Live-cell STED microscopy enables 50 nm resolution imaging with preserved cell proliferation

Area of Science:

• Cell biology
• Microscopy techniques
• Biophysics

Background:

• Stimulated emission depletion (STED) microscopy offers super-resolution imaging by overcoming the diffraction limit.
• Its high spatiotemporal resolution and nontoxic labeling enable live-cell imaging.
• Concerns exist regarding potential phototoxicity due to high laser doses and repeated excitation-depletion cycles.

Purpose of the Study:

• To evaluate the invasiveness and potential phototoxic effects of live-cell STED microscopy.
• To validate STED microscopy for routine use in live-cell imaging applications.

Main Methods:

• Assessed long-term cell proliferation and survival over 20 hours after high-resolution STED imaging (50 nm resolution, 775 nm depletion beam).
• Monitored mitotic timing to detect any delays in STED-imaged cells compared to controls.
• Measured short-term cellular stress by analyzing cytosolic calcium levels during and after STED imaging.

Main Results:

• No significant differences in proliferation or mortality rates were observed between STED-imaged and control cells across multiple human cell lines (U2OS, HeLa, RPE-1).
• STED imaging of various cellular structures (nuclear pore complex, Golgi, actin, mitochondria) did not induce significant mitotic delay.
• No significant short-term stress responses, indicated by cytosolic calcium levels, were detected.

Conclusions:

• Live-cell STED microscopy, even with high-resolution imaging, demonstrates minimal invasiveness.
• The technique is validated as a non-phototoxic tool for super-resolution imaging in living cells.
• STED microscopy is suitable for long-term live-cell studies without compromising cellular health or function.