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【转帖】【分享帖】【英文资料】激光共聚焦显微镜的应用
【转帖】【分享帖】【英文资料】激光共聚焦显微镜的应用
Applications of confocal microscopy
Detection of apoptosis
Assymetry of plasma membranes is disturbed when a cell undergoes apoptosis. In normal, cycling cells, phosphatidylserine is found exclusively in the inner leaflet of the plasma membrane. In apoptotic cells phosphatidylserine is also found in the outer leaflet of the membrane and can be detected by annexin binding.
Top: a schematic representation of the principle of detection of apoptosis by staining of plasma membranes with annexin. Left: a microscope image of a group of normal cells and one apototic cell which binds annexin (green) on the surface. Annexin is labeled with fluorescein.
In apoptosis condensation and fragmentation of chromatin occurs. Subsequently, nuclei loose their round or oval shape, bud and become fragmented - apoptotic bodies are formed.
Nuclei of normal human fibroblasts in culture, in early (left) and advanced (right) spontaneous apoptosis.
Apoptosis caused in HL60 cells by camptothecin (topoisomerase inhibitor). Images of nuclei in early (top, left), mid (top, right) and late (bottom) apoptosis.
A schematic view of a nucleus in a cell undergoing apoptosis: healthy cell (top, right), early apoptosis (top, left), advanced apoptosis (bottom, right), late apoptosis (bottom, left).
Cell cycle analysis
Subsequent stages of the cell division cycle. Histone H2B was tagged with green fluorescent protein (GFP). This made it possible to obtain fluorescence confocal microscopy images of chromatin.
Interphase nuclei
Prophase nuclei
Anaphase nuclei
Telophase nucleus
A schematic representation of changes of chromatin content and architecture of nuclei in cell cycle.
Assesment of cell viability
Cell damage may be manifested by a loss of plasma membrane integrity. Two popular tests are used to reveal and measure membrane damage: a test with fluorescein diacetate (FDA) and a propidium iodide exclusion test. FDA enters cells and is hydrolised to free fluorescein and acetate. Fluorescein is not membrane permeable and is trapped in intact cells. thus, cells with intact membranes acquire green fluorescence. Damaged cells are not fluorescent in this test. PI cannot cross intact membranes and is excluded from undamaged cells. If the integrity of plasma membranes is compromised, PI enters cell interior and binds to nucleic acids - RNA and DNA. Upon binding, the intensity of PI fluorescence increases many-fold. Thus, in this test damaged cells fluoresce red, while intact cells are not detected.
A schematic illustration of the principle of PI/FDA cell viability assay
A video shows a group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.
Visualisation of cytoskeleton
Actin (left) and tubulin (right) in keratocytes, visualized by immunofluorescence.
A schematic representation of cytoskeleton in a keratocyte. Actin fibres are depicted as straight, thick lines (red), whereas microtubules are drawn as twisted, thin lines (green) surrounding the nucleus.
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【转帖】【分享帖】【英文资料】激光共聚焦显微镜的应用
【转帖】【分享帖】【英文资料】激光共聚焦显微镜的应用
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sr9971 (2014-11-01 16:16:54)
Analysis of architecture of extracellular matrix
Extracellular matrix (ECM) is a complicated three-dimensional macromolecular assembly of several kinds of collagens, elastin, glycoproteins, glycosaminoglycans, and proteoglycans. It defines the histoarchitecture specific for every organ, provides cells with information and a mechanical scaffold for adhesion and migration. ECM controls various physiological phenomena including acquisition and maintenance of differentiated phenotypes during embryogenesis, development of form (morphogenesis), vessel formation (angiogenesis), wound healing, and tumor metastasis.
HTB-140 melanoma cells cultured within collagen type i (model of extracellular matrix). The cells were staind with vital dye FM-1-43 (red color). Collagen fibers were visualized using backscattered light (gray color).
