. Single 230 × 230 μm frame (Fig. 4a) can contain 20 to 40 individual blood cells moving simultaneously along their trajectories, leaving the observation area, then returning to it. If we take a set of sequential observation frames for a time interval of 30 seconds and mark the recorded cells with dots in each of them, we get a picture of cell movement (Fig. 4b). It displays statistics of particle registration in different parts of the frame. As video-observation shows, this time interval provides approximately double the time reserve allowing for the passage of the slowest moving cell through an entire frame. In addition to the morphology of the vascular network, this brief analysis makes it possible to qualitatively judge the average speed of cell movement: it is lower in regions where the dots (in Fig. 4b) are located more densely. It is also possible to draw qualitative conclusions about the most “popular” trajectories of movement: they are seen most distinctly. It is interesting to note that even being in one vessel, the trajectories of the cells do not fill it completely, leaving voids.
Fluorescent staining in an in vivo experiment provides extensive opportunities for observations, for example, of the pharmacokinetics of drugs, including parameters of extravasation from blood flow to tissues [27] and drug delivery [28], the work of immune cells [29], microcirculation in tumor tissues [30].
Conclusion
The study of biological processes using light is widespread today and is routinely used in both in vitro and ex vivo techniques. However, just in the same way as photography is inferior in the number of details to a video file, and video recording is inferior to real vision, the standard microscopy of cells or tissues contains only a small part of the rich picture of biological processes. Intravital microscopy makes it possible to make progress in overcoming the shortcomings of standard techniques. We have implemented the research technique in one of its basic variants and demonstrated the possibility of observing subcutaneous tissue layers, including blood vessels and individual blood cells in real time. Test experiments have shown that the processes occurring in biological tissues can be recorded for a long time, up to weeks. Assessing the potential of the implemented technique, we count on broad cooperation and the introduction of this promising method into everyday research practice both in areas of basic research and actual biomedical practice, for example, to study the pharmacokinetics of existing and developing drugs, to assess their interaction with body cells (including immune cells), active and passive delivery processes of drugs.
Credits
The work was performed within the framework of the topic of the state assignment of the Ministry of Education of the Russian Federation “Laser Technologies for Biomedical Applications” No. 122122600055-2 in terms of conducting optical observations using the dorsal chamber method and the topic of the state assignment of the Kurchatov Institute Research Center in terms of conducting research using luminescent spectroscopy. The design of the dorsal chamber and the stage was developed within the framework of research under contract No. 749-ЭA‑24-НИР dated 06/25/2024 between the P. N. Lebedev Physical Institute of the Russian Academy of Sciences and the B. V. Petrovsky National Research Center of Surgery.
М. Е. Stepanov 1, 2, А. А. Vlasov 1, P. А. Demina 1, 2, 3, 4, R. А. Akasov 1, 2, 3, G. Babaeva 5, V. I. Yusupov 3, 6, Т. V. Egorova 1, 6, К. R. Karimullin 6, А. N. Generalova 3, 4, А. V. Naumov 1, 6, Е. V. Khaydukov 1, 2, 3, 4, 7
Moscow State Pedagogical University, Moscow, Russia.
Petrovsky National Research Center of Surgery, Moscow, Russia.
Kurchatov Complex of Crystallography and Photonics, National Research Center “Kurchatov Institute”, Moscow, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia.
Research Institute of Molecular and Cellular Medicine, Peoples’ Friendship University of Russia, Moscow, Russia.
P. N. Lebedev Physical Institute of the Russian Academy of Sciences, Troitsk Branch, Moscow, Troitsk, Russia.
D. I. Mendeleev Russian University of Chemical Technology, Moscow, Russia.
The article shows the potential of practical use of the dorsal skin fold optical microscopy method as an effective diagnostic technology for biosystems. It has been experimentally proved that even in the basic formulation, the presented method allows obtaining a large amount of useful research data in conditions as close as possible to native ones.
Keywords: life sciences, optical clearing agents, light-field microscopy in white light, dark-field fluorescence microscopy
The article is received: 14.11.2024
The article is accepted:02.12.2024
Introduction
In life sciences, a large amount of important information can be obtained only by directly studying biosystems, considering all complex internal processes and interactions. Under in vivo conditions, while maintaining intercellular interactions, transport of substances and signals, one can hope to obtain a real picture of the processes occurring in biological tissues. A living organism consists of trillions of cells [1], where each individual cell is a chemical factory that synthesizes and disposes of some substances and stimuli to produce other ones and transfers them further down the chain to one of the trillions of recipients, or maybe all at once. And all this in absolute “silence”, using only the language of physico-chemical “gestures”. Yet, it is necessary and possible to study this intricate system, and photons turn out to be the main tool here, allowing information to be recorded in an intuitive way, on the one hand, and realizing sufficient resolution when analyzing a living system, on the other hand. Optical spectroscopy and microscopy methods are among the most effective ways to study the structure and dynamics of complex molecular systems, nanostructures and materials based on them [2–9] and are widely used for diagnostics and therapy in biology and medicine [10–14].
