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

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.

Thermal, Photochromic and Dynamic Properties of Water-Soluble Spiropyrans

“…Apart from their exceptional photochromic features, spiropyran compounds or rather their open‐ring MC isomers are known to fluoresce . Therefore, the emission properties of the three investigated BIPS derivatives were monitored by means of excitation wavelength‐dependent fluorescence contour plots (Figure ) that were measured under identical experimental conditions.…”

Rapid amplitude-modulation of a diarylethene photoswitch: en route to contrast-enhanced fluorescence imaging

“…Note that this scheme is not applicable using other frequently employed photoswitches that almost exclusively presents uorescence in the colored isomeric form, such as the spiropyrans used in the previously reported cell studies. 10,14 We would also like to emphasize that the absorption band of the uorescent form Dasy(o) extends beyond 400 nm as opposed to most other photochromic switches where the colorization reaction has to be triggered at 365 nm or shorter. This implies conveniently that the line at 405 nm can be used in the microscopy experiments and also a reduced phototoxicity.…”

Kinetics of Reactive Modules Adds Discriminative Dimensions for Selective Cell Imaging

“…Several fluorescent labels can be reversibly switched between two states of different brightnesses by alternating illumination with two light sources of appropriate wavelengths. OLID has exploited this property for dynamic contrast generation in biological environments . More precisely, after several cycles of optical switching, the modulated fluorescence signal issued from the target is isolated from the non‐modulated background by cross‐correlation with a reference waveform.…”