Excitation spectral microscopy for highly multiplexed fluorescence imaging and quantitative biosensing

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Multiplexing in fluorescence microscopy is limited by broad spectral widths.
• Current spectral imaging methods often reduce throughput due to spectral dispersion.

Purpose of the Study:

• To develop a novel fluorescence imaging technique for high-throughput, multiplexed live-cell imaging.
• To overcome spectral overlap limitations and enable simultaneous detection of multiple fluorophores.

Main Methods:

• Utilized excitation wavelength scanning with an acousto-optic tunable filter and a fixed emission band.
• Developed spectral unmixing algorithms to quantify fluorophore abundances.
• Applied the technique to live-cell imaging of biosensors and cellular processes.

Main Results:

• Achieved simultaneous imaging of up to six subcellular targets with low crosstalk (<1%) and high temporal resolution (~10 ms).
• Enabled quantitative imaging of mitochondrial pH and macromolecular crowding.
• Demonstrated multiplexed pH imaging with other organelles/proteins to study mitophagy.

Conclusions:

• Excitation spectral microscopy offers a powerful approach for highly multiplexed fluorescence imaging in live cells.
• This method provides high sensitivity and spatiotemporal resolution without fluorescence dispersion.
• The technique has significant potential for complex biological pathway elucidation.