*2.1.2 Non-invasive imaging of the bee*

Traditionally, the morphological classification of bees has been conducted with the aid of dissecting microscopes which use light. The technique is understandably limited when used for amber inclusions, particularly with specimens preserved in opaque pieces. Light microscopy was used in this experiment in an attempt to describe its limitations with opaque specimens.

#### **Figure 3.**

*A piece of semi-clear, brown amber from the Dominican Republic (Early Miocene: Burdigalian), with many inclusions. The stingless bee is at the widest end (arrow) [44].*

#### **Figure 4.**

*Air bubbles, fractures and general thickness of the amber prevent adequate visualisation of the metasoma, posterior mesosoma and wings. Image taken under optimal optical conditions (increasing or decreasing light intensity further degraded image quality) [44].*

#### *2.1.3 Light microscopy*

For light microscopy, the bee was viewed using a Leica MZ12 stereomicroscope, Leica Microsystems GmbH Ernst-Leitz-Strasse 17–37 35578 Wetzlar. The Leica MZ12 has distortion-free 109 eyepieces with a resolution of 375 line-pairs per mm.

Ideally, because of the thickness, air bubble inclusions and **Figure 4**, it would have been better to slice the fractures present in the amber piece prior to light microscopy examination. However, the sample was intentionally preserved to enable visualisation of the other biological inclusions using DR in future studies.

The colour of the bee was brown to dark-brown; however, it is possible that the bee was black when alive and that the cuticular melanin was altered over time. Moreover, newly moulted adult stingless bees are often lighter in coloration and so the more brownish colour of the specimen cannot be considered diagnostic. Gross external morphological features of the bee such as the chaetae, coxae, trochanter and tibiae were visible to about the level of the mesothorax. The air bubbles, fractures and general thickness of the amber piece prevented adequate visualisation of the more posterior including a lack of detail of the wings **Figure 4**. Increasing light intensity created image degradation due to light diffracting from cracks, air bubbles and generalised opacity of the amber. Decreasing light intensity made it difficult to optically visualise the bee's morphological features.

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**Figure 6.**

**Figure 5.**

*Volume rendering image of the holotype worker of Proplebeia abdita in Early Miocene* 

*(Burdigalian) Dominican amber. Wings (W), flagellomeres (F), base of trochanter (T), tibiae (Tb), tarsi (Ts), the corbicula (C) of the metatibia and the broadened metabasitarsi (Bm) are all well visualised [44].*

*A schematic diagram of sample positioning for DR. Essentially, the only preparation required is that the sample* 

*(bee) is positioned securely on the sample stage so that it remains motionless during the scan [44].*

*Diagnostic Radioentomology*

*2.1.4 DR scanning*

*DOI: http://dx.doi.org/10.5772/intechopen.89005*

Elettra Light Source Synchrotron in Trieste (Italy).

We need to keep in mind that this experiment was conducted during early testing phases for the potential applications of X-rays to insect morphology. Therefore, to assess their potential, three different apparatuses were used. X-ray MicroCT scans were performed a commercial benchtop system, a custom designed X-ray scanner and the facility for MicroCT available at the SYRMEP beamline of the

For a full description of these methods see [45]. Prior to scanning, the sample was placed in a 20.5 mm cylindrical sample holder between the X-ray source and the image detector **Figure 5**. This simple positioning procedure for the scanning phase of a DR examination can be adapted for all X-ray apparatuses. Scanning produces 2D images which are then converted to 3D images with specialised software.

As with light microscopy, gross external morphological features of the bee such as the Chaetae, the articulations of the coxae, trochanters, tibiae, and tarsi, including the corbiculae of the metatibiae and the broadened metabasitarsi, were well visualised in the 3D reconstructions **Figure 6**. In addition, gross internal structures, such as the brain (including details of its anatomical regions), direct and indirect flight muscles and a loaded rectum were accurately represented, **Figure 7**. **Video 2** (Ancient bee Proplebeia abdita trapped in amber approximately 20 million years ago) will highlight these features and also provide an understanding of what can be achieved during DR, 3D processing. Considering the specimen's age (16–19 Ma), the brain of this bee was