Confocal image of HepG2 cells culture within collagen type I. The image is based on backscattered light. Additionaly to 3D volume rendering, depth is coded with colors - from red denoting proximity to violet meaning farness.
3D volume rendering of an image demonstrating the structure of picro-sirius red stained sis (small intestinal submucosa - natural ECM).
sr9971 (2014-11-01 16:17:39)
Cardiomyocytes in a functioning heart contract in a coordinated manner. These cells communicate via gap junctions. In a healthy cardiomyocyte gap junctions are located only at the distant ends where the cell communicates with two neighbours.In a Falot cardiomyocyte gap junctions are distributed randomly over the surface of the cell. this anomaly in a distribution of gap junctions is suspected to play a role in Falot tetralogy.
Images of cardiomyocytes (blue) taken from a heart of healthy donor (left) and a donor suffering from Falot tetralogy (right). Connexin - a protein in gap junctions was stained by immunofluorescence (yellow-red).
A schematic image of gap junctions in an assembly of cardiomyocytes in normal state and in Fallot teratology.
Measurement of mitochondrial electric potential
Mitochondria in a normal human fibroblast in culture, stained with JC-1 fluorescent probe. JC-1 accumulates in mitochondria due to high membrane potential. It fluoresces green in low energy mitochondria. In mitochondria or parts of mitochondria with high membrane potential JC-1 is accumulated to a higher concentration and forms J-aggregates that fluoresce red.
The movie: distribution of high and low energy regions in mitochondria and the movement of large and small mitochondria in a living cell.
Measurement of cell redox capacity
Reduction of a tetrazolium salt, mtt, to a corresponding formazan is often used a convenient test of cell metabolic activity. It is also used as an alternative to a clonogenic assay. This series of images shows mitochondria (green) in hepatoma cells in culture and formation of MTT-formazan following addition of culture medium supplemented with MTT. Crystals of MTT appeared as blue dots and were detected using reflected light confocal microscopy.
Schematic illustration of the process of formation of MTT-formazan deposits in relation to mitochondria stained with fluorescent dye (TMRE).
Cells prior to the experiment; M - mitochondria, N - nuclei
Image of mitochondria loaded with the fluorescent dye (nuclei and cytoplasm can not bee seen) Following addttion of a tetrazolium salt (MTT) fluorescence of the mitochondrial marker decreases (probably due to the displacement of the dye by non-fluorescent MTT).
Cells reduce MTT to the respective formazan. deposits of the formazan (blue) are imaged in backscattered light. Following 30 minutes of incubation with mtt cells are loaded with deposits of MTT-formazan (blue). the fluorescence of the mitochondrial marker is undetectable.
The movie: formation of MTT formazan in HepG2 cells with mitochondria stained with TMRE.
Detection of phagocytosis
These images show macrophages in culture, following a 24 hour-long incubation with silica particles. Some particles were phagocytozed by macrophages. Cells were fixed with formaldehyde and stained with propidium iodide (PI). PI stains RNA and DNA, thus the transmitted light image shows only cell shapes, while a fluorescence (red) and dark field reflected light confoal microscopy (green) images reveal cytoplasm, nuclei, nucleoli (red) and silica particles (green) ingested by a macrophage.
Phagocytosis of silica particles by macrophages in culture. Left: fluorescence of nuclei (red) and light reflected by silica particles (green). Right: transmitted light.
Schematic view of phagocytosis of silica particles by macrophages. Silica particles are marked in green, nucleic acids in the cells (DNA in nuclei and RNA in cytoplasm) marked in red.
These images show a group of human monocytes in culture, following incubation with bacteria. Mitochondria were stained fluorescently green, while bacteria were stained fluorescently red. Transmitted light image shows cell shapes, while a corresponding confocal fluorescence image shows mitochondria and phagocytozed bacteria.
