Hyperspectral two-photon excitation microscopy using visible wavelength.

Area of Science:

• Biomedical Optics
• Microscopy
• Spectroscopy

Background:

• Hyperspectral imaging provides rich spectral information crucial for biological samples.
• Traditional methods can be slow or lack spectral resolution.
• Two-photon excitation microscopy offers advantages in depth penetration and reduced phototoxicity.

Purpose of the Study:

• To develop and demonstrate a novel hyperspectral imaging technique using visible-wavelength two-photon excitation microscopy.
• To achieve simultaneous excitation and detection of multiple fluorescent proteins.
• To enable efficient and rapid spectral unmixing for probe localization in living cells.

Main Methods:

• Utilized visible-wavelength two-photon excitation microscopy with line illumination and slit-confocal detection.
• Employed a 530 nm femtosecond pulsed laser for simultaneous excitation of fluorophores.
• Integrated linear spectral unmixing algorithms for data analysis.

Main Results:

• Successfully acquired hyperspectral images of living HeLa cells with high spectral resolution (160 data points per spectrum).
• Achieved simultaneous detection of three distinct fluorescence spectra with rapid acquisition (1 ms per line).
• Demonstrated accurate localization of fluorescent probes within cells using spectral unmixing.

Conclusions:

• The developed hyperspectral imaging technique is efficient for capturing spectral information from biological samples.
• The combination of nonlinear excitation and slit detection provides excellent depth discrimination capabilities.
• This method offers a powerful tool for multicolor imaging and analysis in cell biology.