Single-chromophore-based photoswitchable nanoparticles enable dual-alternating-color fluorescence for unambiguous live cell imaging.

Synthesis and characterization of spiropyran photoswitchable dyes

The photoswitchable spiropyran derivatives were synthesized according to literature procedures 36 , and herein their characterization data are summarized below. 3′, 3′-dimethyl-6-nitro-1′-(4-vinylbenzyl)-spiro[chromene-2, 2′-indoline] (SP) MALDI-TOF MS: m/z = 425 [M+H] +; 1 H NMR (300 MHz, CDCl 3 ) δ = 1.30(3H, s), 1.34(3H, s), 4.20(1H, d, 17Hz), 4.50(1H, d, 17Hz), 5.20(1H, d, 10Hz), 5.71(1H, d, 17Hz), 5.91(1H, d, 10Hz), 6.35(1H, d, 8Hz), 6.66(1H, dd, 2 and 3Hz), 6.72(1H, m), 6.85–6.91(2H, m), 7.04–7.14 (2H, m), 7.22–7.26(2H, m), 7.33–7.35(2H, m), 8.00–8.05(2H, m). 5′-methoxy-3′, 3′-dimethyl-6-nitro-1′-(4-vinylbenzyl)-spiro[chromene-2, 2′-indoline] (MSP) MALDI-TOF MS: m/z = 455 [M+H] +; 1 H NMR (300 MHz, CDCl 3 ) δ = 1.31(3H, s), 1.32(3H, s), 3.76(3H, s), 4.11(1H, d, 17Hz), 4.44(1H, d, 17Hz), 5.22(1H, d, 10Hz), 5.72(1H, d, 17Hz), 5.91(1H, d, 10Hz), 6.22(1H, d, 8Hz), 6.59(1H, dd, 2 and 3Hz), 6.68(1H, m), 6.75(1H, d, 2Hz), 6.78–6.88 (2H, m), 7.23(1H, d, 3Hz), 7.33–7.36(2H, m), 7.98(1H, d, 3Hz), 8.02–8.05(2H, m). 5′-methoxy-3′, 3′-dimethyl-6-cyano-1′-(4-vinylbenzyl)-spiro[chromene-2,2′-indoline] (MCSP) MALDI-TOF MS: m/z = 435 [M+H] +; 1 H NMR (300 MHz, CDCl 3 ) δ = 1.29(3H, s), 1.31(3H, s), 3.76(3H, s), 4.09(1H, d, 17Hz), 4.43(1H, d, 17Hz), 5.22(1H, d, 10Hz), 5.72(1H, d, 17Hz), 5.87(1H, d, 10Hz), 6.20(1H, d, 8Hz), 6.56(1H, dd, 2 and 3Hz), 6.64(1H, m), 6.69(1H, d, 2Hz), 6.74–6.80 (2H, m), 7.22(1H, m), 7.25(2H, m), 7.36–7.38(2H, m), 7.40(1H,d, 2Hz). 3′, 3′-dimethyl-6-cyano-1′-(4-vinylbenzyl)-spiro [chromene-2, 2′-indoline] (CSP) MALDI-TOF MS: m/z = 405 [M+H] +; 1 H NMR (300 MHz, CDCl 3 ) δ = 1.29(3H, s), 1.33(3H, s), 4.18(1H, d, 17Hz), 4.48(1H, d, 17Hz), 5.22(1H, d, 10Hz), 5.72(1H, d, 17Hz), 5.86(1H, d, 10Hz), 6.33(1H, d, 8Hz), 6.66(1H, dd, 2 and 3Hz), 6.75(1H, m), 6.78–6.90(2H, m), 7.04–7.13 (2H, m), 7.22–7.25(2H, m), 7.33–7.40(4H, m).

Synthesize and characterize nanoparticles with embedded spiropyran dyes Reagents

All reagents and solvents were purchased from Aldrich Chemical Co. and used as received except stated otherwise. Acrylamide (A) and 4, 4′-azobis-(4-cyano-valeric acid) (ABVA) was recrystallized before use. Styrene (ST, 99%), acrylic acid (AA) and butyl acrylate (BA) were distilled under reduced pressure. Emulsion Polymerization Emulsion polymerization was carried out according to the literature procedure as reported elsewhere 28 – 30 . UV-analysis of the organic phase (CHCl 3 ) extracted from the emulsion mixture suggests that more than 95% of the initial spiropyran co-monomer was incorporated into the final nanoparticles. The as-synthesized nanoparticles showed no obvious aggregation as measured by dynamic light scattering. Characterization 1 H NMR spectra were recorded in CDCl 3 using a JEOL JNM-AL300 instrument. Dynamic light scattering (DLS) measurements were carried out on a Beckman-Coulter N4 instrument at fixed scattering angles of 62.6° and 90° using the 632.8 line of a He-Ne laser as the excitation source; standard polystyrene microspheres were used to calibrate the instrument. The average particle sizes and size distributions were obtained from the autocorrelation decay functions by CONTIN analysis using standard software package supplied by Beckman-Coulter. A JEOL 1010 transmission electron microscope (TEM) operated at 100 kV was employed to obtain TEM images. The microscope sample was prepared by placing a drop of the polymer dispersion on a carbon-coated Cu grid, followed by solvent evaporation at room temperature.

