General analysis and optimization strategy to suppress autofluorescence in microscope lenses

Lu, Xiang; Zhang, Yueqian; Groß, Herbert

doi:10.1364/ao.58.007404

Cited by 4 publications

(1 citation statement)

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“…Limited reports exist on these effects: there are some studies of autofluorescence from optical glasses or biomolecules but they are few and mainly concern excitation in the UV range. [17][18][19][20][21] Even if the disturbing autofluorescence background from optical components have sometimes been noticed in the literature regarding different kind of optical devices, [22][23][24][25] this issue was not explored thoroughly, e.g. by investigating quantitative effects in specific applications.…”

Section: Introductionmentioning

confidence: 99%

Optimized Two‐Color Single‐Molecule Tracking of Fast‐Diffusing Membrane Receptors

Schirripa Spagnolo,

Moscardini,

Amodeo

et al. 2023

Advanced Optical Materials

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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.

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