High-contrast fluorescence imaging in fixed and living cells using optimized optical switches.

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

Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues

The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis

Summary of the structures of the BIPS derived probes prepared for the red-shifting of the SP and MC spectra.

<p>Summary of the structures of the BIPS derived probes prepared for the red-shifting of the SP and MC spectra.</p

High-Contrast Fluorescence Imaging in Fixed and Living Cells Using Optimized Optical Switches

<div><p>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.</p></div

Optical switching of C₁₂-TzBIPS (Fig. 3C) in living NBT-II cells.

<p><b>A</b>), Image montage showing MC fluorescence signal of MC-state of C₁₂-TzBIPS within a field of living cells over 3 cycles of optical switching using a low power objective; <b>B</b>), Higher magnification view of the optical switching of MC-fluorescence of C₁₂-TzBIPS within a single cell in the same sample; <b>C</b>), Intensity trace of MC-fluorescence corresponding to the yellow boxed region in (5A); <b>D</b>), Intensity trace of MC-fluorescence within the yellow boxed region in (5B). <b>E</b>), Image montage 6 cycles of rapid optical switching of C₁₂-TzBIPS in living cells within a narrow field of view; <b>F</b>), Intensity trace of the MC-fluorescence averaged over the entire image field (<b>E</b>).</p

Spectroscopic properties of BIPS probes in ethanol.

a<p>Reference compound 6-NO₂-BIPS;</p>b<p>Photochemistry of <b>1e</b> and <b>1g</b> is too low to be measured;</p>c<p>The MC-state of <b>2f</b> does not convert back to the SP-state even with light irradiation;</p>d<p>The MC-state of <b>1f</b> is thermally stable in the dark at room temperature but it converts back to SP state upon exposure to green light;</p>e<p>Colorability of the probe after exposure to 365 nm light for 30 s;</p>f<p>Colorability of the probe after exposure to 405 nm light for 60 s.</p

Schematic representations of optical switching reactions and the modulation of MC fluorescence.

<p>(<b>A</b>), Optically-induced transitions between the SP and MC states of BIPS. (<b>B</b>), Modulation of the MC-fluorescence signal in response to orthogonal control of the SP and MC states.</p