Laser confocal microscopy or transillumination microscopy to study the cell–scaffold interfaces or to count cells or follow their trajectory during scaffold population require the use of thin samples or suffer from spatial resolution, respectively. One major constraint when it comes to imaging scaffolds and cells seeded within, is the limited penetration depth for most microscopy techniques. Our knowledge of biomaterials, their composition and interaction has evolved tremendously from the application of modern microscopy techniques. Microscopy technologies probably represent the most adequate means to fulfil such criteria, with light microscopy even having the advantage of not interfering with the biological sample (cells and scaffolds) for a wide range of wavelengths and illumination intensities. On the other hand, it is likewise important to confer reliable parameters for the cell–scaffold interaction, desirably online and for extended time periods. In any case, the structural requirements towards the implemented scaffolds regarding biocompatibility, durability, ‘homing suitability’, cellular attractibility and mechanical resistance need to be met and are constantly being improved. Other, more unconventional recent TE approaches, suggest omitting the retrieval of source cells for external seeding and aim to use the patient's own cells and cytokine environment to populate an implanted external scaffold. This seems to play an important role not only during the early differentiation phase of organogenesis, but may also serve as a reprimable programme during wound healing later in life. These chemical and physical microenvironments may alter the signalling behaviour of ingrowing cells to steer their maturation and differentiation potency. In the human body, the extracellular matrix (ECM) and its characteristic cytokine and growth factor profiles may change during spread of precursor cells. To promote an adequate microenvironment, not only is the continuous development of more biocompatible scaffold compositions a key requisite, but also the provision of more extended scaffold architectures and designs to account for the three-dimensional aspect of cell seeding and proliferation. One major constraint of this approach, however, has been the creation of artificial organotropic environments within small-scale bioreactors to promote cell proliferation into a simplified organomimetic construct, yet largely without vascularization or the complex cellular–matrix coupling usually found within the body. The cell-centric approach of seeding cells onto a supportive scaffolding material has by far been the most broadly investigated attempt to reconstruct tissues or repair rather smaller defects. Tissue engineering (TE) has become an emerging field for regenerative medicine, and bringing together patient cells and biocompatible artificial scaffolds for the production of matrix and subsequent revascularization has been often coined as ‘the holy grail’ on the path to tissue repair and personalized medicine. This review provides an overview of the powerful and constantly evolving field of multiphoton microscopy, which is a powerful and indispensable tool for the development of artificial tissues in regenerative medicine and which is likely to gain importance also as a means for general diagnostic medical imaging. coherent anti-Stokes Raman scattering, third harmonic generation). Finally, some insight is given into state-of-the-art three-photon-based imaging methods (e.g. These cover imaging of autofluorescence and fluorescence-labelled tissue and biomaterial structures, SHG-based quantitative morphometry of collagen I and other proteins, imaging of vascularization and online monitoring techniques in TE. Besides our own cell encapsulation, cell printing and collagen scaffolding systems and their NLOM imaging the most current research articles will be reviewed. After introducing classical imaging methodologies such as ♜T, MRI, optical coherence tomography, electron microscopy and conventional microscopy two-photon fluorescence (2-PF) and second harmonic generation (SHG) imaging are described in detail (principle, power, limitations) together with their most widely used TE applications. With NLOM techniques, biomaterial matrices, cultured cells and their produced extracellular matrix may be visualized with high resolution. This review focuses on modern nonlinear optical microscopy (NLOM) methods that are increasingly being used in the field of tissue engineering (TE) to image tissue non-invasively and without labelling in depths unreached by conventional microscopy techniques.