  The tumor microenvironment (TME) features distinct hallmarks, including
acidosis, hypoxia, reactive oxygen species (ROS) generation, and altered ion
fluxes, which are crucial targets for early cancer biomarker detection, tumor
diagnosis, and therapeutic strategies. A variety of imaging and sensing
techniques have been developed and employed in both research and clinical
settings to visualize and monitor cellular and TME dynamics. Among these,
ratiometric fluorescence-based sensors have emerged as powerful analytical
tools, providing precise and sensitive insights into the TME and enabling
real-time detection and tracking of dynamic changes. In this comprehensive
review, we discuss the latest advancements in ratiometric fluorescent probes
designed for optical mapping of pH, oxygen, ROS, ions, and biomarkers within
the TME. We elucidate their structural designs and sensing mechanisms, as well
as their applications in in vitro and in vivo detection. Furthermore, we
explore integrated sensing platforms that reveal the spatiotemporal behavior of
complex tumor cultures, highlighting the potential of high-resolution imaging
techniques combined with computational methods. This review aims to provide a
solid foundation for understanding the current state of the art and the future
potential of fluorescent nano- and microparticles in the field of cellular
microenvironment sensing.
