This lecture deals with signal processing methods dedicated to hyperspectral data recorded in confocal fluorescence microscopy and atomic force microscopy (AFM), with applications to biological imaging. We first address two typical inverse problems arising in confocal microscopy, namely hyperspectral image restoration (or deconvolution) and hyperspectral unmixing. The second part of the lecture is dedicated to the analysis of force curves and force-volume images recorded in AFM. Their interpretation is done using physico-chemical models, e.g., electrostatic and mechanical models. The quantitative estimation of parameters from experimental force curves is based on fully automated tools. This leads us to reconstruct a set of images at the nanoscale, each corresponding to a specific physico-chemical parameter.