  Fluorescence microscopic imaging in centimeter-deep tissue has been highly
sought-after for many years because much interesting in vivo micro-information,
such as microcirculation, tumor angiogenesis, and metastasis, may deeply locate
in tissue. In this study, for the first time this goal has been achieved in
3-centimeter deep tissue with high signal-to-noise ratio (SNR) and picomole
sensitivity under radiation safety thresholds. These results are demonstrated
not only in tissue-mimic phantoms but also in actual tissues, such as porcine
muscle, ex vivo mouse liver, ex vivo spleen, and in vivo mouse tissue. These
results are achieved based on three unique technologies: excellent near
infrared ultrasound-switchable fluorescence (USF) contrast agents, a sensitive
USF imaging system, and an effective correlation method. Multiplex USF
fluorescence imaging is also achieved. It is useful to simultaneously image
multiple targets and observe their interactions. This work opens the door for
future studies of centimeter-deep tissue fluorescence microscopic imaging.
