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Morphological Methods
Apart from simply looking at an object there is an abundance of techniques that may be applied for investigating forms. Studying complex vascular systems, specifically the excretory system of vertebrates, poses several problems. A blood vessel is essentially a tube that – when it is devoid of blood – may be (crudely) interpreted as a hole in the tissue. Such a hole – like every hole – is defined by the absence of matter, i.e., animal tissue. Consequently, this poses the question of studying the form of something that is not there – a logical paradox. However, there are ways to overcome this problem – e.g., by filling the hole (vessel) with something like dyes or casting resin.
Investigating the blood supply in a body is even more challenging in several ways. The most obvious problem is that a highly dynamic, living system – that responds rapidly to many variables like e.g., temperature, pressure, oxygen level, etc., – usually has to be studied in a static condition. For example capillaries and other very small vessels are prone to collapse when the circulation seizes. Low temperature can shut down peripheral vasculature and practically all preparatory steps result in dimensional changes , either dilation or shrinkage. Controlling all these variables and visualizing even minute blood vessels in a life-like state (in widely differing vertebrate species) is a demanding task. Fortunately, several standard methods have proven to be useful. Ideally, in order to study e.g., the renal vascular bed, several of these methods should be combined.
High quality dissections are essential for many reasons, if nothing more than to find and process your target quickly. While this is usually less of a problem in mammals, it is surprising that good topographical anatomy sources are rather rare for non-mammalian vertebrates even in the 21th century. Anyway, a good dissection is the beginning of all anatomical understanding and may allow interesting observations.
Boa constrictor, left kidney – ventral view, vessels injected with contrast media (injection by H. Splechtna & W Grünberg).
Most classical structural investigations involve transmitted light- and/or electron microscopy. Sectioning, however, involves several rather elaborate and time-consuming procedures as well as some experience in the interpretation of the images (esp. with TEM). Also, shrinkage and inevitable artifacts like e.g. compression from the sectioning knife, have to be considered. However, many details become accessable even down to the molecular level.
Anaconda glomerulus - semi-thin section in light microscopy
Microscopic sections are expedient for visualizing the interior of the cells and their two-dimensional pattern. However, spatial information has to be derived by rather time consuming 3D-reconstructions. Still, measurements of processed tissues are of limited value due to object shrinkage.
Anaconda glomerulus – serial sections rendered to 3D image
Unlike conventional light microscopy, supra-vital microscopy and confocal laser scanning microscopic methods are capable of showing living, only minimally altered systems – at least in theory. However, surgical access of the optics to the surface of the kidney is required – with all of the drawbacks that such a severe intrusion may have and furthermore, the observable depth in the organ is rather small. So - at least at the moment - morphological studies on living matter are rather limited.
Nevertheless, with critical-point or freeze dried preparations in a scanning electron microscope (SEM), the surface of structures can be studied in high resolution:
Anaconda glomerulus – critical point dried in a SEM
However, not all objects are suitable for this method and only surfaces, either natural or obtained by fracturing, can be studied.
The micro-corrosion casting technique is a special method for three-dimensional visualization of vascular or other “hollow” structures. Although this technique requires some experience, it allows studying complicate vascular patterns in a life-like form.
Anaconda glomerulus – corrosion cast in SEM
Inherently, however, the structure itself, namely the surrounding tissue is lost during the corrosion casting procedure. Consequently, this method shows the anatomical inversion (“negative”) of the critical-point dried preparation above.
Considering all the pros and cons of the different methods it seems the best choice to combine them - and finally add information from biochemistry, physiology, ethology, ecology , thus allowing functional considerations based on structural data. Even then – an old approved axiom still applies:
The whole is more than the sum of it's parts!
(brought into kidney context by Bodil Schmidt-Nielsen)
However, this axiom may be extended – returning to our beginning question about studying vessels (tubes) that are essentially holes in a tissue:
The hole ist more than the absence of parts!