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.


TABLE OF CONTENTS General Experimental Methods S2
Design and Synthesis of Compounds 1-5 S2-S20

General Experimental Methods
All reactions were carried out under an atmosphere of dry nitrogen. Glassware were oven-dried prior to use. Unless otherwise indicated, common reagents or materials were obtained from commercial source and used without further purification. Dry distilled THF and CH 2 Cl 2 were obtained from Acros and used as received. Flash column chromatography was performed using silica gel 60 (70-230 mesh). Analytical thin layer chromatography (TLC) was carried out on Merck silica gel plates with QF-254 indicator and visualized by UV. Absorbance spectra were recorded on a Shimadzu UV-1601PC spectrophotometer at room temperature. Fluorescence spectra were obtained on a AMINCO-Bowman Series 2 spectrofluorometer with 4 nm excitation and emission slit width at room temperature.
1 H and 13 C NMR spectra were recorded on a Bruker 400 (400 MHz 1 H; 100 MHz 13 C) spectrometer at room temperature. Chemical shifts were reported in ppm relative to the residual solvent signal (CDCl 3 : 99.8 % D contains 0.05% v/v TMS,  7.26 ppm 1 H;  77.00 ppm 13 C).
Photochemistry: a hand-held UV lamp (UVGL-25) was used as the 365 nm light source.

Design of red-shifted BIPS derivatives
There is increasing scepticism amongst biologists about using near ultraviolet light to generate fluorescent signals from a labeled cell. The main issues underlying this trend are: living cells undergo a UV-triggered stress response that can interfere with the study at hand and, most commercial confocal fluorescence microscopes do not allow for laser or wde-field excitation of the field with light lower than 400 nm. The challenge in exploiting unique properties of optical switch probes in cell and tissue biology then is to shift the SP-absorption band beyond 400 nm, while maintaining robust and high-fidelity switching between the SP and MC states.
Our strategy in red-shifting the SP-absorption band from a typical valiue of 350 nm to beyond 400 nm is to maintain the core aromatic structure of BIPS while extending bond conjugation at different sites on the aromatic scaffold. We reasoned that this strategy would lead to a red-shifting of the SP-absorption band while providing a quantitative evaluation and insight into how these spectral shifts affect quantum yields for excite state transitions between SP and MC. The alkynyl substituent is chosen to extend the conjugation because (i) it is compact, engages in -bonding with the indoline and pyran moeities and only modestly increases the mass of BIPS; (ii) the alkyne group is easily introduced into BIPS by using a palladium catalyzed Sonogashira coupling reaction; (iii) red-shifting of the absorption wavelength of aromatic probes with alkynyl groups has already been demonstrated for BODIPY; (iv) alkynyl groups are readily functionalized eg. by direct attachment to an aryl moiety or by conversion to a triazole via click chemistry. Here we describe the synthesis of BIPS molecules harboring alknyl groups at different sites and carry out a systematic investigation of the effect of the alkyne substituent on the spectroscopic and photochromic properties for each substitution. Alkyne substitutions are carried out using the 6-NitroBIPS with the exception of 8-nitro-BIPS (compound 1f).
Several alkynyl BIPS with promising photochromic properties are chosen for further functionalization with anthracene or triazole in order to improve their photoswitching performance. In particular, 9-Alkynyl-anthracene has a strong absorbtion band at 405 nm and is easily prepared from the alkynyl-BIPS while the triazole group will also increase -conjugation and is readily generated from the alkyne by using click chemistry.

Synthesis of BIPS derivatives
The synthesis of new BIPS probes is summarized in Scheme S1-2 and detailed in the experimental procedures. The alkyne substituted BIPS 1a-1h are prepared from the corresponding Iodo-BIPS precursors 4a-4h (bromo-BIPS for 4f) by a Sonogashira coupling reaction. The iodo-BIPS are synthesized by condensation of corresponding indoline and salicylaldehyde. The aldehyde fragment for BIPS 4a-4d is a commercially available compound 2-hydroxy-5-nitrobenzaldehyde. The indoline fragments for 4a-4d are synthesized from the corresponding Iodo-anilines using reported conditions. It is noteworthy that the two indolines 5a and 5c, which can be separated by chromatography, are generated from 3-iodoaniline. For 4e-4h, we note that the indoline fragment is commercially available and the substituted salicylaldehydes are prepared according to reported procedures. Unfortunately, several reactions conditions employed in the preparation of 5-iodo-3nitrosalicylaldehyde all lead to the formation of inseparable mixtures. a b Scheme S1. Illustrative synthesis of (a) 4a -4d; (b) 4e -4h.
However, the reaction did not go to completion for the bromo-BIPS 4f even after prolonged reaction time and elevated temperature although sufficient material is isolated for spectroscopic studies. The trimethylsilyl group in compounds 1a-h is removed by tetrabutylammonium fluoride (TBAF) to generate the free acetylene derivative. Coupling of the deprotected compounds with 9-bromoanthracene was unsuccessful due to dimerization of the free acetylene BIPS. Thus compounds 2a and 2f are prepared from the treaction of the BIPS halide with 9-alkyne-anthracene using Sonogashira coupling. The low yield for these compounds is due to incomplete reactions and decomposition of the starting materials at high temperature.
Cu(I)-catalyzed cycloaddition of the free acetylene BIPS and tert-butyl azidoacetate afford the triazole substituted BIPS 3a and 3f in moderate yields. A derivative 3fa with a long hydrocarbon chain for labeling of cell membranes is also prepared by a similar method.

