To gain insight into the fundamental processes determining the motion of macromolecules in polymeric matrices, the dynamical hindrance of polymeric dextran molecules diffusing as probe through a polyacrylamide hydrogel is systematically explored. Three complementary experimental methods combined with Brownian dynamics simulations are used to study a broad range of dextran molecular weights and salt concentrations. While multi-parameter fluorescence image spectroscopy (MFIS) is applied to investigate the local diffusion of single molecules on a microscopic length scale inside the hydrogel, a macroscopic transmission imaging (MTI) fluorescence technique and nuclear magnetic resonance (NMR) are used to study the collective motion of dextrans on the macroscopic scale. These fundamentally different experimental methods, probing different length scales of the system, yield long-time diffusion coefficients for the dextran molecules which agree quantitatively. The measured diffusion coefficients decay markedly with increasing molecular weight of the dextran and fall onto a master curve. The observed trends of the hindrance factors are consistent with Brownian dynamics simulations. The simulations also allow us to estimate the mean pore size for the herein investigated experimental conditions. In addition to the diffusing molecules, MFIS detects temporarily trapped molecules inside the matrix with diffusion times above 10 ms, which is also confirmed by anisotropy analysis. The fraction of bound molecules depends on the ionic strength of the solution and the charge of the dye. Using fluorescence intensity analysis, also MTI confirms the observation of the interaction of dextrans with the hydrogel. Moreover, pixelwise analysis permits to show significant heterogeneity of the gel on the microscopic scale.