Abstract
We present the design, synthesis and characterization of new functionalized fluorescent optical switches for rapid, all-visible light-mediated manipulation of fluorescence signals from labelled structures within living cells, and as probes for high-contrast optical lock-in detection (OLID) imaging microscopy. A triazole-substituted BIPS (TzBIPS) is identified from a rational synthetic design strategy that undergoes robust, rapid and reversible, visible light-driven transitions between a colorless spiro- (SP) and a far-red absorbing merocyanine (MC) state within living cells. The excited MC-state of TzBIPS may also decay to the MC-ground state emitting near infra-red fluorescence, which is used as a sensitive and quantitative read-out of the state of the optical switch in living cells. The SP to MC transition for a membrane-targeted TzBIPS probe (C12-TzBIPS) is triggered at 405 nm at an energy level compatible with studies in living cells, while the action spectrum of the reverse transition (MC to SP) has a maximum at 650 nm. The SP to MC transition is complete within the 790 ns pixel dwell time of the confocal microscope, while a single cycle of optical switching between the SP and MC states in a region of interest is complete within 8 ms (125 Hz) within living cells, the fastest rate attained for any optical switch probe in a biological sample. This property can be exploited for real-time correction of background signals in living cells. A reactive form of TzBIPS is linked to secondary antibodies and used, in conjunction with an enhanced scope-based analysis of the modulated MC-fluorescence in immuno-stained cells, for high-contrast immunofluorescence microscopic analysis of the actin cytoskeleton.
Highlights
Photochemical manipulation of organic molecules has been used for reversible and irreversible control of the two states of photochromic molecules for more than a century [1,2,3,4], with recent attention shifting towards the design of both synthetic and genetically-encoded photochromes for applications in biology [2,4]
In the ‘‘scope’’ approach to optical lock-in detection (OLID) imaging [24], we exploit our earlier finding that the 405 nmmediated change in the fluorescence signal from the MC-state is a sufficiently sensitive indicator of optical switching of the triazole-substituted BIPS (TzBIPS) probe in a pixel, and is discriminated when analyzed over multiple cycles of optical switching compared to the corresponding response of the signal from background probes and noise
Summary We have detailed the rational design, synthesis and characterization of new fluorescent optical switches based on the BIPS photochrome for all-visible light driven control of MC-fluorescence in living systems
Summary
Photochemical manipulation of organic molecules has been used for reversible and irreversible control of the two states of photochromic molecules for more than a century [1,2,3,4], with recent attention shifting towards the design of both synthetic and genetically-encoded photochromes for applications in biology [2,4]. Multiple cycles of optical switching between the SP and MC states of C12-TzBIPS can be brought about in living cells and quantified through measurements of the MC fluorescence signal (Figure 5C).
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