Phagocytosis of bacteria by monocytes in culture. Images of transmitted light (right) and fluorescence (left) of bacteria (red) and mitochondria (green); cell contours are shown as white lines.
sr9971 (2014-11-01 16:18:08)
Applications of confocal microscopy
Detection of apoptosis
Assymetry of plasma membranes is disturbed when a cell undergoes apoptosis. In normal, cycling cells, phosphatidylserine is found exclusively in the inner leaflet of the plasma membrane. In apoptotic cells phosphatidylserine is also found in the outer leaflet of the membrane and can be detected by annexin binding.
Top: a schematic representation of the principle of detection of apoptosis by staining of plasma membranes with annexin. Left: a microscope image of a group of normal cells and one apototic cell which binds annexin (green) on the surface. Annexin is labeled with fluorescein.
In apoptosis condensation and fragmentation of chromatin occurs. Subsequently, nuclei loose their round or oval shape, bud and become fragmented - apoptotic bodies are formed.
Nuclei of normal human fibroblasts in culture, in early (left) and advanced (right) spontaneous apoptosis.
Apoptosis caused in HL60 cells by camptothecin (topoisomerase inhibitor). Images of nuclei in early (top, left), mid (top, right) and late (bottom) apoptosis.
A schematic view of a nucleus in a cell undergoing apoptosis: healthy cell (top, right), early apoptosis (top, left), advanced apoptosis (bottom, right), late apoptosis (bottom, left).
Cell cycle analysis
Subsequent stages of the cell division cycle. Histone H2B was tagged with green fluorescent protein (GFP). This made it possible to obtain fluorescence confocal microscopy images of chromatin.
Interphase nuclei
Prophase nuclei
Anaphase nuclei
Telophase nucleus
A schematic representation of changes of chromatin content and architecture of nuclei in cell cycle.
Assesment of cell viability
Cell damage may be manifested by a loss of plasma membrane integrity. Two popular tests are used to reveal and measure membrane damage: a test with fluorescein diacetate (FDA) and a propidium iodide exclusion test. FDA enters cells and is hydrolised to free fluorescein and acetate. Fluorescein is not membrane permeable and is trapped in intact cells. thus, cells with intact membranes acquire green fluorescence. Damaged cells are not fluorescent in this test. PI cannot cross intact membranes and is excluded from undamaged cells. If the integrity of plasma membranes is compromised, PI enters cell interior and binds to nucleic acids - RNA and DNA. Upon binding, the intensity of PI fluorescence increases many-fold. Thus, in this test damaged cells fluoresce red, while intact cells are not detected.
A schematic illustration of the principle of PI/FDA cell viability assay
A video shows a group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.
Visualisation of cytoskeleton
Actin (left) and tubulin (right) in keratocytes, visualized by immunofluorescence.
A schematic representation of cytoskeleton in a keratocyte. Actin fibres are depicted as straight, thick lines (red), whereas microtubules are drawn as twisted, thin lines (green) surrounding the nucleus.
sr9971 (2014-11-01 16:18:36)
Analysis of architecture of extracellular matrix
Extracellular matrix (ECM) is a complicated three-dimensional macromolecular assembly of several kinds of collagens, elastin, glycoproteins, glycosaminoglycans, and proteoglycans. It defines the histoarchitecture specific for every organ, provides cells with information and a mechanical scaffold for adhesion and migration. ECM controls various physiological phenomena including acquisition and maintenance of differentiated phenotypes during embryogenesis, development of form (morphogenesis), vessel formation (angiogenesis), wound healing, and tumor metastasis.
HTB-140 melanoma cells cultured within collagen type i (model of extracellular matrix). The cells were staind with vital dye FM-1-43 (red color). Collagen fibers were visualized using backscattered light (gray color).
Confocal image of HepG2 cells culture within collagen type I. The image is based on backscattered light. Additionaly to 3D volume rendering, depth is coded with colors - from red denoting proximity to violet meaning farness.
3D volume rendering of an image demonstrating the structure of picro-sirius red stained sis (small intestinal submucosa - natural ECM).
