One solution is to manipulate the tissue blocks before thin-sectioning so that cross-sections will not be favored there should be an equal chance to get any section, cross-, longitudinal-, or all possible orthogonal-sections.
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For example if the cardiac myocytes are sectioned in such a way that longitudinal orientation is favored, the capillaries will also be mostly longitudinal, and their surface will be over-estimated because more intersections are counted than if the interaction between probe (line) and tissue (surface) was isotropic. If you use a probe that is a surface and count the intersections with the vessels (see using image as probe for length), and vertical or isotropic sections are not used, you are in danger of over- or under-estimating the surface area. It is pointed out that ‘the orientation of the capillaries in cardiac muscle is related to the orientation of the cardio myocytes’ (Mühlfeld, 2.3, first paragraph, first sentence). Another example given is estimating the length of capillaries in the heart (Mühlfeld. see Merz probe) will make less intersections with the muscle surface than if the probe tissue interaction were isotropic in 3-D space. If papillary muscles are only sectioned and visualized in cross section, the surface area will be underestimated because the probe (consisting of lines, e.g. A cautionary example is given involving the determination of the surface area of papillary muscles in cross-section.
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This means for thin sections, a process to ensure that the tissue itself is isotropic must be employed, and it is recommended to use the ‘isector’ or ‘orientator’ to ‘spin’ tissue blocks to make their contents isotropic (see isotropic and vertical sections). No orientation in 3-D space between the probe and the tissue being probed can be favored.
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No assumptions about shape, size, or distribution of structures can be used (see design based stereology). The need to understand sources of bias in quantitative 3D microscopy is stressed (Mühlfeld, Table 1).
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Therefore the goal of this review is to make clear how to estimate ‘hard, biologically useful information’ regarding number, length, surface, or volume of structures within a well-defined region –in a way that ‘should be simple and provide a precise estimate of the real value at low cost’ in other words, efficiently by encouraging the use of design based stereology described as the ‘gold standard in quantitative microscopy’ (Mühlfeld, Current status, second paragraph). But it is also pointed out that the use of unbiased stereology is rare for cardiac research (Mühlfeld, abstract). Examples of important questions are given: ‘Is there a significant loss of cardiomyocytes during progression from ventricular hypertrophy to heart failure?’ and ‘Does a specific treatment reduce the degree of fibrosis in the heart?’. The authors characterize the use of unbiased stereological methods in biomedical research as aiming to ‘obtain quantitative information about three-dimensional (3D) features of tissues, cells, or organelles from two-dimensional physical or optical sections’. The suggestions given in this review for unbiased stereology on thin sections of cardiac tissue are valid but we will also include suggestions for using thick sections, like in the image above, because probes that can be used on thick sections do not require isotropic or vertical sections. Alt, Heart and Vascular Institute, Department of Medicine, Tulane University, New Orleans, LA, USA The two different colored arrows follow two individual cardiac myocytes through the z-axis, large arrows indicate a cross section through the nucleus. Section was processes with CLARITY, followed by immunofluorescence against troponin (red) and connexin 43 (yellow), and imaged with an Olympus DSU microscope.
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However, recently developed histological techniques can allow for good visualization with thick sections.Įighty micron thick section from mouse heart. The use of thin sections is recommended to ‘improve resolution and allow us to visualize internal structural detail’ (Mühlfeld, 2.1, first paragraph, second sentence). The aim of this review is to encourage those doing cardiac research to use unbiased stereology as is done in other fields such as Pulmonary, Kidney, Placenta, and Neuroscience (Mühlfeld, 2010, third paragraph). Mayhew (2010) A review of state-of-the-art stereology for better quantitative 3D morphology in cardiac research.