Stereology spaceballs software#
Our software package, StereoInvestigator, is used, giving the “advantage that all calculations are taken care of.” (Wälchli, et al., “Imaging and quantification, fourth paragraph”).
The fractionator method could have been used if the volume fraction could have been tracked. The NvVref method used requires that an estimate of numerical density (Nv) such as surface per volume, is multiplied by the reference volume (Vref) to avoid the volume-reference trap. The easiest way to measure is to count the interactions between vascular features and geometric probes - i.e., how many times does a test point fall onto the volume of a vessel, how many times does the length of a vessel intersect with a test area or how many vessel branch points are contained in a volume probe? (Wälchli, et al., “Imaging and quantification, fourth paragraph”).
(Wälchli, et al., “Imaging and quantification, second paragraph” also see, “Advantages of the protocol compared with alternatives, fourth and fifth bullet-point”). This is in stark contrast to the biologically difficult-to-interpret but commonly used variety of measures such as ‘percent of the area of a section occupied by vessels’, ‘number of pixels occupied in an image’ or ‘measures of length or diameter’ in 2D projections. Provide direct information about morphological 3D parameters of interest - e.g., the length of vessels, the number of vessel branch points or the vessel volume fraction. It is stressed that design-based stereological methods This is fine for all of these analyses except for estimating number, where a higher numerical aperture lens (0.13 or 0.14) should be used. Analysis of perfused and non-perfused vessel structure using unbiased stereology to determine, volume fraction, length and number of branch points and endothelial tip cells was done with a 20x objective (Wälchli, et al., “Imaging and quantification”). Confocal laser-scanning microscopy image stacks were taken. Systematic random sampling for choosing the inter-section and the intra-section interval is also recommended. 5).” (Wälchli, et al., “Experimental design, last paragraph, also see, PROCEDURE, step 17”). The authors ”recommend the use of relatively thick (e.g., 40-μm) brain sections to obtain reliable 3D information about the blood vessel tree and to quantify vessel parameters such as vessel volume fraction, vessel length or vessel branch points ( Fig. Brains were removed and sectioned coronally into three slabs and fixed and then treated for isolectin B4 (IB4) that binds to endothelial cells, thus revealing both perfused and non-perfused vessels. Evans Blue (EB) that binds to serum albumin was injected intracardially on post natal day eight to label perfused blood vessels but not non-perfused vessels. Perfused blood vessels and non-perfused blood vessels were differentiated to look at ‘perfusion status’. The post-natal mouse brain is an interesting model system to study angiogenesis since it is ongoing after birth (Wälchli, et al., “Toward mature, perfused blood vessels: the postnatal mouse brain as a model”). In other words, instead of having to use isotropic or vertical sections when making length and surface estimates as is the case for thin sections, the authors’ preferred orientation, in this case coronal (Wälchli, et al., “PROCEDURE, step 33, first critical step”), can be used! Isotropic probes can be used in thick sections when estimating length and surface, eliminating the need to make the tissue isotropic.
Stereology spaceballs how to#
Unlike the paper on thin sections reviewed here (Mühlfeld, 2014) however this review (Wälchli, et al., 2015) considers how to quantify vasculature in thick, forty micron sections instead of thin, two to three micron sections. The next manuscript we will look at is also a very well written guide to using unbiased stereology to estimate cardinal parameters of the vasculature. Vogel (2015) Quantitative Assessment of Angiogenesis, Perfused Blood Vessels and Endothelial Tip Cells in the Postnatal Mouse Brain.
Review of Publication: Wälchli, T., Mateos, J.M., Weinman, O., Babic, D., Regli, L., Hoerstrup, S.P., Gerhardt, H., Schwab, M.E., and J.