Evaluation of laser ablation microtomy for correlative microscopy of hard tissues

Abstract

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 μm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy.

Regulation of the Bone Vascular Network is Sexually Dimorphic

“…Samples were treated to remove bone matrix and mineral by successive application of 2N HCl and 7% chlorine sodium hypochlorite solutions, removing calcified tissue and leaving a solid PMMA cast. To enable visualization of soft tissue, the surfaces of polished blocks and casts were stained using either ammonium triiodide or iodine vapor …”

“…To enable visualization of soft tissue, the surfaces of polished blocks and casts were stained using either ammonium triiodide or iodine vapor. (23,24) Osteoblast cell culture Long bone osteoblasts (LOBs) were isolated from 4-day-old (P4) male and female VEGF fl/fl mice using 10 mg/mL collagenase and 4 mM EDTA, in a four-step process. (25) Endothelial cell culture Primary murine bone marrow endothelial cells were purchased from Generon (Slough, UK) and cultured according to manufacturer's guidelines.…”

The Bone Cartilage Interface and Osteoarthritis

“…12 Cartilage in bone, Trabecular excrescences (adipocytic bone moulding) Sharpey fibres in trabeculae, tidemark reduplication. a 10um section prepared by Laser Ablation Microtomy [ 48 ] of PMMA block of distal femur removed at operation for arthroplasty. The light microscopy slide was imaged by 20 kV 3D BSE-SEM (grey components; uncoated, SEM chamber pressure 50 Pa) and transmitted light microscopy after MacNeal’s tetrachrome staining (blue components).…”

“…The most excellent LM sections are to be obtained with the technique of laser ablation microtomy (LAM) [ 48 ]. With LAM we have permanent preparations which can be studied with all SEM, LM and X-ray methods.…”

Nontraditional Machining Process In Healthcare Applications

“…In (Boyde, 2018), laser ablation method was evaluated in terms of problems arising from bone, joint and dental tissue where thin and intact parts were needed. (Šugár et al, 2019) was about developing osseointegration of the titanium implants.…”