The Experts below are selected from a list of 276 Experts worldwide ranked by ideXlab platform
Shinichi Ohno - One of the best experts on this subject based on the ideXlab platform.
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Renaissance of morphological studies: the examination of functional structures in living Animal Organs using the in vivo cryotechnique
Anatomical Science International, 2017Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Yurika Saitoh, Nobuo TeradaAbstract:Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living Animal Organs, as well as the locations of their molecular components. Recently, the live imaging of Animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living Animal Organs are compatible with those obtained from live images of the same Organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living Animal Organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components—such as rapidly changing signaling molecules, membrane channels, or receptors—in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.
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Biomedical Significance and Development of “IVCT”
In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs, 2016Co-Authors: Shinichi OhnoAbstract:Preparation procedures after resection of Animal tissues are commonly a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, thick or thin sectioning, and dye or metal staining steps. During such preparation steps, various kinds of inevitable artifacts always modify their original morphology. Some problems are both molecular movement and structural changes of cells and tissues during the fixation time. Another problem is that dynamic morphological images in vivo are difficult to be captured by the conventional chemical fixation. Then, a strong effort of morphologists has been made to avoid such technical artifacts during the conventional preparation steps. The quick-freezing (QF) method was introduced for biological specimens at the middle of the twentieth century. For the final morphological purpose, various preparation procedures can be chosen after the quick-freezing. One of them is freeze-substitution (FS) fixation, in which the frozen specimens are usually incubated in cooled organic solvents containing chemical fixatives at about −80 °C. However, the freeze-substituted specimens are known to be affected to some extent by the organic solvents. Another deep-etching (DE) replication method has been developed, in which replica membranes of freeze-fractured and deeply etched tissues are obtained by rotary-shadowing with platinum metal at lower temperatures below −100 °C under high vacuum conditions. However, some pieces of tissues have to be always resected and taken out from living Animal Organs. Thus, the dynamically changing morphology of living Animal Organs is hardly investigated by the conventional QF method. To overcome these technical problems, it is necessary to avoid the tissue resection step of living Animal Organs and directly freeze them in vivo under normal blood circulation. The IVCT was an original technique to directly cryofix living Animal Organs without separating their tissues from blood circulation. The most significant point of IVCT is that normal blood circulation into living Animal Organs is strictly preserved at the exact moment of freezing.
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How to Perform “IVCT”
In Vivo Cryotechnique in Biomedical Research and Application for Bioimaging of Living Animal Organs, 2016Co-Authors: Shinichi OhnoAbstract:The isopentane-propane (IP) cryogen (−193 °C) was manually poured over exposed Animal Organs by the original in vivo cryotechnique (IVCT), which were simultaneously cryocut with a precooled cryoknife in liquid nitrogen (−196 °C) with a group of three persons. The in vivo frozen Animal Organs were cracked off from the Animal body in the liquid nitrogen and then plunged into the liquid nitrogen. To perform the IVCT more easily, the “in vivo cryoapparatus” has been commercially available all over the world. The IVCT was originally performed with a handmade “in vivo cryoapparatus.” The operation manual about the new “in vivo cryoapparatus” is briefly described in this chapter. When the IVCT is used only for light microscopic observation, it can be more simply performed without any special cryoapparatus. So the simple “in vivo” freezing method is performed only by pouring the IP cryogen (−193 °C) directly onto living Animal Organs. This simple cryoprocedure usually allows us to make an easy performance of IVCT at a light microscopic level. In the prepared such specimens, good morphological preservation is within less than a few hundred micrometers away from the frozen tissue surface at a light microscopic level. The procedures to prepare the mixed isopentane-propane cryogen were described in this chapter.
