shows a 3D rendering of an Z-stack of a hepatocyte nucleus with surrounding cytoplasm and it has a quarter of the volume “cut-away” for clarity and is shown at two angles. On the left is the original Z-stack prior to deconvolution and on the right is the deconvolved volume showing the greater nuclear chromatin detail in 3D following deconvolution.
Figure 1 Partial volume of tissue including a whole hepatocyte nucleus before (left) and after (right) deconvolution. The volume in both cases is shown from two different angles (top and bottom). In the predeconvolved volume the smearing of structural details (more ...)
shows the results of the quantitative measurements of chromatin texture on all 12 cells. The difference between performing this measurement on the whole 3D volume as compared to a single central 2D section through each nucleus is that the 3D measurement makes the distinction between the two types of nuclei more obvious (for 3D measurements the Mann-Whitney P value is <0.01, whereas it is <0.05 for the 2D measurements). This also makes intuitive sense as in 3D one measures the chromatin distribution across the whole nucleus and so, for measurements which are insensitive to orientation, this will be the same for a given nucleus. However, when making this measurement on a 2D section through a nucleus, the value will be insensitive to the orientation of that section but can be expected to be different for any other section taken through the same nucleus at a different level or angle. Thus making measurements on 2D sections or projections bring with them an inherent level of variability that may be viewed as “noise” superimposed on the “signal” which does not exist if the measurement is made on the whole 3D structure. This, in turn, makes discrimination between different morphological groups more difficult if one restricts measurements to the 2D case (as with routine tissue sections or cytology preparations). This also has similar implications for the qualitative visual discrimination of 3D patterns from 2D samples as elaborated in the next paragraph.
Figure 2 Chromatin texture measurement on all 12 cells used in this study by the third moment of Hu. The value of the third moment calculation is shown on the ordinate versus the cell measured on the abscissa (the 6 plasma cells followed by the 6 hepatocytes). (more ...)
shows the results of one of the qualitative experiments where a single plasma cell nucleus, being reconstructed in 3D, is subsequently viewed in 2D projection from various angles. This is analogous to a cytology preparation of a whole cell lying on a slide—any given cell may be oriented on the slide in a random orientation. From this figure it is clear that, even though the cell is one and the same, it appears (from the cytologist's viewpoint) as if we are actually looking at 5 different cells because the 2D projection pattern of the same 3D chromatin arrangement is different depending on the angle of projection. For simple geometrical shapes (such as a cube) or familiar 3D forms (such as a cup or a face) we can intuitively recognise the 3D object from a multitude of its projections—the cubical frame at the top of will be obvious to most observers as the projection of the same 3D object from different angles even though the actual arrangement of the lines differs in the various projections. However, this intuitive sense is not present when one considers unfamiliar and irregular 3D objects such as the chromatin of the interphase nucleus. This has another implication in reverse. That is to say, if we had the freedom to view nuclear chromatin from all angles (such as is provided via the 3D imaging methods described in this paper) and we had 5 separate cells in a sample with identical chromatin (which might be neoplastic clones of each other, e.g.) then we could identify that those cells were identical (and hence possibly neoplastic). However, if we were restricted to seeing those cells fixed as they lie on a cytology slide at all different angles we would see 5 different 2D projection chromatin patterns similar to those shown in and we could not know they were identical. Thus the freedom to examine cells in 3D can enhance our morphological discriminatory abilities to detect both subtle differences between cells () and also similarities between cells () which could be of significant utility in comparative microanatomical studies.
Figure 3 The freedom to view cells in 3D, as provided by the reconstruction methods in this paper, gives us greater morphological discriminatory abilities such that we can detect when cells that look different in 2D projections or slices (such as in cytology and (more ...)
As described in many classical texts, a very typical morphological feature of a mature plasma cell nucleus is its “clock face” or “cart wheel” chromatin pattern. This refers to the appearance of peripheral separate clumps of chromatin against the nuclear membrane (the “numerals” of the clock face) which often have a tapering extension facing radially inwards (the “spokes” of the cart wheel) with a central clump (the hub of the cart wheel or the centre of the clock face where the hands attach). Whereas these descriptors refer to a typical 2D pattern, the fact that this pattern is so characteristic implies a 3D structure that can display this pattern from almost any angle of projection (the orientation of plasma cells on a slide is not uniform yet they characteristically project this similar pattern) as well as being typical even when the plasma cell nucleus is cut in partial section through any plane at any angle (as is the case with histological preparations). This raises the interesting question of which 3D structure can give rise to a similar 2D pattern under such a diverse set of projections and partial sections through any angle? After studying the 6 plasma cell nuclei in 3D (including “flying into” the nucleus and observation of chromatin from the inside in interactive VRML—see supplementary movie 1 of the supplementary material available online at doi: 10.1155/2012/898707 for an example of this type of exploration) I have attempted to summarise the 3D architectural basis for the characteristic “clock face” pattern. I must emphasise that what follows is a stylised simplified representation of the chromatin but does capture the essence of the 3D arrangement of the plasma cell chromatin for the purpose of explaining this morphological feature.
