Renal‐Clearable Organic Probes From D–A–D Type Aza‐BODIPY Fluorophores for Multiphoton Deep‐Brain Imaging

Abstract Bright near‐infrared (NIR) fluorescent probes play an important role in in vivo optical imaging. Here, renal‐clearable nanodots prepared from Aza‐BODIPY are reported fluorophores for multiphoton brain imaging. The design of donor–acceptor–donor (D–A–D) type conjugated structures endowed the fluorophores with large three‐photon absorption cross‐section for both 1620 and 2200 nm excitation. The side chain modification and lipid encapsulation yield ultrasmall nanodots (≈4 nm) and a high fluorescence quantum yield (≈0.35) at 720 nm emission in the aqueous phase. The measured three‐photon action cross‐section of a single Aza‐BODIPY fluorophore in the nanodots is ≈30 times higher than the commonly used Sulforhodamine 101 dye. Three‐photon deep brain imaging of subcortical structures is demonstrated, reaching a depth of 1900 µm below the brain surface in a live mouse study. The nanodots enabled blood flow measurement at a depth of 1617 µm using line scanning three‐photon microscopy (3PM). This work provides superior fluorescent probes for multiphoton deep‐brain imaging.