figure 1: Rowiak TissueSurgeon

figure 2: Principle of laser microtomy (here 3D cut). Femtose-cond laser is focussed into the depth of the specimen, where photodisruption leads to tissue separation.  

figure 3: Menlo Systems BlueCut femtosecond Laser used for the experiment.

figure 4: Sectioned sample of bone. 160 nJ pulse energy at the sample.

figure 5: Sectioned sample of teeth. 180 nJ pulse energy at the specimen; section thickness: 50 µm.

figure 6: Microscopic image of sectioned bone.

Introduction

Traditional sample preparation for non-decalcified hard tissue histology requires time consuming methods. For good embedding results, fresh hard tissue needs to be fixed and properly dehydrated afterwards. The embedding process itself can last up to one week. Afterwards the resulting blocks need to be trimmed and pre-sectioned. For ground sections, the surface resulting from pre-sectioning has to be polished and is then mounted to a microscope slide.

In most cases, the time consuming and laborious preparation steps are unnecessary when using ROWIAK TissueSurgeon (figure 1). Fixed or even fresh hard tissue is mounted on a microscope slide with special glues designed for hard tissue. This mounted sample is cut plan-parallel to the surface of the slide and the section is ready for staining. The whole process only takes about five minutes per sample. Bone for example can be cut between min. 10 μm and max. 100 μm section thickness. Also cutting of 3D-shapes is possible (figure 2).

Material and Methods

Rowiak TissueSurgeon, equipped with the BlueCut laser from Menlo Systems (figure 3) was used to prepare thin sections of dried and embedded bone and teeth. 

The BlueCut laser is based on a fully fiber integrated laser oscillator amplifier setup with intermediate pulse picking unit. This results in a flexible but robust system. The balanced pulse stretching and compression results in pulses with excellent temporal quality. The intrinsically good spatial beam quality (M² < 1.25 in both axis) leads to high energy per unit square.

The femtosecond laser delivered pulses at a repetition rate between 1 MHz and 5 MHz to utilize the  high processing speed of the tissue surgeon.  The pulse energy was adjusted up to 1 µJ. In conjunction with the short duration of the pulses of 300 fs, this allowed to process both teeth and bone. 

Results and Conclusion

Sectioning of bone and teeth with Rowiak TissueSurgeon equipped with  the Menlo Systems BlueCut laser showed equal quality to standard setup and to sections performed with classical methods (e.g. ground sections). 

Tissue separation was easy so that complete sections especially from relatively large specimens remained on the microscope slide (figure 4). No relevant heating artifacts occurred. The dark spots that can be seen onto the section in figure 4 are residues from polishing and can be wiped away with ethanol. 

Figure 5 shows a section of a tooth. In particular the enamel (white part) could be cut completely which is one of the most difficult biological materials due to the high light scattering behavior. 

Figure 6 shows a H & E stained section of bone imaged with a 40x microscope objective. The Quality of section allowed to take images with good quality at cellular level.

In conclusion the experiments showed that the Menlo BlueCut laser is a good option for Rowiak TissueSurgeon, allowing to section hard tissue with good quality.