A key factor in current approaches for tissue and organ regeneration relies on enhancing (stem) cell-material interactions to obtain the same original functionality. Different approaches include delivery of biological factors, functionalization of biological factors onto 3D scaffolds surface, engineering surface properties (e.g. via topography modifications), and controlling bulk and structural chemical and mechanical properties of the cell-laden biomaterial porous constructs that are developed for regeneration. Although these strategies have proved to augment cell activity on biomaterials, they are still characterized by limited control in space and time, which hampers the proper regeneration of complex tissues. Here, we present a few examples where integration of biofabrication platforms allowed the generation of a new library of biological constructs with tailored biological, physicochemical, and mechanical cues at the macro, micro, and nano scale. These biological constructs are characterized by tailored cell-material interactions able to influence the activity of stem cells, thereby sustaining the regeneration of complex tissues. From these examples as well as from the study of other scientists, converging technologies seems to be a powerful route towards designing of biological constructs with instructive properties able to control cell activity for the regeneration of functional tissues. Future efforts should aim at further improving technology integration to achieve a fine control on stem cell fate by biomaterial and scaffolds design at multiple scales. This will enable the regeneration of complex tissues including vasculature and innervation, which will result in enhanced in vivo integration with surrounding tissues. By doing so, the gap from tissue to organ regeneration will be reduced, bringing regenerative medicine technologies closer to the clinics.

The Nonlinear Bio-Imaging Lab, in collaboration with the FAIR CHARM consortium, is developing new nonlinear imaging platforms to meet diverse biological challenges. In this talk, I will present recent results from two complementary systems: the SWIM microscope, designed for deep, label-free imaging, and SLIDE, designed for ultrafast volumetric acquisition.

Using SWIM, we are developing AI-assisted cell detection from THG images of stained histological samples of human skin, and are now extending this approach to unstained ex vivo biopsies as a step toward in vivo 3D skin microscopy. We also apply SWIM to label-free imaging of mucus in lung models, where we visualize mucus volume, structure, and flow behavior without the need for fluorescent tracers.

With SLIDE, we demonstrate detection of stem cells in whole, unfiltered blood. Harmonic nanoparticle-labeled cells are imaged in flow using a diffractive scanning geometry driven by a wavelength-swept FDML laser. AI segmentation and downstream analysis distinguish isolated cells from aggregates, supporting future applications for in vivo tracking of rare cells circulating in the bloodstream.

Both systems are based on parametric excitation of harmonic signals, but differ in optical design and laser source. SWIM uses a tunable femtosecond OPA source (1250–1800 nm) for deep tissue imaging via 3P and THG processes, while SLIDE uses narrowband pulses and diffractive scanning to achieve 40 Hz 3D imaging at subcellular resolution. Together, these platforms broaden the reach of multiphoton microscopy in both depth and speed, from static 3D tissue structure to dynamic processes.