Large Stokes-shift bioorthogonal probes for STED, 2P-STED and multi-color STED nanoscopy

Area of Science:

• Chemical Biology
• Microscopy
• Organic Synthesis

Background:

• Super-resolution microscopy requires advanced fluorescent probes for detailed cellular imaging.
• Existing probes may have limitations in spectral properties or bioorthogonal compatibility.

Purpose of the Study:

• To synthesize and evaluate novel red-emitting tetrazine-functionalized fluorescent probes.
• To demonstrate their utility in various super-resolution microscopy techniques, including STED and 2P-STED.
• To showcase their application in bioorthogonal labeling of cellular targets.

Main Methods:

• Synthesis of four red-emitting tetrazine-functionalized fluorescent probes (CBRD 1-4).
• Application in Stimulated Emission Depletion (STED) microscopy for bioorthogonal labeling of actin, keratin-19, and TOMM20.
• Live-cell imaging of insulin receptors using genetically encoded non-canonical amino acids.
• Utilizing probes with two-photon excitation (2P-STED) and multi-color STED imaging.

Main Results:

• Successful synthesis of four red-emitting fluorescent probes with large Stokes-shifts.
• Demonstrated applicability in super-resolution microscopy via bioorthogonal labeling of cytoskeletal and mitochondrial proteins.
• Achieved super-resolved imaging of insulin receptors in live cells.
• Probes showed compatibility with common depletion lasers and enabled 2P-STED for enhanced resolution.
• Successful three-color STED imaging using one of the developed probes.

Conclusions:

• The developed tetrazine-functionalized fluorescent probes are highly suitable for advanced super-resolution microscopy.
• Their large Stokes-shift, wide spectral bands, and bioorthogonal capabilities facilitate versatile cellular imaging.
• These probes offer improved spatial resolution and multicolor imaging possibilities in live and fixed cells.