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Morphofunctional Merits of an In Vivo Cryotechnique for Living Animal Organs: Challenges of Clinical Applications from Basic Medical Research
Acta histochemica et cytochemica, 2015Co-Authors: Shinichi OhnoAbstract:Recent advances in molecular and genetic techniques have led to establishment of new biomedical fields; however, morphological techniques are still required for a more precise understanding of functioning cells and tissues. Conventional preparation procedures involve a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, sectioning, and staining steps. In these steps, technical artifacts modify original morphologies of the cells being examined. Furthermore, difficulties are associated with capturing dynamic images in vivo using conventional chemical fixation. Therefore, a quick-freezing (QF) method was introduced for biological specimens in the 20th century. However, specimens have to be resected from living Animal Organs with blood supply, and their dynamical morphologies have not been investigated in detail using the QF method. In order to overcome these issues, the tissue resection step of Organs had to be avoided and samples needed to be frozen under blood circulation. Our in vivo cryotechnique (IVCT) was an original technique to cryofix samples without resecting their tissues. The most significant merit of IVCT is that blood circulation into Organs is preserved at the exact moment of freezing, which has been useful for arresting transient physiological processes of cells and tissues and maintaining their components in situ.
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Morphofunctional Significance of “In Vivo Cryotechnique” for Analyses of Cells and Tissues in Living Animal Organs
Microscopy and Microanalysis, 2011Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.
Hiroshi Kikuchi - One of the best experts on this subject based on the ideXlab platform.
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Correction to: Motion analysis for better understanding of psychomotor skills in laparoscopy: objective assessment-based simulation training using Animal Organs
Surgical Endoscopy, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:This article was updated to correct the labeling of Fig. 6.
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Motion analysis for better understanding of psychomotor skills in laparoscopy: objective assessment-based simulation training using Animal Organs
Surgical Endoscopy, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:Background Our aim was to characterize the motions of multiple laparoscopic surgical instruments among participants with different levels of surgical experience in a series of wet-lab training drills, in which participants need to perform a range of surgical procedures including grasping tissue, tissue traction and dissection, applying a Hem-o-lok clip, and suturing/knotting, and digitize the level of surgical competency. Methods Participants performed tissue dissection around the aorta, dividing encountered vessels after applying a Hem-o-lok (Task 1), and renal parenchymal closure (Task 2: suturing, Task 3: suturing and knot-tying), using swine cadaveric Organs placed in a box trainer under a motion capture (Mocap) system. Motion-related metrics were compared according to participants’ level of surgical experience (experts: 50 ≤ laparoscopic surgeries, intermediates: 10–49, novices: 0–9), using the Kruskal–Wallis test, and significant metrics were subjected to principal component analysis (PCA). Results A total of 15 experts, 12 intermediates, and 18 novices participated in the training. In Task 1, a shorter path length and faster velocity/acceleration/jerk were observed using both scissors and a Hem-o-lok applier in the experts, and Hem-o-lok-related metrics markedly contributed to the 1st principal component on PCA analysis, followed by scissors-related metrics. Higher-level skills including a shorter path length and faster velocity were observed in both hands of the experts also in tasks 2 and 3. Sub-analysis showed that, in experts with 100 ≤ cases, scissors moved more frequently in the “close zone (0 ≤ to
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correction to motion analysis for better understanding of psychomotor skills in laparoscopy objective assessment based simulation training using Animal Organs
Surgical Endoscopy and Other Interventional Techniques, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:Our aim was to characterize the motions of multiple laparoscopic surgical instruments among participants with different levels of surgical experience in a series of wet-lab training drills, in which participants need to perform a range of surgical procedures including grasping tissue, tissue traction and dissection, applying a Hem-o-lok clip, and suturing/knotting, and digitize the level of surgical competency. Participants performed tissue dissection around the aorta, dividing encountered vessels after applying a Hem-o-lok (Task 1), and renal parenchymal closure (Task 2: suturing, Task 3: suturing and knot-tying), using swine cadaveric Organs placed in a box trainer under a motion capture (Mocap) system. Motion-related metrics were compared according to participants’ level of surgical experience (experts: 50 ≤ laparoscopic surgeries, intermediates: 10–49, novices: 0–9), using the Kruskal–Wallis test, and significant metrics were subjected to principal component analysis (PCA). A total of 15 experts, 12 intermediates, and 18 novices participated in the training. In Task 1, a shorter path length and faster velocity/acceleration/jerk were observed using both scissors and a Hem-o-lok applier in the experts, and Hem-o-lok-related metrics markedly contributed to the 1st principal component on PCA analysis, followed by scissors-related metrics. Higher-level skills including a shorter path length and faster velocity were observed in both hands of the experts also in tasks 2 and 3. Sub-analysis showed that, in experts with 100 ≤ cases, scissors moved more frequently in the “close zone (0 ≤ to < 2.0 cm from aorta)” than those with 50–99 cases. Our novel Mocap system recognized significant differences in several metrics in multiple instruments according to the level of surgical experience. “Applying a Hem-o-lok clip on a pedicle” strongly reflected the level of surgical experience, and zone-metrics may be a promising tool to assess surgical expertise. Our next challenge is to give completely objective feedback to trainees on-site in the wet-lab.
