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bioRxiv · 10.64898/2026.01.19.700264

Membrane-Promoted J-Aggregation of BODIPY Dimers Enables Spontaneous Blinking in Green and Red Channels for Live-Cell Nanoscopy

Pfister, S.·Walter, S.·Didier, P.·Collot, M.
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Abstract

BODIPY dyes are widely used in bioimaging, yet their aggregation behavior within the plasma membrane (PM) remains poorly exploited for single-molecule localization microscopy (SMLM). Here, we design a series of green-emitting PM-targeted BODIPY dimers engineered to undergo spontaneous and transient H- and J-aggregation. By tuning the linker length between the fluorophores, we identify dimers that form intramolecular H-aggregates in polar media and emissive J-aggregates ({lambda}em {approx} 535 nm) through membrane-driven intermolecular interactions. In lipid bilayers, all dimers aggregate above a probe/lipid ratio of 1/100, exclusively generating J-aggregates. In live-cell SMLM, the monomeric MB-488 provides high event numbers via diffusion-driven emission, whereas dimers exhibit stable blinking and yield brighter red-shifted J-aggregate events with improved localization precision. Red-channel events localize to specific PM regions, suggesting preferential J-aggregation within distinct membrane microdomains. HaloTag constructs targeted to cell-surface PDGFR further confirm that intramolecular J-aggregation is possible but strongly amplified by the membrane through intermolecular collisions. These results demonstrate that green BODIPYs and their J-aggregates enable robust live SMLM in green and red channels, and reveal the PM as a privileged environment for emissive J-aggregation.

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Pfister, S., Walter, S., Didier, P., Collot, M.. 2026-01-21. Membrane-Promoted J-Aggregation of BODIPY Dimers Enables Spontaneous Blinking in Green and Red Channels for Live-Cell Nanoscopy. https://doi.org/10.64898/2026.01.19.700264

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