Blinking Fluorescent Probes for Tubulin Nanoscopy in Living and Fixed Cells

ACS Chem Biol. 2021 Nov 19;16(11):2130-2136. doi: 10.1021/acschembio.1c00538. Epub 2021 Nov 4.

Abstract

Here we report a small molecule tubulin probe for single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy and MINFLUX nanoscopy, which can be used in living and fixed cells. We explored a series of taxane derivatives containing spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR) and found that the linker length profoundly affects the probe permeability and off-targeting in living cells. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and the 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameter of ≤50 nm was routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin allows a complementary use of different nanoscopy techniques for investigating microtubule functions and developing imaging methods. For the first time, we resolved the inner microtubule diameter of 16 ± 5 nm by optical nanoscopy and thereby demonstrated the utility of a self-blinking dye for MINFLUX imaging.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Fluorescent Dyes
  • Humans
  • Microscopy / methods*
  • Microtubules / chemistry
  • Microtubules / physiology
  • Molecular Structure
  • Osteosarcoma
  • Rhodamines / chemistry
  • Single Molecule Imaging
  • Single-Cell Analysis
  • Taxoids / chemistry*
  • Tubulin / chemistry*

Substances

  • Fluorescent Dyes
  • Rhodamines
  • Taxoids
  • Tubulin
  • cabazitaxel