Nobuhiko Ohno - One of the best experts on this subject based on the ideXlab platform.
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Renaissance of morphological studies: the examination of functional structures in living Animal Organs using the in vivo cryotechnique
Anatomical Science International, 2017Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Yurika Saitoh, Nobuo TeradaAbstract:Medical and biological scientists wish to understand the in vivo structures of the cells and tissues that make up living Animal Organs, as well as the locations of their molecular components. Recently, the live imaging of Animal cells and tissues with fluorescence-labeled proteins produced via gene manipulation has become increasingly common. Therefore, it is important to ensure that findings derived from histological or immunohistochemical tissue sections of living Animal Organs are compatible with those obtained from live images of the same Organs, which can be assessed using recently developed digital imaging techniques. Over the past two decades, we have performed immunohistochemical and morphological studies of the cells and tissues in living Animal Organs using a novel in vivo cryotechnique. The use of a specially designed liquid cryogen system with or without a cryoknife during this cryotechnique solved the technical problems that inevitably arise during the conventional preparation methods employed prior to light or electron microscopic examinations. Our in vivo cryotechnique has been found to be extremely useful for arresting transient physiological processes in cells and tissues and for maintaining their functional components—such as rapidly changing signaling molecules, membrane channels, or receptors—in situ. The purpose of the present review is to describe the basic mechanism underlying cryotechniques and the significance of our in vivo cryotechnique. In addition, it describes various morphological or immunohistochemical findings, observations made using quantum dots, and a Raman cryomicroscopy-based method for assessing oxygen saturation in the erythrocytes flowing through intestinal tissues.
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Morphofunctional Significance of “In Vivo Cryotechnique” for Analyses of Cells and Tissues in Living Animal Organs
Microscopy and Microanalysis, 2011Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.
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Significance of 'in vivo cryotechnique' for morphofunctional analyses of living Animal Organs.
Journal of electron microscopy, 2010Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:Our final goal of morphological and immunohistochemical studies is that all findings examined in Animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues of Animals is necessary for describing the functional morphology of living Animal Organs. It is generally accepted that morphological findings of various Organs were easily modified by stopping their blood supply. There had been a need to develop a new preparation technique for freezing the living Animal Organs in vivo and then obtaining acceptable morphology and also immunolocalization of original components in functioning cells and tissues. We already developed the 'in vivo cryotechnique' (IVCT) not only for their morphology, but also for immunohistochemistry of many soluble components in various living Animal Organs. All physiological processes of cells and tissues were immediately immobilized by IVCT, and every component in the cells and tissues was maintained in situ at the time of freezing. Thus, the ischaemic or anoxic effects on them could be minimized by IVCT. Our specially designed cryoknife with liquid cryogen has solved the morphological and immunohistochemical problems which are inevitable with the conventional preparation methods at a light or electron microscopic level. The IVCT will be extremely useful for arresting transient physiological processes and for maintaining any intracellular components in situ, such as rapidly changing signal molecules, membrane channels and receptors.
