rus
TS_pub
technospheramag
technospheramag
ТЕХНОСФЕРА_РИЦ
Issue #1/2024
A.I.Akhmetova, T.O.Sovetnikov, E.O.Zorikova, I.V.Yaminsky
SCANNING PROBE MICROSCOPY OF SUBSTANTIA NIGRA
SCANNING PROBE MICROSCOPY OF SUBSTANTIA NIGRA
DOI: 10.22184/1993-8578.2024.17.1.26.31
The response of cells to mechanical signals plays a key role in biological processes such as organ development, tissue regeneration, aging and cancer development. Changes in mechanical properties, including stiffness and viscosity, show how cells and tissues react to stress and how their biological functions depend on it. Scanning probe microscopy (SPM) is a versatile tool for quantitative characterization of the mechanical properties of tissues and cells in vivo. Data on the mechanical properties of biological objects obtained using atomic force and capillary microscopy can be associated with biological processes and pathologies in tissues.
The response of cells to mechanical signals plays a key role in biological processes such as organ development, tissue regeneration, aging and cancer development. Changes in mechanical properties, including stiffness and viscosity, show how cells and tissues react to stress and how their biological functions depend on it. Scanning probe microscopy (SPM) is a versatile tool for quantitative characterization of the mechanical properties of tissues and cells in vivo. Data on the mechanical properties of biological objects obtained using atomic force and capillary microscopy can be associated with biological processes and pathologies in tissues.
INTRODUCTION
Scanning probe microscopy includes the whole family of techniques where surface profile is recorded using a probe: it is not only atomic force microscopy, but also other types of microscopy: tunnelling, capillary, resistive, and others.
In AFM, topography and mechanical information of a sample are determined by measuring interaction between the sample surface and the cantilever probe during line-by-line scanning of the sample. The sub-nanometre resolution in AFM allows measuring the mechanical properties of biological objects: stiffness, viscosity and adhesion, so that it is possible to match the properties images of surface and subcellular structures of different types of tissue from diseased and healthy donors. Scanning capillary microscopy is a unique tool that allows studying 3D morphology of living cells and tissues in liquid with nanometre precision.
Quantitative visualisation in AFM and measurement of mechanical properties of tissues and cells are of particular interest for in vivo assessment of biological objects and thus require non-destructive preparation to preserve them in vitro for data collection in normality and disease [1].
AFM has been used in study of heart and aortic tissues to detect mechanobiological changes associated with physiological and pathological processes such as regeneration, cardiac development, diabetes mellitus, and coronary heart disease [2]. However, so far preparation of tissues for microscope measurement remains a rather difficult task due to presence of topographic heterogeneities, uniqueness of sample preparation techniques for a particular tissue type.
In scanning capillary microscopy (SCM), a capillary filled with electrolyte acts as a probe. The capillary is positioned by measuring the magnitude of the ionic current flowing through the tip of the capillary. The uniqueness of capillary microscopy lies in the non-contact nature of scanning – the capillary stops above the sample surface when the current drops by ~0.5% of the specified value [3].
Using SСM we determined changes in morphometric characteristics of endothelial cells during experimental septicopaemia: endothelial cells decreased the adhesion area to substrate, which was determined by a decrease in the area of cell projection, and the cell membrane was smoothed. However, endothelial cell stiffness was paradoxically unchanged compared with controls. Over time, neutrophil migration led to more significant changes in endothelial cells: first, shallow perforations in membranes were formed, which healed rather quickly, then stress fibrils were formed and, finally, endothelial cells died and multiple perforations were formed on their surface [4].
However, despite the advances in SPM [5], the obtained data are still difficult to use in clinical diagnosis and medicine.
MATERIALS AND METHODS
Tissue slices of Substantia nigra from donor without neurological pathology and Parkinson’s patients were examined using a developed scanning capillary microscopy unit.
The excised biomaterial fragments were placed in 10% neutral formalin solution for fixation, with the volume of fixing liquid exceeding the volume of biomaterial fragments 10 times. Fixation in formalin was carried out at room temperature for 24-48 hours. After one day the fixing liquid solution was changed.