The standard in the field of observation is in vitro cell microscopy (from Latin “in glass”), when a cellular specimen is studied in real time under high magnification in artificially created conditions. However, it is not difficult to understand that most of the information associated with signals from other cells (endocrine – from distant cells, paracrine – from neighboring cells) is completely lost with this approach. A complete picture of the mutual influence of cells on each other can be preserved using ex vivo (lat. “from life”) histological approaches, when a fixed thin tissue sample is analyzed, but at the same time, there is obviously a loss of data on the development of processes over time.
From what has been said, in modern biology and medicine, approaches are extremely in demand that provide long-term monitoring of a living organism in real time at the level of its individual cells and/or cell systems, which is quite difficult to implement due to the limited penetration of radiation from the optical range of the spectrum in biological tissues. However, there are several methods to work around this problem. For example, clarification methods [15] can be used when the transparency of tissues (mainly integuments) for incident light increases due to modification of their structural and optical properties by immersion in biocompatible optical clearing agents that equalize the optical properties of diffusers. It is possible to do otherwise and shift radiation into spectral regions in which it weakly interacts with body tissues – into the so-called spectral “transparency windows” of tissues [15, 16] located in the near infrared or X-ray spectral range. Finally, you can get around the problem by choosing the geometry of the experiment to create a real window for observation. All these methods, combined under the general name “intravital microscopy” [17] (microscopy of biosystems), today make it possible to achieve significant progress in life sciences, since they do not disrupt the picture of complex intercellular interaction [18] and provide real-time observation at the level of individual cells and cellular structures [19].
In this paper, we demonstrate an example of an implementation of an in vivo method for observing laboratory mice using an original dorsal skinfold design. In this design, the fold of skin on the animal’s back is lifted above the main surface of the body and fixed by a special camera with an optically transparent window, which makes it possible to conduct observations with a total duration of up to several weeks [20]. It is shown that even in the most basic configuration, using an inverted optical microscope, it is possible to observe subcutaneous layers of biological tissues and blood vessels (including capillaries), as well as register the processes occurring in them at the level of individual cells in real time.
1. The method of experiment
The experimental scheme is shown on Fig. 1. Microscopic studies were performed using an inverted fluorescence microscope Motic AE31E with an incandescent lamp as a white light source and a mercury lamp for fluorescence imaging. For additional labeling of biological tissues, a fluorescent dye Cy‑5‑amine with absorption (emission) maxima at wavelengths of 646 (662) nm was used. An appropriate set of spectral filters was used to excite and detect fluorescence. A Raptor Photonics camera (digital FALCON EMCCD FA285-CL) was used to register optical signals.
The dorsal chamber and the table compatible with a standard microscope were designed by us independently and made of biocompatible polycarbonate on a 3D printer [21]. The key difference of the dorsal chamber we used is the minimized weight of the device. It is well known that commercially available titanium chambers reach up to 30–40% of the animal’s weight, which causes not only excessive injury to animals [22], but also leads to significant physiological changes in the observation process, for example, due to the production of the stress hormone corticosterone [23].
To prepare for the experiment, the dorsal chamber was surgically mounted on the mouse’s back with fixation using a 5–0 monofil thread. All manipulations with animals were performed using general injection anesthesia by administering a combination of Zoletil-Xylazine drugs (20–40 mg/kg of Xylazine intramuscularly and 5–10 mg/kg of Zoletil intraperitoneally 10 minutes after Xylazine). To reduce light scattering, a thin layer of skin was resected on one side of the skinfold up to the muscular fascia. Microscopy was performed using a set of standard 4–40× micro lenses. The resulting images and videos were processed in the ImageJ program.