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In Vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:The final goal of immunohistochemical studies is that all findings examined in Animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues is necessary for describing the functional morphology of living Animal Organs. It is generally accepted that morphological findings of various Organs are easily modified during the conventional preparation steps. The quick-freezing method, by which resected tissues are quickly frozen, reduces morphological artifacts resulting in significant findings of native cells and tissues. However, tissues have to first be resected from living Animal Organs for quick-freezing. We have developed an "in vivo cryotechnique" for immunohistochemistry of some components in living Animal Organs. All physiological processes are immediately immobilized in the ice crystals by the "in vivo cryotechnique," and every components of the cells and tissues are maintained in situ at the time of freezing. Thus, ischemic or anoxic effects are minimized on immunohistochemical localization of the components. Another new "cryobiopsy" technique will be useful for capturing time-dependent morphological changes in the same Animal including humans and for maintaining intracellular components.
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Application of in vivo cryotechnique to the examination of cells and tissues in living Animal Organs.
Histology and histopathology, 2006Co-Authors: Noboyuki Terada, Nobuhiko Ohno, Takeshi Baba, Yuu Fujii, Shinichi OhnoAbstract:When all biological materials in cells and tissues of living Animal Organs are quickly and promptly frozen, immunolocalization of their components and structural features in situ is necessary to understand their in vivo functioning states. However, these direct morphological analyses were difficult to achieve by conventional chemical fixation methods during the last century. A new cryofixation method, named the "in vivo cryotechnique", in which the normal blood circulation in living Animals is always retained at the moment of freezing, has become a powerful tool to visualize the real native morphology of cells and tissues with functional meaning. The "in vivo cryotechnique" can usually be combined with a wide range of subsequent preparation techniques, and can thereby enable us to perform various direct analyses on biological samples, reflecting the physiological functions of living Animal Organs.
Koki Ebina - One of the best experts on this subject based on the ideXlab platform.
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Correction to: Motion analysis for better understanding of psychomotor skills in laparoscopy: objective assessment-based simulation training using Animal Organs
Surgical Endoscopy, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:This article was updated to correct the labeling of Fig. 6.
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Motion analysis for better understanding of psychomotor skills in laparoscopy: objective assessment-based simulation training using Animal Organs
Surgical Endoscopy, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:Background Our aim was to characterize the motions of multiple laparoscopic surgical instruments among participants with different levels of surgical experience in a series of wet-lab training drills, in which participants need to perform a range of surgical procedures including grasping tissue, tissue traction and dissection, applying a Hem-o-lok clip, and suturing/knotting, and digitize the level of surgical competency. Methods Participants performed tissue dissection around the aorta, dividing encountered vessels after applying a Hem-o-lok (Task 1), and renal parenchymal closure (Task 2: suturing, Task 3: suturing and knot-tying), using swine cadaveric Organs placed in a box trainer under a motion capture (Mocap) system. Motion-related metrics were compared according to participants’ level of surgical experience (experts: 50 ≤ laparoscopic surgeries, intermediates: 10–49, novices: 0–9), using the Kruskal–Wallis test, and significant metrics were subjected to principal component analysis (PCA). Results A total of 15 experts, 12 intermediates, and 18 novices participated in the training. In Task 1, a shorter path length and faster velocity/acceleration/jerk were observed using both scissors and a Hem-o-lok applier in the experts, and Hem-o-lok-related metrics markedly contributed to the 1st principal component on PCA analysis, followed by scissors-related metrics. Higher-level skills including a shorter path length and faster velocity were observed in both hands of the experts also in tasks 2 and 3. Sub-analysis showed that, in experts with 100 ≤ cases, scissors moved more frequently in the “close zone (0 ≤ to
