Label-Free Imaging of Candida Albicans Using Raman Spectroscopy

Objectives: Candida albicans is referred to as a ‘keystone’ commensal organism within the oral cavity as it is found in most healthy individuals. However, overgrowth of this species can lead to oral diseases such as candidiasis. Under certain physiological conditions, C. albicans is able to form biofilms with development of hyphae providing a scaffold for disease-associated bacteria to adhere to, exacerbating other oral inflammatory conditions such as periodontitis. The main objective of this study was to investigate Raman spectroscopy as a rapid and sensitive analytical technique for the detection and characterization of Candida albicans in the oral cavity, particularly in its planktonic and biofilm forms. The aims were to profile structural and biochemical differences in fungal cells and biofilms, in the hope of improving our understanding of their pathogenicity and developing this technique for diagnostic purposes. Methods: C. albicans planktonic cells were cultured overnight and biofilms grown for 4 and 24 hours, then analysed using spontaneous Raman spectroscopy and stimulated Raman scattering (SRS) microscopy. These methods provided spectral profiles and structural information, which were further assessed using chemometric analysis to identify biochemical variations. Results: Spontaneous Raman spectroscopy produced distinct spectral profiles for planktonic cells and biofilms, revealing differences in their biochemical composition whilst identifying essential components of the fungal cell. SRS microscopy provided structural data, showing variations in protein and lipid abundance between the planktonic cells and biofilms. Chemometric analysis also revealed differences in the lipid content of pseudo-hyphae and hyphae, demonstrating clear separation based on fungal morphology, a key change in phenotype, characteristic of a shift from oral health to disease in C. albicans-related infections. Conclusions: Raman spectroscopy is a promising tool for the characterisation of fungal pathogens such as C. albicans. It offers rapid, detailed structural and biochemical insights, with potential applications in point-of-care diagnostics, tracking microbial metabolism, and antimicrobial susceptibility testing.