2. Light-field microscopy
in white light
The results obtained using the brightfield technique are illustrated in Fig. 2. The technique makes it possible to observe various components of subcutaneous tissues: the vascular bed, as well as rounded subcutaneous fat cells (adipocytes). At the same time, the morphology of the vascular network can be traced almost to the scale of capillaries: the red marks show the sequential branching of a large vessel up to terminal arterioles (~15 μm). The detailed picture makes it possible to study many pathophysiological processes. For example, vascular growth and development (angiogenesis) can be investigated in response to damage or chemical stimulus, or pathological formation in the dorsal window area. In addition, the model makes it possible to directly observe vasodilation and vasoconstriction (respectively, dilation or narrowing of the lumen of blood vessels) under chemical or thermal influences or inflammation, which is extremely valuable, since usually such data are studied only indirectly [24]. Clear distinctness of vasa vasorum vessels which are thin vessels branching directly from large ones and feeding their walls and nearby tissues (branches are marked in Fig. 2a in green), – can be used to study some common vascular pathologies. For example, violations in their work hypothetically can cause inflammation in the walls of large vessels and lead to the development of atherosclerosis [25].
As can be seen from Fig. 2b, adipose (fat) tissue is also available for research. This allows us to study physiology, as well as diseases associated with metabolic disorders, including diabetes mellitus [26]. We calculated the statistics of maximum adipocyte diameters (Fig. 2b) and found that it corresponds to the normal distribution law, while the average size of the adipose tissue element is 42 ± 12 μm.
3. Dark-field fluorescence microscopy
Widefield fluorescence microscopy makes it possible to significantly expand the range of bioprocesses under study and to obtain valuable data in real time. Within a few seconds after the introduction of fluorescent dye into the bloodstream of a small animal, large vessels begin to noticeably fluoresce around the dorsal skinfold with a maximum intensity at times of 3 to 5 minutes. The coloring details can be evaluated on Fig. 3, where the same area of the dorsal window is shown in two modes at low magnification (4× lens).
Fig. 3b shows that at a characteristic scale of 20–30 μm, approximately corresponding to the size of adipocytes, a thin mesh structure of the colored capillary network appears in the fluorescent mode. At a higher magnification (magnification of the 40× lens), it becomes possible to observe the trajectories of individual blood components (Fig. 4) continuously circulating through arterioles and capillaries. Single 230 × 230 μm frame (Fig. 4a) can contain 20 to 40 individual blood cells moving simultaneously along their trajectories, leaving the observation area, then returning to it. If we take a set of sequential observation frames for a time interval of 30 seconds and mark the recorded cells with dots in each of them, we get a picture of cell movement (Fig. 4b). It displays statistics of particle registration in different parts of the frame. As video-observation shows, this time interval provides approximately double the time reserve allowing for the passage of the slowest moving cell through an entire frame. In addition to the morphology of the vascular network, this brief analysis makes it possible to qualitatively judge the average speed of cell movement: it is lower in regions where the dots (in Fig. 4b) are located more densely. It is also possible to draw qualitative conclusions about the most “popular” trajectories of movement: they are seen most distinctly. It is interesting to note that even being in one vessel, the trajectories of the cells do not fill it completely, leaving voids.
Fluorescent staining in an in vivo experiment provides extensive opportunities for observations, for example, of the pharmacokinetics of drugs, including parameters of extravasation from blood flow to tissues [27] and drug delivery [28], the work of immune cells [29], microcirculation in tumor tissues [30].
Conclusion
The study of biological processes using light is widespread today and is routinely used in both in vitro and ex vivo techniques. However, just in the same way as photography is inferior in the number of details to a video file, and video recording is inferior to real vision, the standard microscopy of cells or tissues contains only a small part of the rich picture of biological processes. Intravital microscopy makes it possible to make progress in overcoming the shortcomings of standard techniques. We have implemented the research technique in one of its basic variants and demonstrated the possibility of observing subcutaneous tissue layers, including blood vessels and individual blood cells in real time. Test experiments have shown that the processes occurring in biological tissues can be recorded for a long time, up to weeks. Assessing the potential of the implemented technique, we count on broad cooperation and the introduction of this promising method into everyday research practice both in areas of basic research and actual biomedical practice, for example, to study the pharmacokinetics of existing and developing drugs, to assess their interaction with body cells (including immune cells), active and passive delivery processes of drugs.
Credits
The work was performed within the framework of the topic of the state assignment of the Ministry of Education of the Russian Federation “Laser Technologies for Biomedical Applications” No. 122122600055-2 in terms of conducting optical observations using the dorsal chamber method and the topic of the state assignment of the Kurchatov Institute Research Center in terms of conducting research using luminescent spectroscopy. The design of the dorsal chamber and the stage was developed within the framework of research under contract No. 749-ЭA‑24-НИР dated 06/25/2024 between the P. N. Lebedev Physical Institute of the Russian Academy of Sciences and the B. V. Petrovsky National Research Center of Surgery.
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