Design of Multiplexed, Live Cell Imaging Experiments Using Excitation Scan-Based Hyperspectral Imaging Microscopy

Area of Science:

• Cellular and Molecular Biology
• Microscopy and Imaging Technologies
• Biophotonics

Background:

• Advances in microscopy and fluorescent probes have improved cellular tracking.
• Real-time multiplexed imaging remains a challenge in live-cell studies.
• Excitation scan-based hyperspectral imaging (HSI) offers potential for multiplexed imaging but lacks standardized protocols.

Purpose of the Study:

• To address the lack of protocols for selecting multiple fluorescent labels for excitation scan-based HSI.
• To outline considerations for choosing fluorescent labels and optimizing HSI parameters.
• To demonstrate the capability of HSI for simultaneous quantification of various cellular components.

Main Methods:

• Developed and utilized a custom-built excitation scan-based HSI microscope with tunable filters (360-550 nm).
• Transfected HEK-293 cells with fluorescent proteins and loaded cells with specific dyes.
• Measured excitation spectra of individual and combined fluorescent labels.

Main Results:

• Successfully quantified the relative abundance and spatial distribution of multiple fluorophores, including NucBlue, AlexaFluor dyes, Cal dyes, and fluorescent proteins (GFP, Cerulean, Turquoise, Venus, tdTomato, mCherry).
• Demonstrated the effectiveness of excitation scan-based HSI for distinguishing and measuring individual and combined labels.
• Established a foundation for selecting optimal label combinations for multiplexed cellular imaging.

Conclusions:

• Excitation scan-based HSI is a powerful technique for multiplexed cellular imaging.
• Standardized protocols for label selection can enhance the adoption and application of HSI.
• This work facilitates more comprehensive real-time analysis of protein dynamics and cellular activities.