Two-photon fluorescent probes for quantitative bio-imaging analysis in live tissues

Two-photon (TP) microscopy (TPM) is an indispensable visualization tool for deep-tissue imaging. TPM uses the low energy light of the near-infrared window as an excitation source of fluorophores which reduces autofluorescence, photodamage, and light scattering, resulting in high resolution fluorescence images potentially beyond several millimeter depths in living systems. Due to the continuous development of the microscope and advances in optical output algorithms, it is crucial to develop innovative strategies for the quantitative analysis of biomedically relevant target analytes in living systems using TP fluorescence probes. In this review, we have highlighted ratiometric TP probe design strategies with representative ratiometric TP sensors in the last decade. We have also summarized the recent progress in the development of TP probes published between 2017 and 2022 for quantitatively detecting analytes such as enzymes, reactive species, bio-thiols, pH, neutral molecules, and metal ions in live cells and tissues. • Current advances in quantitative bio-imaging in live tissue using ratiometric two-photon fluorescent probes are reviewed. • Special attention is given to designing strategies involving ratiometric bio-imaging such as ICT, FRET, and internal reference-based approaches. • Identification of specific analytes and applications in deep live tissues, in vivo, and biomedical research are discussed. • Insights into the current state, existing challenges, and future direction in developing ratiometric two-photon probes are rationally envisioned.