Tissue-plastinated vs. celloidin-embedded large serial sections in video, analog and digital photographic on-screen reproduction: a preliminary step to exact virtual 3D modelling, exemplified in the normal midface and cleft-lip and palate

Abstract

This study analyses tissue-plastinated vs. celloidin-embedded large serial sections, their inherent artefacts and aptitude with common video, analog or digital photographic on-screen reproduction. Subsequent virtual 3D microanatomical reconstruction will increase our knowledge of normal and pathological microanatomy for cleft-lip-palate (clp) reconstructive surgery. Of 18 fetal (six clp, 12 control) specimens, six randomized specimens (two clp) were BiodurE12-plastinated, sawn, burnished 90 microm thick transversely (five) or frontally (one), stained with azureII/methylene blue, and counterstained with basic-fuchsin (TP-AMF). Twelve remaining specimens (four clp) were celloidin-embedded, microtome-sectioned 75 microm thick transversely (ten) or frontally (two), and stained with haematoxylin-eosin (CE-HE). Computed-planimetry gauged artefacts, structure differentiation was compared with light microscopy on video, analog and digital photography. Total artefact was 0.9% (TP-AMF) and 2.1% (CE-HE); TP-AMF showed higher colour contrast, gamut and luminance, and CE-HE more red contrast, saturation and hue (P < 0.4). All (100%) structures of interest were light microscopically discerned, 83% on video, 76% on analog photography and 98% in digital photography. Computed image analysis assessed the greatest colour contrast, gamut, luminance and saturation on video; the most detailed, colour-balanced and sharpest images were obtained with digital photography (P < 0.02). TP-AMF retained spatial oversight, covered the entire area of interest and should be combined in different specimens with CE-HE which enables more refined muscle fibre reproduction. Digital photography is preferred for on-screen analysis.

Fig. 1

TP-AMF sample section from specimen P (21st week), imaged using a video camera (a), analog photography (b) and digital photography (c). The section depicts the subalar upper lip region. Left is anterior, above is right. Maxillary bone, oropharynx, temporal bone and inner ear together with the cerebellum are depicted.

Fig. 2

A clp TP-AMF section from specimen E (23rd week), imaged using a video camera (a), analog photography (b) and digital photography (c). The section depicts the clefting upper lip, alveolus and hard palate, and also the nasopharynx, left inner ear and cerebellum. Orientation as in Fig. 1. Stain artefact is seen as small black taints, e.g. at the left buccal fat pad.

Fig. 3

Normal CE-HE section example (specimen A, 13th week) imaged using a video camera (a), analog photography (b) and digital photography (c). Orientation as to Figs 1 and 2. Both alae, maxilla, oral cavity, soft palate (transverse), oropharynx, inner ear and medulla oblongata are transected. Disruption artefact runs from right anterior to left posterior in parallel orientated streaks.

Fig. 4

A clp CE-HE example (specimen M, 20th week) imaged by video camera (a), analog photography (b) and digital photography (c). Orientation as in Figs 1–3. The clefting lip, hard and soft palate, and oropharynx are shown; the cerebrum is missing. Small bilateral disruption artefact can be seen at the pterygoid fossa.

Fig. 5

Artefact distribution (yellow = celloidin, blue = biodur) dependent upon whether the initial third of the total slices is cut from the embedded tissue block (I. section), the middle third (II. section) or the last third (III. section). The highest artefact density was encountered in coronary-frontal sectioning of a CE specimen (see Fig. 8b,I) and in macroscopically macerated upper (L) and lower (B) specimen sections. Artefact assessment was performed by four independent observers and averaged values were counted.

Fig. 6

Statistical distribution of the colours from Figs 1–4. The colour histograms show the primary colours (RGB), luminance (black) and saturation (violet). The total range of a tone value is 0–255 (x-axis), where 0 is the blackest black at the left end. The height of each bin (y-axis) is proportional to the maximum recorded intensity for any pixel.

Fig. 7

Digital camera images from the left midface of all four examples: (a) Fig. 1(c), (b) Fig. 2(c), (c) Fig. 3(c), (d) Fig. 4(c). Muscle fibres of the orbicularis oris, nasalis, zygomaticus and levator labii superioris can be discerned with better contrast with HE stain than in azure-methylene blue-fuchsin. By contrast, tissue plastination shows no disruption artefact as in HE (c,d). Figures orientated as in Figs 1–4.

Fig. 8

Frontal example sections depicting the cavum nasi, maxilla, upper lip, cheek, cavum oris, lower lip and mandible. (a) TP section from specimen Q, 32nd week; (b) CE example shows considerably more artefacts. Artefact area (green) and total specimen area (black) are segmented and labelled, specimen I, 18th week.