Label-free imaging using two-photon autofluorescence of reduced form nicotinamide adenine dinucleotide phosphate, or NAD(P)H, provides nondestructive, high-resolution, 3D visualization of cellular activities in living systems. Due to light scattering, however, this imaging technique typically can only penetrate as far as 300 μm into living tissues. To enable deep imaging of thick tissues, researchers at MIT implemented multimode fiber-based, three-photon excitation of NAD(P)H with a low repetition rate and high peak power. They used living, engineered, human multicellular microtissues as test samples. With this approach, the researchers more than doubled the standard depth limit of NAD(P)H imaging, extending it beyond 700 μm. They achieved deep and dynamic simultaneous localization and mapping (dSLAM) microscopy for structural and metabolic imaging of intact, living biosystems. The dSLAM microscopy technique attained a high peak power exceeding 0.5 megawatts (MW) at a band of 1100 nm, plus or minus 25 nm. This was achieved by adaptively modulating multimodal, nonlinear pulse propagation with a compact fiber shaper. An initial image (left), and the optimized image using the new technique. Courtesy of Kunzan Liu et al. The fiber shaper enabled the researchers to tune the color and pulses of laser light to minimize scattering and maximize the signal, so they could see further into the tissue and capture clearer images. The eightfold increase in pulse energy at 1100 nm allowed faster imaging of monocyte behaviors in living multicellular models. The new, noninvasive imaging technique could help biomedical researchers study the body’s immune responses in living tissue and develop new medicines. The ability to capture the metabolic dynamics of living biosystems is essential for basic biomedical research and laboratory testing. The enhanced depth provided by dSLAM microscopy, combined with the improved imaging speed, could help fuel new investigations into complex cellular interactions. The flexibility provided by the modular design — a step-index multimode fiber with a slip-on fiber shaper — makes this imaging methodology suitable for demanding in vivo and in vitro imaging applications, including cancer research, immune responses, and tissue engineering. Advanced microscopy technologies have led to a better understanding of biology, with each technique offering unique advantages for specific applications. With its noninvasive, deep tissue imaging capabilities, dSLAM microscopy could serve as a complementary tool to other imaging techniques for studying cellular dynamics in living tissues. It requires minimal sample preparation and no exogenous labels. Continued advancements in beam optimization, system design, and streamlined data analysis could make dSLAM microscopy increasingly accessible to biomedical researchers, as a valuable addition to the existing arsenal of microscopy techniques for investigating living tissues. “It opens new avenues for studying and exploring metabolic dynamics deep in living biosystems,” professor Sixian You said. The research was published in Science Advances (www.doi.org/10.1126/sciadv.adp2438).