Significance: Follicular thyroid carcinoma carries a substantially poor prognosis due to its unique biological behavior and less favorable outcomes. In particular, fine-needle aspiration (FNA) biopsies, which play a key role in screening thyroid nodules, cannot differentiate benign from malignant follicular neoplasm.

Aim: We report on the use of hyperspectral Raman microscopy in combination with chemometric analysis for identifying and classifying single cells obtained from clinical samples of human follicular thyroid neoplasms.

Approach: We used a method intended to simulate the FNA procedure to obtain single cells from thyroid nodules. A total of 392 hyperspectral Raman images of single cells from follicular thyroid neoplasms were collected.

Results: Malignant cells were identified based on their intrinsic Raman spectral signatures with an overall diagnostic accuracy of up to 83.7%.

Conclusions: Our findings indicate that hyperspectral Raman microscopy can potentially be developed into an ancillary test for analyzing single cells from thyroid FNA biopsies to better stratify “indeterminate” nodules and other cytologically challenging cases.