**4. Conclusions**

Confocal Raman microscopy showed the potential to be applied to study process-related microstructural modifications in high moisture spreadable dairy products, such as cream cheese. With Raman microscopy it was possible to observe structural di fferences, especially in terms of phase separation and water pockets re-organization. On the other hand, under the conditions reported in this work, with confocal laser scanning microscopy better results were obtained, in terms of fat and protein structure visualization. The use of confocal Raman microscopy allows one to better distinguish the e ffect of freezing-thawing processes of the two di fferent cream cheeses. This was coherent with the low resolution of Raman microscopy obtained with the measurement parameters applied in this study; the parameters were limited in order to avoid any possible sample damage or modification that could be expected by a higher extent of heat generated by the laser source in the case of a slower but more resolute scan. Therefore, it was not possible to visualize the presence and distribution of sugars and di fferent stabilizers, which probably contributed to the di fferent freezing stability of the cheeses. It is also important to note that the Raman microscopy data complemented the information derived from low resolution NMR.

These first results sugges<sup>t</sup> that confocal Raman microscopy may become in the future an additional key analytical tool because it can provide relatively rapid, non-destructive and online food quality evaluation, that could be complementary to confocal laser scanning microscopy.

Further studies need to be carried out on the quantitative assessment of components and ingredients, by image analysis techniques and/or by further elaborating spectral information.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/5/679/s1, Figure S1–S12: Raman spectra of reference samples: (1) Carrageenan, (2) Sorbic acid, (3) Citrus fibre, (4) Gelatine, (5) α-lactalbumin, (6) β-lactoglobulin (7) Whey protein concentrate, (8) Sucrose, (9) Lactose, (10) Skim milk powder, (11) Micellar casein isolate, (12) Calcium caseinate, Figure S13–S14: Raman fingerprint region (500-1800 cm-1) of protein components: (13) Calcium caseinate, (14) Whey protein concentrate. (A) Phenylalanine's benzene ring breathing (1000-5 cm-1); (B) Amide III (1270 cm-1); (C) Tyrosine (1614 cm-1); (D) Amide I (1650-70 cm-1), Figure S15: Supplementary Confocal Raman micrographs of cream cheeses. Cream cheese A before freezing (A), after freezing (B). Cream cheese B before freezing (C), after freezing (D). Red: fat. Green: protein. Blue: Water, Figure S16. Supplementary Confocal laser scanning micrographs of cream cheeses. Cream cheese A before freezing (A), after freezing (B). Cream cheese B before freezing (C), after freezing (D). Red: fat. Green: protein. Table S1: Reference samples used to acquire Raman spectroscopy peaks.

**Author Contributions:** Conceptualization, M.A., U.A., L.W. and M.C.; methodology, M.A.; software, M.A., T.A.M.R.; formal analysis, M.A.; investigation, M.A., T.A.M.R., B.M.; resources, G.M., U.A. and M.C.; data curation, M.A.; writing—original draft preparation, M.A.; writing—review and editing, G.M., U.A., T.A.M.R., L.W. and M.C.; visualization, M.A.; supervision, G.M. and M.C.; project administration, U.A., L.W. and M.C.; funding acquisition, G.M. and M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This research did not receive any specific gran<sup>t</sup> from funding agencies, in the public, commercial or non-for-profit sectors. Authors would like to acknowledge the University of Parma and iFOOD for the support of M. A. mobility grant.

**Conflicts of Interest:** The authors declare no conflict of interest.
