PMC:2570081 / 3929-7204 JSONTXT

{"project":"TEST0","denotations":[{"id":"18982097-226-234-722363","span":{"begin":844,"end":848},"obj":"[\"15589093\"]"},{"id":"18982097-230-238-722364","span":{"begin":864,"end":868},"obj":"[\"17870180\"]"},{"id":"18982097-229-237-722365","span":{"begin":1660,"end":1664},"obj":"[\"18353998\"]"}],"text":"The problems\nThe above analyses are still subject to several major limitations. There are two main obstacles at the light microscope level. First, it is necessary to use relatively thin tissue slices to visualize labeled neurons, frequently in the order of a few microns, in contrast to the hundreds of microns or even millimeters over which neuronal processes may expand. Currently, this problem can only be overcome by using serial sections to reconstruct the cell in 3D. The second major limitation is that the processes are not always easy to trace and they may be lost in the background noise at times. Together, these drawbacks make it very laborious and time-consuming to obtain meaningful measurements from neurons and thus, automated techniques must be developed to assist in the reconstruction of axons and dendrites (Schmitt et al., 2004; Macke et al., 2008). There are also two important problems at the electron microscope level. First, obtaining long series of ultrathin sections is extremely time-consuming and difficult, often making it impossible to completely reconstruct neurons or their processes (particularly axons). This makes electron microscopy impractical to study large numbers of individual neurons in detail, or to reconstruct large volumes of tissue. Therefore, most studies are based on the analysis of a few cells, limiting the value of the general conclusions drawn from these observations. Hence, the development of automated electron microscopy techniques will represent another important step in the study of neuronal circuits (e.g., serial section scanning electron microscopy using focused ion beam milling: Knott et al., 2008). A second problem that is often disregarded is the difficulty in studying synaptic inputs to labelled neurons. Most techniques used to visualize neurons produce intense, homogeneous intracellular staining, which unfortunately mask post-synaptic densities (such as the deposit produced by the chromogen 3,3' diaminobenzidine tetrahydrochloride). This makes it difficult or in many cases impossible to identify synaptic inputs when the presynaptic element is also labelled. This is what I call in laboratory jargon “arrowed synapses”, those that are only seen when indicated by an arrow in a figure. Thus, this problem represents another important challenge and techniques to label neurons without masking their ultrastructure must be used (e.g., techniques that employ gold particles).\nFurthermore, full reconstruction of whole brains at the electron microscope level is possible in some invertebrates, or for relatively simple nervous systems. However, even for a small mammal like the mouse it is impossible to fully reconstruct the brain at this level. The magnification needed to visualize chemical synapses yield microscopy images of at least 10 x 10 microns in size (higher magnification is needed in the case of electrical synapses, making these fields even smaller), so to reconstruct only 1 mm3 would require the assembly of a stack of around 106 images. Full reconstructions of a small region of the mammalian brain are feasible, but structures like the cerebral cortex which can reach a surface area of 2,200 cm2 and a thickness that varies between 1.5 to 4.5 mm in humans is certainly not possible."}

