Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Stimulated emission depletion (STED) microscopy allows visualization of cellular structures below the optical diffraction limit.
• Simultaneous imaging of multiple species within cells is a growing demand in biological research.
• Existing multispecies STED techniques face challenges in spectral resolution and sample preparation.

Purpose of the Study:

• To develop a novel, beneficial approach for multispecies STED nanoscopy.
• To enable simultaneous investigation of multiple species with closely spaced spectral properties.
• To improve the efficiency and reduce artifacts in multispecies STED imaging.

Main Methods:

• Utilizing three excitation wavelengths in pulsed interleaved excitation (PIE) mode.
• Employing a linear unmixing algorithm based on reference patterns for image decomposition.
• Introducing the image correlation map (ICM) to identify single-species regions for reference pattern generation.

Main Results:

• Successfully probed multiple species with fluorescent labels having absorption maxima as close as 13 nm.
• Demonstrated high-speed species separation quality for up to three species.
• Eliminated the need for cumbersome preparation of additional reference samples.

Conclusions:

• The proposed method offers a powerful approach for multispecies STED nanoscopy.
• Spectral exploitation and advanced algorithms enable high-fidelity separation of closely labeled species.
• This technique advances the capability for simultaneous, high-resolution imaging in cellular biology.