Journal of Microscopy, July 2018, Vol.271(1), pp.17-30
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 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. There are several types of tissues in bones and teeth which provide mechanical support, are normally calcified, hard and tough, and difficult to slice finely, which must be done to understand normal and altered structure and function and relationships to surrounding soft cellular parts which maintain them. Classically, hard tissues are softened by removing the mineral component so that they may be cut with a knife: crystals cannot be so cut, because they cannot be bent. But if we ‘decalcify’, we remove a main structural component which we need to understand. It has been next to impossible to have thin slices where all the parts are intact and properly connected. We investigated cutting sections by laser machining. In extensive pilot studies, we have shown that the method works to produce slide‐mounted samples which we can study by both light and electron microscopy. The sections produced are thinner than the ‘voxel’ in most laboratory X‐ray microtomography systems. It should be born in mind that the resolution of the light microscope is still far greater than that of even the highest resolution, synchotron XMT and that this is best obtained in the present context from the use of thin sections. Serial slicing can be achieved with as little as 20 μm waste between sections. Productivity is high: one square centimetre blocks can be cut in 10 min. The basic procedure tested to date has used PMMA embedded material, but this could be any resin, including LR White and glycol methacrylate. A flat block face is produced by micromilling or polishing. The block face can be studied by SEM and confocal at this stage. The block face and the underlying few microns are to be contained in the first section. The block face is stuck to a standard glass light microscope slide with a cyanoacrylate adhesive. We focus through the glass slide, the cyanoacrylate adhesive, the block face, and a further increment into the specimen. The focussed, pulsed laser beam is scanned along a 1 mm line, and this swathe is translated in an snake scan to cover the entire area of the block, which 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.
Bone ; Enthesis ; Ligament ; Osteoarthritis ; Osteoporosis ; Tendon