This digital resource is a scientific article published in Nature Methods, focusing on deep tissue two-photon microscopy. Two-photon microscopy is a specialized fluorescence imaging technique that excels at imaging deep within scattering biological tissues, a significant advantage over traditional widefield or confocal microscopy. The article likely provides a comprehensive review of the principles, advantages, and applications of this advanced imaging modality. The article explains that two-photon microscopy overcomes the limitations of light scattering in biological tissues by using longer excitation wavelengths (typically near-infrared) and a nonlinear excitation process, which confines fluorescence excitation to the focal plane. This results in reduced phototoxicity and photobleaching, allowing for deeper penetration and prolonged imaging of live samples. It discusses key parameters influencing imaging depth, such as excitation wavelength and tissue optical properties. Researchers in neuroscience, developmental biology, and oncology widely use deep tissue two-photon microscopy for in vivo imaging of cellular dynamics, neuronal activity, and tumor microenvironments. The technique enables high-resolution imaging several hundred microns deep in living animals, providing crucial insights into complex biological processes in their native context. The article may also delve into advanced aspects such as adaptive optics for aberration correction, specialized laser systems (e.g., ytterbium-doped fiber lasers), and the use of specific fluorophores optimized for two-photon excitation. It serves as a valuable guide for researchers aiming to implement or optimize deep tissue imaging experiments, offering practical considerations for achieving robust and reliable results in challenging biological systems.