Phasor-based multi-harmonic unmixing forin-vivohyperspectral imaging

Area of Science:

• Biomedical optics
• Spectroscopy
• Cellular imaging

Background:

• Hyperspectral imaging (HSI) is crucial in biomedical science but faces challenges in spectral component unmixing and quantification.
• Traditional methods require specific spectroscopic parameters of fluorescent species, limiting their application.

Purpose of the Study:

• To develop and validate a phasor-based multi-harmonic unmixing method for HSI that simplifies spectral component quantification.
• To demonstrate the method's feasibility for analyzing multiple spectral components, including autofluorescence, in biological samples.

Main Methods:

• The phasor-based multi-harmonic unmixing method was developed, requiring only empirical measurements of pure species.
• Simulations were performed to assess the method's accuracy for up to 6 components, including an unknown autofluorescence component.
• The method was tested in living cells using commercial dyes for organelle labeling and a solvatochromic probe (LAURDAN) for membrane dynamics.

Main Results:

• Simulations confirmed the method's feasibility for up to 5 components and its robustness with autofluorescence in typical confocal imaging conditions.
• The method accurately separated 5 common commercial dyes used for organelle labeling in living cells.
• The study successfully monitored and quantitatively compared membrane order in the Golgi apparatus, mitochondria, and plasma membrane within the same cell using LAURDAN.

Conclusions:

• The phasor-based multi-harmonic unmixing method offers a simplified approach to HSI data analysis, eliminating the need for specialized spectroscopic knowledge.
• This technique democratizes HSI, expanding its use in biomedical research for accurate spectral component unmixing and quantitative analysis of cellular processes.