High Spatial Resolution Imaging of a Structure of Interest in a Specimen

Traditional optical microscopy is constrained by the diffraction limit, which restricts resolution to about half the wavelength of light used. Advancements have introduced techniques like STORM and PALM, which utilize switchable fluorescent proteins and dyes to achieve super-resolution imaging. However, these methods are limited by the availability and performance of photoactivatable compounds. GSDIM and the novel high-resolution imaging technique using non-switchable dyes address these limitations by employing alternative mechanisms for fluorescence modulation, thereby enhancing resolution without the need for specialized dyes.

GSDIM:

This patented technology involves switching fluorophores to a metastable dark state and recording those that return to the ground state. Continuous widefield illumination and a high-speed camera capture high-resolution images. The technique works with various fluorophores in different environments, providing flexibility and enhancing imaging capabilities. The method significantly improves resolution, achieving details less than 30 nm.

High Spatial Resolution Imaging with Non-Switchable Dyes:

This method enhances spatial resolution by exploiting the natural transitions of non-switchable dyes between their electronic ground and dark states. By carefully managing the illumination intensity, a significant fraction of the fluorophores is switched to the dark state, allowing the remaining fluorophores to be imaged with high precision. This technique avoids the need for specialized switchable dyes, making it broadly applicable and easier to implement.

GSDIM:

• Utilizes ordinary fluorophores, eliminating the need for photoactivatable compounds.
• Achieves super-resolution with simple continuous illumination and camera setup.
• Compatible with various dyes, enhancing versatility.
• Enables dual-color imaging with standard fluorophores.
• Expands the conceptual range and applicability of far-field optical nanoscopy.

This innovative imaging technology unmatched flexibility by working effectively with general fluorophores, unlike many super-resolution techniques that require specialized fluorescent dyes or proteins. This flexibility greatly facilitates broader application across various research fields where specific fluorophores may not be readily available or are prohibitively expensive. This approach not only reduces operational costs but also simplifies experimental setups, making advanced imaging more accessible and versatile.

High Spatial Resolution Imaging with Non-Switchable Dyes:

• Avoids the limitations associated with switchable fluorescent dyes.
• Enhances spatial resolution beyond the diffraction limit.
• Utilizes common, non-specialized dyes.
• Reduces complexity and cost of implementation.

This method optimizes the control of fluorophore excitation states through meticulously managed illumination. This precision allows for a reduction in the number of light sources and system complexity required, offering significant advantages in applications such as medical diagnostics and field applications where simplicity and reliability are crucial. By enhancing the efficiency of the imaging process, our technology streamlines workflows and increases throughput, providing a practical solution that delivers high-resolution imaging without the need for complex infrastructure.

GSDIM:

• Live-cell imaging for studying dynamic biological processes.
• High-resolution mapping of cellular structures.
• Dual-color imaging for differentiating multiple cellular components.
• Research in cell biology, neuroscience, and developmental biology.
• Super-resolution imaging in clinical diagnostics.

High Spatial Resolution Imaging with Non-Switchable Dyes:

• High-precision imaging of biological specimens.
• Enhanced imaging of fixed cells and tissue samples.
• Advanced research in molecular biology and biochemistry.
• Applications in materials science for imaging nanostructures.

Development of new diagnostic imaging techniques.

GSDIM: priority date: 27.04.2007: EP2605003B1, JP523869B2, CN10148479B, US8084754
High Spatial Resolution Imaging with Non-Switchable Dyes: priority date: 20.05.2029: US8174692, CN102037347B, EP2291641B2, JP587257B2

Fölling, J., Bossi, M., Bock, H. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5, 943–945 (2008). https://doi.org/10.1038/nmeth.1257