a, Across nine morphological stages of the development of lung SCC, freshly frozen samples were assayed for gene-expression profiling. The two methodological axes are visualized separately; one flow chart focuses on the detection of gene-expression patterns (left) and one flow chart focuses on the in-depth immune characterization (right) from gene expression and multispectral imaging. On the basis of gene co-expression, molecular phenotypes and gene modules were defined and functionally characterized. Immune-gene signatures and deconvolution methods were used for quantitative assessment of different immune-cell types. Relevant immune cells were investigated in more depth using multiplex immunohistochemistry and multispectral imaging. b, Bronchoscopy of in situ carcinoma tissue with white light (left) and with 400-nm wavelength illumination, under which the bronchial epithelium appears in green (right). Although it is difficult to distinguish the in situ carcinoma from normal tissue under white light, the in situ carcinoma displays a lack of green fluorescence compared to the normal epithelial tissue under fluorescence bronchoscopy.