Reversible chemical reactions for single-color multiplexing microscopy

Area of Science:

• * Advanced optical microscopy and bioimaging.
• * Chemical biology and fluorescent probe development.

Background:

• * Standard fluorescence microscopy faces limitations in simultaneously imaging multiple structures due to restricted multiplexing channels.
• * Existing methods like spectral multiplexing and fluorescence-lifetime imaging have inherent channel constraints.

Purpose of the Study:

• * To introduce a novel chemical multiplexing approach to expand the number of imaging channels in fluorescence microscopy.
• * To demonstrate the feasibility of controlling fluorescent dye emissive states via chemical reactions for sequential imaging.
• * To show the orthogonality and compatibility of chemical multiplexing with existing techniques.

Main Methods:

• * Development and application of a reversible chemical switching mechanism for a fluorescent copper sensor.
• * Sequential imaging of distinct biological structures within the same spectral channel using chemical control.
• * Combination of chemical multiplexing with spectral multiplexing using two different fluorescent dyes.

Main Results:

• * Successfully demonstrated reversible switching of a fluorescent dye, enabling successive imaging of two structures in one channel.
• * Established that chemical multiplexing is orthogonal to established imaging modalities.
• * Achieved simultaneous imaging of four structures using only two spectrally distinct channels by combining chemical and spectral multiplexing.

Conclusions:

• * Chemical control over fluorescent dye emission offers a powerful strategy to increase imaging channel capacity in fluorescence microscopy.
• * This method overcomes limitations of spectral multiplexing, particularly for demanding techniques like stimulated emission depletion microscopy.
• * The developed chemical multiplexing approach provides a versatile tool for advanced bioimaging applications.