After fixation, the biomaterial fragments removed from the fixing fluid were washed in running water for 2 hours. Next, histological wiring of the biomaterial through alcohols of ascending concentration, xylene-paraffin and liquid paraffin was performed by automated isopropyl wiring using an automatic processor Leica TP 1020. The paraffin-impregnated biomaterial fragments were embedded in special warmed casting moulds and poured with commercial paraffin melted at 60 °C on a Leica EG 1160 casting station to form paraffin blocks. A plastic histological cassette was used as a base for the paraffin block. The finished blocks were cooled.
Histological sections 3–4 µm thick were made from paraffin blocks on a Leica RM 2235 rotary microtome.
Prepared slices (two from each tissue fragment) for light microscopy after spreading on a water bath were placed on slides and dried on a heating table.
Before staining, these samples were freed from paraffin by running them through a battery of solvents (xylene, alcohol). The specimens were stained with haematoxylin and eosin in strict accordance with the instructions of the dye sets and according to the standard procedure. For the conclusion of histological preparations, a transparent preserving medium (e.g., Canada or fir balsam) was applied to the stained section, not covered with cover glass.
The study was performed on a FemtoScan Xi scanning capillary microscope [6], and data processing was performed in FemtoScan Online software [7].
Capillary microscopy was used to obtain 3D morphology of the tissue surface and to detect visual differences in the samples.
Tissue samples from donor without neurological pathology are characterised by a more structured surface, presence of characteristic depressions with craters and pronounced edges. Morphology of slices from Parkinson’s patients is visually more homogeneous, has no characteristic drops with even edges. At the same time general roughness of healthy tissue samples was higher than in Parkinson’s patients: the average value of roughness was Ra 196 ±33 nm, Rq 250 ± 48 nm, similar parameters for samples from Parkinson’s patients: Ra 143 ± 17 nm, Rq 181 ± 19 nm.
When comparing morphology of the two specimens, characteristic differences are noticeable, but more statistical data on the study of tissue slices, including those obtained with AFM, are required. An important indicator of this area of Substantia nigra is also specimens conductivity, which can be measured using AFM [8]. The question of the relationship between changes in tissue morphology of patients and direct signs of Parkinson’s disease remains unresolved.
CONCLUSION
As part of the work, slices of Substantia nigra were obtained from a healthy donor and a Parkinson’s patient. The samples were examined using scanning capillary microscopy. The 3D morphology of the slices was evaluated. It was visually shown that the tissue slices from a donor without neurological pathology have a more branched and rougher surface compared to samples from Parkinson’s patients.
ACKNOWLEDGMENTS
The work was performed under the state order with the financial support of Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software was provided by Advanced Technologies Center, www.nanoscopy.ru.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Scanning probe microscopy includes the whole family of techniques where surface profile is recorded using a probe: it is not only atomic force microscopy, but also other types of microscopy: tunnelling, capillary, resistive, and others.
In AFM, topography and mechanical information of a sample are determined by measuring interaction between the sample surface and the cantilever probe during line-by-line scanning of the sample. The sub-nanometre resolution in AFM allows measuring the mechanical properties of biological objects: stiffness, viscosity and adhesion, so that it is possible to match the properties images of surface and subcellular structures of different types of tissue from diseased and healthy donors. Scanning capillary microscopy is a unique tool that allows studying 3D morphology of living cells and tissues in liquid with nanometre precision.
Quantitative visualisation in AFM and measurement of mechanical properties of tissues and cells are of particular interest for in vivo assessment of biological objects and thus require non-destructive preparation to preserve them in vitro for data collection in normality and disease [1].
AFM has been used in study of heart and aortic tissues to detect mechanobiological changes associated with physiological and pathological processes such as regeneration, cardiac development, diabetes mellitus, and coronary heart disease [2]. However, so far preparation of tissues for microscope measurement remains a rather difficult task due to presence of topographic heterogeneities, uniqueness of sample preparation techniques for a particular tissue type.
In scanning capillary microscopy (SCM), a capillary filled with electrolyte acts as a probe. The capillary is positioned by measuring the magnitude of the ionic current flowing through the tip of the capillary. The uniqueness of capillary microscopy lies in the non-contact nature of scanning – the capillary stops above the sample surface when the current drops by ~0.5% of the specified value [3].
