Recent advances in label-free imaging techniques based on nonlinear optical microscopy to reveal the heterogeneity of the tumor microenvironment.

The tumor microenvironment (TME) is a complex and dynamic network that significantly influences cancer progression. Understanding its intricate components, including the extracellular matrix (ECM), stromal cells, immune cells, and vascular endothelial cells, is crucial for developing effective cancer therapies. Conventional diagnostic methods, while essential, have limitations in sensitivity, specificity, and invasiveness. Label-free multimodal nonlinear optical (MNLO) microscopy offers a promising alternative, enabling detailed imaging without external labels. Techniques such as second harmonic generation (SHG), third harmonic generation (THG), coherent anti-Stokes Raman scattering (CARS), and two-photon fluorescence (TPF) provide complementary insights into the TME. SHG is particularly effective for imaging collagen fibers, while CARS highlights lipid-rich structures, and THG and TPF offer high-resolution imaging of cellular and subcellular structures. These modalities reveal crucial information about tumor progression, including changes in collagen organization and lipid metabolism, and allow for the study of cellular interactions and ECM remodeling. Multimodal setups, combining SHG, CARS, THG, and TPF, enable comprehensive analysis of the TME, facilitating the identification of early-stage cancerous changes and tracking of tumor progression. Despite the advantages of MNLO microscopy, such as reduced photodamage and the ability to image live tissues, challenges remain, including the complexity and cost of the setups. Addressing these challenges through technological advancements and optimization can enhance the applicability of MNLO microscopy in clinical diagnostics and cancer research, ultimately contributing to improved cancer diagnosis, prognosis, and treatment strategies.