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correction to motion analysis for better understanding of psychomotor skills in laparoscopy objective assessment based simulation training using Animal Organs
Surgical Endoscopy and Other Interventional Techniques, 2020Co-Authors: Koki Ebina, Takashige Abe, Madoka Higuchi, Jun Furumido, Naoya Iwahara, Masafumi Kon, Kiyohiko Hotta, Shunsuke Komizunai, Yo Kurashima, Hiroshi KikuchiAbstract:Our aim was to characterize the motions of multiple laparoscopic surgical instruments among participants with different levels of surgical experience in a series of wet-lab training drills, in which participants need to perform a range of surgical procedures including grasping tissue, tissue traction and dissection, applying a Hem-o-lok clip, and suturing/knotting, and digitize the level of surgical competency. Participants performed tissue dissection around the aorta, dividing encountered vessels after applying a Hem-o-lok (Task 1), and renal parenchymal closure (Task 2: suturing, Task 3: suturing and knot-tying), using swine cadaveric Organs placed in a box trainer under a motion capture (Mocap) system. Motion-related metrics were compared according to participants’ level of surgical experience (experts: 50 ≤ laparoscopic surgeries, intermediates: 10–49, novices: 0–9), using the Kruskal–Wallis test, and significant metrics were subjected to principal component analysis (PCA). A total of 15 experts, 12 intermediates, and 18 novices participated in the training. In Task 1, a shorter path length and faster velocity/acceleration/jerk were observed using both scissors and a Hem-o-lok applier in the experts, and Hem-o-lok-related metrics markedly contributed to the 1st principal component on PCA analysis, followed by scissors-related metrics. Higher-level skills including a shorter path length and faster velocity were observed in both hands of the experts also in tasks 2 and 3. Sub-analysis showed that, in experts with 100 ≤ cases, scissors moved more frequently in the “close zone (0 ≤ to < 2.0 cm from aorta)” than those with 50–99 cases. Our novel Mocap system recognized significant differences in several metrics in multiple instruments according to the level of surgical experience. “Applying a Hem-o-lok clip on a pedicle” strongly reflected the level of surgical experience, and zone-metrics may be a promising tool to assess surgical expertise. Our next challenge is to give completely objective feedback to trainees on-site in the wet-lab.
Yasuhisa Fujii - One of the best experts on this subject based on the ideXlab platform.
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Morphofunctional Significance of “In Vivo Cryotechnique” for Analyses of Cells and Tissues in Living Animal Organs
Microscopy and Microanalysis, 2011Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.
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Significance of 'in vivo cryotechnique' for morphofunctional analyses of living Animal Organs.
Journal of electron microscopy, 2010Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:Our final goal of morphological and immunohistochemical studies is that all findings examined in Animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues of Animals is necessary for describing the functional morphology of living Animal Organs. It is generally accepted that morphological findings of various Organs were easily modified by stopping their blood supply. There had been a need to develop a new preparation technique for freezing the living Animal Organs in vivo and then obtaining acceptable morphology and also immunolocalization of original components in functioning cells and tissues. We already developed the 'in vivo cryotechnique' (IVCT) not only for their morphology, but also for immunohistochemistry of many soluble components in various living Animal Organs. All physiological processes of cells and tissues were immediately immobilized by IVCT, and every component in the cells and tissues was maintained in situ at the time of freezing. Thus, the ischaemic or anoxic effects on them could be minimized by IVCT. Our specially designed cryoknife with liquid cryogen has solved the morphological and immunohistochemical problems which are inevitable with the conventional preparation methods at a light or electron microscopic level. The IVCT will be extremely useful for arresting transient physiological processes and for maintaining any intracellular components in situ, such as rapidly changing signal molecules, membrane channels and receptors.
