Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT-TIRFm setups on the signal-to-noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two-color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT.

Optimized two-color single-molecule tracking of fast-diffusing membrane receptors

Schirripa Spagnolo, Chiara;Moscardini, Aldo;Amodeo, Rosy;Beltram, Fabio;Luin, Stefano
2023

Abstract

Single particle tracking (SPT) combined with total internal reflection fluorescence microscopy (TIRFm) is an outstanding approach to decipher mechanisms on the cell membrane at the nanoscale. Multicolor configurations, needed to investigate interactions, are still hindered by several challenges. This work systematically and quantitatively analyzes the impact of necessary elements of SPT-TIRFm setups on the signal-to-noise ratio (SNR), which must be optimized especially in dynamic studies needing minimally invasive dyes for biomolecule labeling. Autofluorescence originating from commonly used optical glass results in the dominant limiting factor in TIRFm, and a cover glass material is tested yielding significant SNR improvements in multichannel TIRFm. Moreover, methodologies are optimized for reducing fluorophore photobleaching in multicolor implementations requiring simultaneous stabilization of multiple dyes. The developed strategies are applied to the fast p75NTR receptors labeled by two fluorophores on the membrane of living cells, achieving reliable, simultaneous two-color SPT, contrary to configurations using standard cover glasses. This work highlights the importance of optical materials suitable for microscopy and with reduced autofluorescence for increasing sensitivity toward ultimate spatiotemporal resolutions. In particular, the present protocols can pave the way for multicolor super-resolved localization and tracking of single molecules by TIRFm, greatly expanding the potential of SPT.
2023
Settore FIS/03 - Fisica della Materia
cover glasses for TIRF microscopy; live cell nanoscopy; multicolor single molecule imaging; optical glass fluorescence; photobleaching reduction
   Nanotechnology for tumor molecular fingerprinting and early diagnosis
   Fondazione Pisa

   PNRR – MISSIONE 4 COMPONENTE 2
   European Union Next-GenerationEU through the PIANO NAZIONALE DI RIPRESA E RESILIENZA

   NQSTI – INVESTIMENTO 1.3
   National Quantum Science and Technology Institute

   THE – INVESTIMENTO 1.5
   Tuscany Health Ecosystem
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11384/137153
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