Visualisation of gap junctions
Cardiomyocytes in a functioning heart contract in a coordinated manner. These cells communicate via gap junctions. In a healthy cardiomyocyte gap junctions are located only at the distant ends where the cell communicates with two neighbours.In a Falot cardiomyocyte gap junctions are distributed randomly over the surface of the cell. this anomaly in a distribution of gap junctions is suspected to play a role in Falot tetralogy.
Images of cardiomyocytes (blue) taken from a heart of healthy donor (left) and a donor suffering from Falot tetralogy (right). Connexin - a protein in gap junctions was stained by immunofluorescence (yellow-red).
A schematic image of gap junctions in an assembly of cardiomyocytes in normal state and in Fallot teratology.
Measurement of mitochondrial electric potential
Mitochondria in a normal human fibroblast in culture, stained with JC-1 fluorescent probe. JC-1 accumulates in mitochondria due to high membrane potential. It fluoresces green in low energy mitochondria. In mitochondria or parts of mitochondria with high membrane potential JC-1 is accumulated to a higher concentration and forms J-aggregates that fluoresce red.
The movie: distribution of high and low energy regions in mitochondria and the movement of large and small mitochondria in a living cell.
Measurement of cell redox capacity
Reduction of a tetrazolium salt, mtt, to a corresponding formazan is often used a convenient test of cell metabolic activity. It is also used as an alternative to a clonogenic assay. This series of images shows mitochondria (green) in hepatoma cells in culture and formation of MTT-formazan following addition of culture medium supplemented with MTT. Crystals of MTT appeared as blue dots and were detected using reflected light confocal microscopy.
Schematic illustration of the process of formation of MTT-formazan deposits in relation to mitochondria stained with fluorescent dye (TMRE).
Cells prior to the experiment; M - mitochondria, N - nuclei
Image of mitochondria loaded with the fluorescent dye (nuclei and cytoplasm can not bee seen) Following addttion of a tetrazolium salt (MTT) fluorescence of the mitochondrial marker decreases (probably due to the displacement of the dye by non-fluorescent MTT).
Cells reduce MTT to the respective formazan. deposits of the formazan (blue) are imaged in backscattered light. Following 30 minutes of incubation with mtt cells are loaded with deposits of MTT-formazan (blue). the fluorescence of the mitochondrial marker is undetectable.
The movie: formation of MTT formazan in HepG2 cells with mitochondria stained with TMRE
sr9971 (2014-11-01 16:18:57)
These images show macrophages in culture, following a 24 hour-long incubation with silica particles. Some particles were phagocytozed by macrophages. Cells were fixed with formaldehyde and stained with propidium iodide (PI). PI stains RNA and DNA, thus the transmitted light image shows only cell shapes, while a fluorescence (red) and dark field reflected light confoal microscopy (green) images reveal cytoplasm, nuclei, nucleoli (red) and silica particles (green) ingested by a macrophage.
Phagocytosis of silica particles by macrophages in culture. Left: fluorescence of nuclei (red) and light reflected by silica particles (green). Right: transmitted light.
Schematic view of phagocytosis of silica particles by macrophages. Silica particles are marked in green, nucleic acids in the cells (DNA in nuclei and RNA in cytoplasm) marked in red.
These images show a group of human monocytes in culture, following incubation with bacteria. Mitochondria were stained fluorescently green, while bacteria were stained fluorescently red. Transmitted light image shows cell shapes, while a corresponding confocal fluorescence image shows mitochondria and phagocytozed bacteria.
Phagocytosis of bacteria by monocytes in culture. Images of transmitted light (right) and fluorescence (left) of bacteria (red) and mitochondria (green); cell contours are shown as white lines.
wood533 (2014-11-01 16:19:26)
sunbent (2014-11-01 16:19:43)
【转帖】【分享帖】【英文资料】激光共聚焦显微镜的应用