Using SСM we determined changes in morphometric characteristics of endothelial cells during experimental septicopaemia: endothelial cells decreased the adhesion area to substrate, which was determined by a decrease in the area of cell projection, and the cell membrane was smoothed. However, endothelial cell stiffness was paradoxically unchanged compared with controls. Over time, neutrophil migration led to more significant changes in endothelial cells: first, shallow perforations in membranes were formed, which healed rather quickly, then stress fibrils were formed and, finally, endothelial cells died and multiple perforations were formed on their surface [4].
However, despite the advances in SPM [5], the obtained data are still difficult to use in clinical diagnosis and medicine.
MATERIALS AND METHODS
Tissue slices of Substantia nigra from donor without neurological pathology and Parkinson’s patients were examined using a developed scanning capillary microscopy unit.
The excised biomaterial fragments were placed in 10% neutral formalin solution for fixation, with the volume of fixing liquid exceeding the volume of biomaterial fragments 10 times. Fixation in formalin was carried out at room temperature for 24-48 hours. After one day the fixing liquid solution was changed.
After fixation, the biomaterial fragments removed from the fixing fluid were washed in running water for 2 hours. Next, histological wiring of the biomaterial through alcohols of ascending concentration, xylene-paraffin and liquid paraffin was performed by automated isopropyl wiring using an automatic processor Leica TP 1020. The paraffin-impregnated biomaterial fragments were embedded in special warmed casting moulds and poured with commercial paraffin melted at 60 °C on a Leica EG 1160 casting station to form paraffin blocks. A plastic histological cassette was used as a base for the paraffin block. The finished blocks were cooled.
Histological sections 3–4 µm thick were made from paraffin blocks on a Leica RM 2235 rotary microtome.
Prepared slices (two from each tissue fragment) for light microscopy after spreading on a water bath were placed on slides and dried on a heating table.
Before staining, these samples were freed from paraffin by running them through a battery of solvents (xylene, alcohol). The specimens were stained with haematoxylin and eosin in strict accordance with the instructions of the dye sets and according to the standard procedure. For the conclusion of histological preparations, a transparent preserving medium (e.g., Canada or fir balsam) was applied to the stained section, not covered with cover glass.
The study was performed on a FemtoScan Xi scanning capillary microscope [6], and data processing was performed in FemtoScan Online software [7].
Capillary microscopy was used to obtain 3D morphology of the tissue surface and to detect visual differences in the samples.
Tissue samples from donor without neurological pathology are characterised by a more structured surface, presence of characteristic depressions with craters and pronounced edges. Morphology of slices from Parkinson’s patients is visually more homogeneous, has no characteristic drops with even edges. At the same time general roughness of healthy tissue samples was higher than in Parkinson’s patients: the average value of roughness was Ra 196 ±33 nm, Rq 250 ± 48 nm, similar parameters for samples from Parkinson’s patients: Ra 143 ± 17 nm, Rq 181 ± 19 nm.
When comparing morphology of the two specimens, characteristic differences are noticeable, but more statistical data on the study of tissue slices, including those obtained with AFM, are required. An important indicator of this area of Substantia nigra is also specimens conductivity, which can be measured using AFM [8]. The question of the relationship between changes in tissue morphology of patients and direct signs of Parkinson’s disease remains unresolved.
CONCLUSION
As part of the work, slices of Substantia nigra were obtained from a healthy donor and a Parkinson’s patient. The samples were examined using scanning capillary microscopy. The 3D morphology of the slices was evaluated. It was visually shown that the tissue slices from a donor without neurological pathology have a more branched and rougher surface compared to samples from Parkinson’s patients.
ACKNOWLEDGMENTS
The work was performed under the state order with the financial support of Physical Department of Lomonosov Moscow State University (Registration subject 122091200048-7). FemtoScan Online software was provided by Advanced Technologies Center, www.nanoscopy.ru.
PEER REVIEW INFO
Editorial board thanks the anonymous reviewer(s) for their contribution to the peer review of this work. It is also grateful for their consent to publish papers on the journal’s website and SEL eLibrary eLIBRARY.RU.
Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Readers feedback
