## 3 Results and Discussion
### Laser Confocal Microscopy  
Laser confocal microscopy was used in order to identify any possible fluorescing species present in the filament or the printed sample and was used to obtain depth profiling data which would not be possible with a simple fluorescence microscopy. In general, micro-chemical analysis combined with large area surface-sensitive spectroscopies and imaging, has proved to be essential for understanding the mechanisms controlling processing-performance relationships [19]. Figure 3 shows a side view of the source filament. Clearly, there are fluorescing species visible (seen as bright green patches) on the surface of the filament, fairly evenly distributed.  
Figure 4 shows a top view of a printed PLA sample. Again, bright fluorescing areas are evident, mostly about 5-30 microns in size (the one larger streak being about \(15\times 115\) microns). However, most of the fluorescent materials are concentrated in the dark voids in the 3D print. Views from a number of regions on the sample surface showed the same thing. Hence, whatever process may have led to the  
Figure 3: Laser confocal image of PLA source filament, collected from a side view with a \(10\times\) objective.  
Figure 2: Scanning electron microscopy image of printed PLA showing micro- and sub-micron-scale roughness.  
formation of voids in the surface most likely also either preserved or preferentially concentrated the fluorescent material.  
In determining what species are fluorescing under irradiation by the 405 nm wavelength laser, we use a combination of vibrational and electron spectroscopies (as detailed below) and X-ray fluorescence (XRF) using a Niton XL3t Thermoscientific system. XRF from the source filament and the printed samples show a number of similarities, as indicated in Table 1.  
While significant P appears in the sample, it is not likely to be the source of fluorescence unless it is present as phosphate. However, the Raman and FTIR spectra (shown below) do not indicate the presence of phosphate, nor is there a significant signal associate with silica. Calcium carbonate, a common additive in processing of polymers, may account for the Ca found (and is in agreement with evidence from Raman and FTIR spectroscopy). However, CaCO\({}_{3}\) alone does not fluoresce under irradiation at 405 nm. It does, however, fluoresce if reacted with organic ligands or if containing trace amounts of transition metal ions [20]. Therefore, it is possible that the fluorescent particles seen represent a form of calcium carbonate which has reacted with organic ligands (from PLA or other unknown additives) or due to association with trace amounts of transition metal contaminants.  
\begin{table}
\begin{tabular}{c c c} \hline
**Element** & **Source Filament (Averaged over Three Samples) (ppm)** & **Printed PLA (Averaged over Three Areas) (ppm)** \\ \hline P & \(2100\pm 50\) & \(1183\pm 70\) \\ Ca & \(528\pm 40\) & \(585\pm 25\) \\ Si & \(310\pm 35\) & \(180\pm 15\) \\ Cr & \(110\pm 10\) & \(61\pm 5\) \\ Ti & \(80\pm 10\) & \(40\pm 10\) \\ Cu & \(35\pm 10\) & \(13\pm 4\) \\ Sn & (below detection) & \(18\pm 5\) \\  & Trace amounts of W, Cd, Zr, Nb, Mo & \\ \hline \end{tabular}
\end{table}
Table 1: Results of XRF measurements from PLA natural source filament and printed PLA.  
Figure 4: Laser confocal image of top surface of a printed PLA Sample.  
The concentration of fluorescent particles in voids may indicate that whatever process resulted in the formation of voids also resulted in preferential retention or formation of CaCO\({}_{3}\)-based particles. It has also been found that inclusion of additives such as pigments in PLA may themselves cause the formation of small voids [20; 21]. As we will discuss, the presence of carbonate may have important implications for biomedical applications of printed PLA structures.