Centimeter-deep tissue fluorescence microscopic imaging with high signal-to-noise ratio and picomole sensitivity

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.