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In Vivo Cryotechniques for Preparation of Animal Tissues for Immunoelectron Microscopy
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Shinichi Ohno, Nobuhiko Ohno, Nobuo Terada, Sei Saitoh, Yurika Saitoh, Yasuhisa FujiiAbstract:The final goal of immunohistochemical studies is that all findings examined in Animal experiments should reflect the physiologically functional background. Therefore, the preservation of original components in cells and tissues is necessary for describing the functional morphology of living Animal Organs. It is generally accepted that morphological findings of various Organs are easily modified during the conventional preparation steps. The quick-freezing method, by which resected tissues are quickly frozen, reduces morphological artifacts resulting in significant findings of native cells and tissues. However, tissues have to first be resected from living Animal Organs for quick-freezing. We have developed an "in vivo cryotechnique" for immunohistochemistry of some components in living Animal Organs. All physiological processes are immediately immobilized in the ice crystals by the "in vivo cryotechnique," and every components of the cells and tissues are maintained in situ at the time of freezing. Thus, ischemic or anoxic effects are minimized on immunohistochemical localization of the components. Another new "cryobiopsy" technique will be useful for capturing time-dependent morphological changes in the same Animal including humans and for maintaining intracellular components.
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"In vivo cryotechnique" for paradigm shift to "living morphology" of Animal Organs
Biomedical Reviews, 2004Co-Authors: Nobuhiko Ohno, Nobuo Terada, Yasuhisa Fujii, Takeshi Baba, Shinichi OhnoAbstract:The morphological study has been one of the major approaches in medical and biological fields. For the last century, the conventional chemical fixation and alcohol dehydration were commonly used as an easy preparation method, but it was frequently pointed out that they usually yield many structural artifacts during their preparation processes. Although both conventional quick-freezing and high-pressure freezing methods, by which Animal tissues are resected and frozen for physical fixation,can reduce such structural artifacts, the tissues have to be removed from living Animal Organs for the freezing. Therefore, such specimens are inevitably exposed to noxious stresses of anoxia and ischemia, exhibiting only dead morphological states of Animal tissues without blood circulation. To the contrary, our "in vivo cryotechnique", by which all cells and tissues in Animal bodies are cryofixed in vivo, can prevent such artifacts of resected specimens. By means of the cryotechnique, it is now possible to reveal the in vivo morphology of cells and tissues in living Animal Organs. Actually, it has been already applied to several Animal Organs, such as kidney, liver, intestine, cerebellum, eye ball, blood vessel, and joint cartilage, and brought new morphological findings, reflecting their physiological significance, which had been difficult to demonstrate by the conventional preparation methods. Moreover, its application to immunohistochemistry has also revealed more precise immunolocalizations of dynamically changing molecules in living Animal Organs, easily translocated by ischemic stresses and anoxia caused during the tissue resection. The "in vivo cryotechnique" allows us to perform novel morphological investigations of "living" morphological states, and develops new medical and biological fields with "living morphology" during this 21st century. Biomedical Reviews 2004; 15: 1-19.
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Ultrastructural Analyses of Living Animal Organs Prepared by "in vivo Cryotechnique" for Electron Microscopy
Microscopy and Microanalysis, 2001Co-Authors: Shinichi Ohno, Nobuo Terada, Yasuhisa Fujii, Ichiro Takayama, H. Ueda, Takeshi BabaAbstract:It is well known that hemodynamic factors, such as blood pressure and flow, exert an important influence on native morphology of various Animal Organs. However, information about the significance of ultrastructures which are revealed by conventional preparation methods has been limited, because routine chemical fixation of cells and tissues takes considerable time, during which their morphology could easily change. Therefore, morphological studies with routine immersion or perfusion fixation have not revealed real ultrastructures of functioning Animal Organs with normal blood circulation.2 The ultimate goal of morphological study is that all features to be examined should reflect the physiological meaning under investigation. For that purpose, the preservation of cells and tissues in functioning Organs is necessary for studies to define their ultrastructures. Most cryotechniques have been based on the use of prior excised tissues. Such small specimens are commonly frozen within several seconds following the excision of the tissues.