{"project":"0_colil","denotations":[{"id":"18982097-15589093-722363","span":{"begin":844,"end":848},"obj":"15589093"},{"id":"18982097-17870180-722364","span":{"begin":864,"end":868},"obj":"17870180"},{"id":"18982097-18353998-722365","span":{"begin":1660,"end":1664},"obj":"18353998"}],"text":"The problems\nThe above analyses are still subject to several major limitations. There are two main obstacles at the light microscope level. First, it is necessary to use relatively thin tissue slices to visualize labeled neurons, frequently in the order of a few microns, in contrast to the hundreds of microns or even millimeters over which neuronal processes may expand. Currently, this problem can only be overcome by using serial sections to reconstruct the cell in 3D. The second major limitation is that the processes are not always easy to trace and they may be lost in the background noise at times. Together, these drawbacks make it very laborious and time-consuming to obtain meaningful measurements from neurons and thus, automated techniques must be developed to assist in the reconstruction of axons and dendrites (Schmitt et al., 2004; Macke et al., 2008). There are also two important problems at the electron microscope level. First, obtaining long series of ultrathin sections is extremely time-consuming and difficult, often making it impossible to completely reconstruct neurons or their processes (particularly axons). This makes electron microscopy impractical to study large numbers of individual neurons in detail, or to reconstruct large volumes of tissue. Therefore, most studies are based on the analysis of a few cells, limiting the value of the general conclusions drawn from these observations. Hence, the development of automated electron microscopy techniques will represent another important step in the study of neuronal circuits (e.g., serial section scanning electron microscopy using focused ion beam milling: Knott et al., 2008). A second problem that is often disregarded is the difficulty in studying synaptic inputs to labelled neurons. Most techniques used to visualize neurons produce intense, homogeneous intracellular staining, which unfortunately mask post-synaptic densities (such as the deposit produced by the chromogen 3,3' diaminobenzidine tetrahydrochloride). This makes it difficult or in many cases impossible to identify synaptic inputs when the presynaptic element is also labelled. This is what I call in laboratory jargon “arrowed synapses”, those that are only seen when indicated by an arrow in a figure. Thus, this problem represents another important challenge and techniques to label neurons without masking their ultrastructure must be used (e.g., techniques that employ gold particles).\nFurthermore, full reconstruction of whole brains at the electron microscope level is possible in some invertebrates, or for relatively simple nervous systems. However, even for a small mammal like the mouse it is impossible to fully reconstruct the brain at this level. The magnification needed to visualize chemical synapses yield microscopy images of at least 10 x 10 microns in size (higher magnification is needed in the case of electrical synapses, making these fields even smaller), so to reconstruct only 1 mm3 would require the assembly of a stack of around 106 images. Full reconstructions of a small region of the mammalian brain are feasible, but structures like the cerebral cortex which can reach a surface area of 2,200 cm2 and a thickness that varies between 1.5 to 4.5 mm in humans is certainly not possible."}

{"project":"2_test","denotations":[{"id":"18982097-15589093-38590122","span":{"begin":844,"end":848},"obj":"15589093"},{"id":"18982097-17870180-38590123","span":{"begin":864,"end":868},"obj":"17870180"},{"id":"18982097-18353998-38590124","span":{"begin":1660,"end":1664},"obj":"18353998"}],"text":"The problems\nThe above analyses are still subject to several major limitations. There are two main obstacles at the light microscope level. First, it is necessary to use relatively thin tissue slices to visualize labeled neurons, frequently in the order of a few microns, in contrast to the hundreds of microns or even millimeters over which neuronal processes may expand. Currently, this problem can only be overcome by using serial sections to reconstruct the cell in 3D. The second major limitation is that the processes are not always easy to trace and they may be lost in the background noise at times. Together, these drawbacks make it very laborious and time-consuming to obtain meaningful measurements from neurons and thus, automated techniques must be developed to assist in the reconstruction of axons and dendrites (Schmitt et al., 2004; Macke et al., 2008). There are also two important problems at the electron microscope level. First, obtaining long series of ultrathin sections is extremely time-consuming and difficult, often making it impossible to completely reconstruct neurons or their processes (particularly axons). This makes electron microscopy impractical to study large numbers of individual neurons in detail, or to reconstruct large volumes of tissue. Therefore, most studies are based on the analysis of a few cells, limiting the value of the general conclusions drawn from these observations. Hence, the development of automated electron microscopy techniques will represent another important step in the study of neuronal circuits (e.g., serial section scanning electron microscopy using focused ion beam milling: Knott et al., 2008). A second problem that is often disregarded is the difficulty in studying synaptic inputs to labelled neurons. Most techniques used to visualize neurons produce intense, homogeneous intracellular staining, which unfortunately mask post-synaptic densities (such as the deposit produced by the chromogen 3,3' diaminobenzidine tetrahydrochloride). This makes it difficult or in many cases impossible to identify synaptic inputs when the presynaptic element is also labelled. This is what I call in laboratory jargon “arrowed synapses”, those that are only seen when indicated by an arrow in a figure. Thus, this problem represents another important challenge and techniques to label neurons without masking their ultrastructure must be used (e.g., techniques that employ gold particles).\nFurthermore, full reconstruction of whole brains at the electron microscope level is possible in some invertebrates, or for relatively simple nervous systems. However, even for a small mammal like the mouse it is impossible to fully reconstruct the brain at this level. The magnification needed to visualize chemical synapses yield microscopy images of at least 10 x 10 microns in size (higher magnification is needed in the case of electrical synapses, making these fields even smaller), so to reconstruct only 1 mm3 would require the assembly of a stack of around 106 images. Full reconstructions of a small region of the mammalian brain are feasible, but structures like the cerebral cortex which can reach a surface area of 2,200 cm2 and a thickness that varies between 1.5 to 4.5 mm in humans is certainly not possible."}