Synthesis of TzBIPS-NHS:
Step 1: Synthesis of compound 7 To a stirred solution of compound 1f (97 mg, 0.23 mmol) in 5 mL of dry THF was added TBAF (1 M in THF, 0.28 mL) at 0 o C. After stirred at 0 o C for 15 min, the reaction mixture was diluted with EtOAc then washed with water and brine. The organic layer was dried over Na 2 SO 4 then concentrated. Step 2: Synthesis of TzBIPS-COOH To a stirred solution of compound 7 (42 mg, 0.12 mmol), Cu(CH 3 CN) 4 PF 6 (9 mg, 0.024 mmol) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 3 mg, 0.006 mmol) in 5 mL of THF was added DIEA (0.21 mL, 1.2 mmol), followed by a solution of 6azido-hexanoic acid (57 mg, 0.36 mmol) in 1 mL of THF. The reaction mixture was stirred at room temperature for 2 h then diluted with EtOAc. The mixture was acidified with 1N HCl then washed with water and brine. The organic layer was dried over Step

3: synthesis of TzBIPS-NHS
To a stirred solution of TzBIPS-COOH (4 mg, 0.008 mmol), dicyclohexcylcarbodiimide (DCC, 2 mg, 0.0096 mmol) and N-hydroxysuccinimide (NHS-OH, 1.2 mg, 0.01 mmol) in 3 mL of THF was added triethylamine (4 L, 0.024 mmol). The reaction mixture was stirred at room temperature overnight then filtered and concentrated. The residue was used directly without further purification.  Kinetic profiles of thermal decay of the MC states in the dark at room temperature:

Optical switching of red-shifted BIPS probes in living cells
The most promising red-shifted optical switch probes 3f is chosen for further testing in living cells. NIH 3T3 cells are incubated with 50 M of probe in medium for 30 min at 37 o C followed by washing with fresh culture medium. Cells loaded with these probes are indistinguishable from control cells, indicating the probes are not cytotoxic. Strong and uniform MC fluorescence is observed for both probes in the cells indicating they penetrate the plasma membrane and are retained within the cytoplasm although in some cases vesicles staining is also found.
Optical switching of the red-shifted BIPS probes in living NIH 3T3 cells is carried out using a Zeiss 700 confocal microscope equipped with 405 nm and 555 nm lasers. A demonstration of optical switching of 3f is shown in Figure S8a. As indicated in the scheme of Figure S8c, a single cycle of optical switching between the SP and MC states of 3f is achieved by first irradiating the red-circled area in the field of view ( Figure S8a) with two sequential scans at 405 nm. Exposure of the sample to 405 nm generates the fluorescent MC state, which is observed on exposing the entire field to 555 nm light. Some of the 555 nm excited MC-molecules may undergo a MC to SP transition. Consequently the MC-fluorescence signal diminishes to almost zero following five sequential scans of the entire field to 555 nm. The intensity of MCfluorescence over a single cycle of optical swiching is only modulated within the redcircled area ie that exposed to both 405 nm and 555 nm ( Figure S8a). Subsequent illumination of SP molecules in the same red circled area with two sequential scans at 405 nm, repopulates the MC state as seen by the increase in MC fluorescence, whereas no red fluorescence is detected outside of the circled area. Frames showing modulation of 3f within the red circled region is shown for two cycles of optical switching ( Figure S8a). The corresponding plot of the MC-intensity within the red circled region within a labeled cell is shown in Figure S8d. Faster cycling times can be realized by increasing the intensity of the 555 nm laser and by reducing the area of the exposed sample.
The robust and highly efficient optical switching of 3f within living cells is realized 27 with power levels for the 405 and 555 nm lasers set at 10 % of the laser capacity.
Even so only 2 scans are required to trigger the almost complete conversion of SP to MC in the 405 nm exposed region and only a handful of scans at 555 nm is required to convet all the MC to the SP state. These poperties suggest that the new red shifted BIPS exhibit superior performance in optical switching compared to previously reported probes. Also significant is that the MC-fluorescence signal reaches almost its initial value in the selected area after two scans of 405 nm irradiation indicating that optical switching of compound 3f is reversible and robust even in living cells. The same sequence of 405 nm and 555 nm scans of a field of cells labeled with 6-NO 2 -BIPS under the same conditions has no effect on the intensity of MC-fluorescence, indicating 405 nm does not trigger the SP to MC transition for this most widely use BIPS probe ( Figure S8b).  Figure 6c); (c) Schematic representation of the laser perturbation sequence used for optical switching in this study; (d) MC-fluorescence intensity profile over multiple cycles of optical switching taken from the cell within the red circle in Figure S8a.