The chromatin in the mature plasma cell was found to be arranged in a variable number of peripheral clump units arranged in rings around the periphery. These peripheral clump units are the basic units of the pattern. Importantly, and perhaps counter-intuitively given the prominence of the central spot to the clock-face pattern, there is no tendency for a central clump (a clump at the 3D centre of the spheroidal nuclear space). In a detailed analysis, there are interesting connecting strands of chromatin in some plasma cell nuclei that pass through or near the centre (see supplementary movie 1) but these are not morphologically prominent as globular clumps neither are they a consistent feature in all plasma cells so do not take part in forming the characteristic central clump of the clock face pattern as seen in 2D projections/sections. The archetypal peripheral chromatin clump unit has an asymmetrical and curved conical shape shown in simplified form in . This has a convex base applied to the nuclear membrane (the convexity follows the inner contour of the nuclear membrane) and a concave conical extension pointing towards the centre of the nucleus. The cross-section of this curved conical clump unit in a plane that is perpendicular to the base-apex axis of the cone is essentially elliptical such that at one angle of view the cone appears broad and blunt-tipped and at right angles to this plane it appears narrow and sharp tipped ().
Figure 4 The basic chromatin structural unit of the plasma cell nucleus (the “clump unit” described in the text). This is essentially an ovoid curved cone. Side views (at 90 degrees to each other) are shown on the top and a plan view is shown bottom (more ...)
The arrangement of the clump units is as a series of intersecting peripheral rings with the clump units in each ring all arranged with their bases abutting the nuclear envelope and tapered ends pointing towards the centre (but with variable rotation about the base-tip axis of the conical clump units). The clumps in each ring are approximately regularly spaced with sparse chromatin in between them. The rings of clump units intersect at various angles to cover the periphery of the nucleus approximately evenly. Where they intersect, two rings share 2 clump units (one at each diameter of the intersection). This arrangement of the bases of the clump units on the nuclear membrane is analogous to the arrangement of the pentagonal black patches on the surface of a standard soccer ball. The typical “clock face” as seen in histology has between 4 and 8 clumps (on average about 5 to 7 clumps) but fewer clumps are seen in 3D (only about 4 or 5 clump units are present in a single circumference) which indicates that parts of clump units from above and below the central plane contribute to the clock-face “numerals” as seen in 2D projections.
As noted above, there is no tendency for a central clump in 3D space. The characteristic central “blob” in the clock-face/cart-wheel pattern seen in histology and cytology is actually a projected image of the peripheral clump unit directly above and/or below the viewing plane as shown in . This 3D geometrical arrangement of clump units explains the various properties of the clock-face pattern as seen in 2D sections/projections.
Figure 5 The plasma cell clock-face or cart-wheel nuclear pattern as seen in 2D sections/projections from almost any angle can be explained by the multiradial arrangement of peripherally placed clump units. Top left shows 7 such units in a circle (some of these (more ...)
- Peripheral clumps are always present in a radial and equally spaced distribution no matter what angle of projection is seen and for most partial slice projections at any angle. This angle-of-projection invariance explains why the feature of a peripheral ring of clock face “numerals” is so characteristic regardless of projection angle or section thickness.
- As the clump unit is conical there is nearly always some amount of central chromatin visible in the perpendicular axis of view even when the nucleus is not whole. This explains the characteristic feature of the central “hub” clump to the clock-face/cart-wheel.
- As the conical clump units are ovoid in cross-section this explains why some clumps appear broad and blunt while others appear thin and tapered. Whilst this is not a feature of the “clock-face” motif it is a typical observation of plasma cells in tissue sections.
Supplementary movies 1 and 2 show fly-into sequences of the 3D structure of a typical plasma cell and hepatocyte nucleus. The grey levels have been inverted in these movies as this aids visual 3D depth cues but these are typical examples of the brightfield 3D data generated in this study. Note how the internal chromatin in the hepatocyte is characterised by more complex multiply branched internal connections in contrast to the plasma cell which has sparse and coarser internal connections. Also note the fine connections between peripheral clumps in the plasma cell which are not obvious from an external inspection as the great contrast of the large clumps throughout the nucleus superimposes on an external view. Thus the ability to fly “into” the nucleus and look at these clumps from the inside out reveals morphological detail not otherwise easily appreciable and is one of the advantages of the methods presented in this study.
Previous attempts at 3D chromatin structure evaluation have been described in the literature. Many of these use fluorescence microscopy and so are not applicable to routine brightfield staining so cannot be used to elucidate chromatin structure as we commonly see it in histology sections or cytology preparations and the structural resolution of the models has been crude relative to the methods used in the current work [12
]. Brightfield 3D microscopy with routine stains has been achieved using a novel tomographic method [13
]. However, this requires the individual cells to be isolated in a capillary tube for imaging and again the models are not as detailed as those presented here. Very detailed models have been generated using electron microscopy techniques [14
] but these do not utilise routine brightfield stains and the altered tissue processing needed for EM all mean that the models obtained by those methods are not suitable for elucidating chromatin features as seen on routine histology preparations. In contrast to all of the above, the method presented in this paper can be used on routine histology preparations with ordinary brightfield light microscopy and has been demonstrated to give detailed 3D models of nuclear chromatin allowing microanatomical analysis of structures seen in everyday microscopy practice. While chromatin has been studied in this paper, the method may also be used to study cytoplasmic and extracellular detail. This type of analysis can explain the basis for morphological features seen in 2D tissue sections and can bridge the gap between light and electron microscopy in 3D reconstruction studies. Such enhanced 3D microanatomical knowledge can help explain the microanatomical basis for histophysiological and pathological processes and has been shown in this paper to enhance our morphological discriminatory abilities which are normally restricted by our 2D views of complex 3D structures at the microscopic level.