(293c) A Wavelength-Induced Frequency Filtering Method for Extending Fluorescent Biological Assays In Vivo

Fluorescence-based assays are employed ubiquitously throughout the life sciences and medicine, however their adaptation and translation to the in vivo environment is fundamentally hampered by unfavorable tissue scattering and intrinsic autofluorescence. Herein, we develop a wavelength-induced frequency filtering (WIFF) and show that it significantly improves the in vivo signal to noise ratio (SNR) of fluorescent probe and sensor components, extending measurements to extremely deep implants up to 5.5±0.1 cm deep in highly scattering tissue phantoms, complex tissues, and SKH1-E mouse models. By modulating the excitation wavelength cyclically across the absorption cross section of any sensing chromophore, the emission waveform can be deconvoluted from the scattering background, autofluorescence and other noise sources. The technique is shown to boost SNR up to 52-fold for ten common fluorescent probes used in biomedical assays and emit across the entire visible range. Near-infrared fluorescent carbon nanotube sensors are also probed with this technique to detect H₂O₂, riboflavin, and ascorbic acid at high fidelity in vivo with 2 Hz temporal resolution in real time. These results based on WIFF open up new avenues of biomedical research by extending a large number of fluorescent sensing assays to previously inaccessible in vivo environments.