⭐ High Impact

Between life and death: strategies to reduce phototoxicity in super-resolution microscopy.

Tosheva Kalina L, Yuan Yue, Matos Pereira Pedro, Culley Siân, Henriques Ricardo

📰 Journal of physics D: Applied physics 📅 2020 📊 71 citations
DOI PubMed

Abstract

Abstract Super-resolution microscopy (SRM) enables non-invasive, molecule-specific imaging of the internal structure and dynamics of cells with sub-diffraction limit spatial resolution. One of its major limitations is the requirement for high-intensity illumination, generating considerable cellular phototoxicity. This factor considerably limits the capacity for live-cell observations, particularly for extended periods of time. Here, we give an overview of new developments in hardware, software and probe chemistry aiming to reduce phototoxicity. Additionally, we discuss how the choice of biological model and sample environment impacts the capacity for live-cell observations.

🔬 Techniques

Airyscan Microscopy Confocal Laser Scanning Microscopy Deconvolution Microscopy Fluorescence Microscopy Lattice Light Sheet Microscopy Light Sheet Fluorescence Microscopy Live Cell Imaging Optogenetics Reversible Saturable Optical Fluorescence Transitions Microscopy Single-Molecule Localization Microscopy Stimulated Emission Depletion Microscopy Stochastic Optical Reconstruction Microscopy Structured Illumination Microscopy Super-Resolution Microscopy Super-Resolution Optical Fluctuation Imaging Total Internal Reflection Fluorescence Microscopy

✨ Fluorophores

Dendra2 EGFP ER-Tracker GFP mEos2 mEos3 mEos3.2 MitoTracker mNeonGreen mScarlet SiR-Actin SiR-Tubulin

🧪 Sample Preparation

Cell Culture Fixation Live Imaging Transfection

🔬 Cell Lines

African Green Monkey Kidney COS-7 HeLa (Henrietta Lacks) Human Osteosarcoma U2OS Primary Neurons Primary T Cells

📷 Detectors

Zeiss Airyscan

🏛️ Research Organizations (ROR)

Affiliated research institutions:

🏛 University College London 🏛 MRC Laboratory for Molecular Cell Biology 🏛 055x5mt09 🏛 Instituto Portuguu00eas Da Qualidade 🏛 The Francis Crick Institute 🏛 Culham Science Centre 🏛 Universidad Catu00f3lica Silva Henru00edquez 🏛 SHL Portugal (Portugal)

📊 Figures

Figure 1.

Figure 1.

Summary of the factors that can be optimised to reduce phototoxicity innSRM.

Figure 2.

Figure 2.

Interactions of light with cellular components leading to phototoxicity. (a)nUV light can trigger apoptosis by inducing Fas receptor-mediated signallingnpathways. (b) UV light can directly damage DNA ...

Figure 3.

Figure 3.

Methods for measuring phototoxicity. (a) u2018Destructive read-outsu2019 arentechniques prohibiting further imaging of the sample. These include blottingnfor phosphorylated forms of proteins present i...

Figure 4.

Figure 4.

Low phototoxicity fluorescent probes and labelling for live-cell SRM. Variousnrecently-developed fluorescent protein (a) and synthetic fluorophore (b)nbased methods for labelling in live-cell super-re...

Figure 5.

Figure 5.

Hardware modalities for conventional and low-phototoxicity SRM. (a)nMicroscopy illumination regimes for conventional fluorescence imaging. (b)nExamples of regimes that reduce light dose to the sample ...

Figure 6.

Figure 6.

Analytics to complement low-phototoxicity imaging regimes. (a) Top: typicalnSMLM images are successfully reconstructed from sparse blinking raw datanacquired under high phototoxic illumination. Bottom...

Figure images are served from the NIH/NLM PubMed Central Open Access Subset or Europe PMC; copyright remains with the publishers and authors.

🏛️ Imaging Facility

🏛️ University College London

💬 Discussion

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