In this two-part paper, we present a novel framework and methodology to analyze data from certain image-based biochemical assays, e.g., ELISPOT and Fluorospot assays. In this first part, we start by presenting a physical partial differential equations (PDE) model up to image acquisition for these biochemical assays. Then, we use the PDEs' Green function to derive a novel parametrization of the acquired images. This parametrization allows us to propose a functional optimization problem to address inverse diffusion. In particular, we propose a non-negative group-sparsity regularized optimization problem with the goal of localizing and characterizing the biological cells involved in the said assays. We continue by proposing a suitable discretization scheme that enables both the generation of synthetic data and implementable algorithms to address inverse diffusion. We end Part I by providing a preliminary comparison between the results of our methodology and an expert human labeler on real data. Part II is devoted to providing an accelerated proximal gradient algorithm to solve the proposed problem and to the empirical validation of our methodology.