Sample Preparation for Microscopy

Cover glasses (Gold Seal No. 1, Fisher) were cleaned by a protocol comprising sonication in 2% Micro 90 for 30 min, rinsing thoroughly with 18 MΩ water, drying in an oven, soaking in Chromerge for 1 h, rinsing thoroughly with 18 MΩ water, and finally heating gently in a methane flame to dryness. After being cooled slowly, the cover glasses were stored in a dust-free container. Nanoparticle samples were prepared by spin-coating (4000 rpm) ~30 μL of a dilute suspension of nanoparticles in spectroscopic grade ethanol onto a cover glass. Samples were diluted sequentially until the density of nanoparticles on the cover glass was suitable for single particle measurements. For cell samples, cover glasses were modified by gluing O-rings onto one side and filling the resulting wells with a DMEM solution. Washed cells were transferred into the well and allowed to settle before inspection.

Microscope Design

Samples were placed on a custom-built stage of an inverted microscope (Zeiss Axiovert 200) equipped with a high numerical aperture oil immersion objective (Zeiss, 100×, 1.3 NA) and an X-Y nanopositioner stage (Mad City Labs). Connected to the side port of the microscope was a spectrometer (Acton Research Corp.) coupled to a liquid nitrogen-cooled CCD detector (Princeton Instruments, Roper Scientific). The spectrometer was equipped with both a mirror for imaging and a grating for spectroscopy. An avalanche photodiode (APD) was used to collect photons from the bottom microscope port. Excitation entering the back port of the microscope was redirected by an appropriate filter set into the back aperture of the objective. Emission light was collected through the same objective and directed to the side or bottom port of the microscope. Cell Growth Transformed human embryonic kidney (HEK 293) cells were grown in 24-well culture plates until cell populations reached ~105 cells per well. A 1 pmol nanoparticle suspension was prepared in 50 μL of DMEM (Dulbecco’s Modified Eagle Medium). In a separate vial, 0.5 μL of Lipofectamine 2000 reagent was mixed with 50 μL of DMEM and incubated for 5 min at room temperature, after which the dilute nanoparticle suspension was added followed by additional 20 min incubation at room temperature.

Delivery of Nanoparticles into Living Cells

Nanoparticles were delivered into cells by adding 100 μL of the nanoparticle-Lipofectamine 2000 suspension to each cell-containing well in the culture plate. After incubating for 5 h at 37°C in a CO 2 incubator, the cells were washed with 500 μL of DMEM and resuspended in 500 μL of DMEM containing 10% FBS (fetal bovine serum).

Wide Field Nanoparticle Imaging

For wide field imaging of nanoparticles, a mercury vapor short arc lamp illuminator (Zeiss HBO 100) was mounted to the back illumination port of the microscope. Two filter sets, each consisting of an excitation filter (ex), dichroic beam splitter (bs), and emission filter (em), were used to isolate either the 365 nm (Zeiss, filter set 2, ex365; bs295; em420) or the 488 nm (Zeiss, filter set 16, ex485/20; bs510; em515) excitation lines from the mercury lamp spectrum. Excitation light was not attenuated. Samples were first illuminated with 365-nm light to convert the photoswitches to their red-fluorescence mero-forms, thereby facilitating focusing of the nanoparticles via CCD imaging. After achieving focus, the 365 nm light was blocked and a new area on the cover glass was moved into the illumination area via the nanopositioner. The new area was illuminated with 365-nm light for 3–4 s to photoswitch spiropyran into fluorescent merocyanine. Next, nanoparticle image acquisition was carried out using 488-nm illumination. The red fluorescence was obtained using a red long pass filter (620 nm) and a CCD detector. All images were collected using a 3-s integration time in WinSpec/32 software. Two-dimensional black and white CCD images were converted into 3-D color surface plots in Igor Pro software (Wavemetrics).

Cell Imaging

For cell imaging, an argon ion laser (488 nm) and hand-held UV lamp were used instead of the mercury lamp because of its heating effects. Cells were first photographed under white light illumination. For MSP-nanoparticle live cell imaging, the red fluorescence channel was obtained using a red long pass filter (620 nm) and a 365-nm pulse to photoswitch on merocyanine followed by 488-nm fluorescence excitation, whereas the green fluorescence channel was acquired using 488-nm excitation and a bandpass filter (510 ± 40 nm). For MCSP-nanoparticle live cell imaging, the red fluorescence channel was obtained using a red long pass filter (620 nm) and a 300-nm pulse to photoswitch on merocyanine followed by 488-nm fluorescence excitation, whereas the blue fluorescence channel was acquired using 365-nm excitation and a long pass filter (420 nm) because 365-nm excitation cannot photoswitch on red fluorescence.

Microscope Design

Samples were placed on a custom-built stage of an inverted microscope (Zeiss Axiovert 200) equipped with a high numerical aperture oil immersion objective (Zeiss, 100×, 1.3 NA) and an X-Y nanopositioner stage (Mad City Labs). Connected to the side port of the microscope was a spectrometer (Acton Research Corp.) coupled to a liquid nitrogen-cooled CCD detector (Princeton Instruments, Roper Scientific). The spectrometer was equipped with both a mirror for imaging and a grating for spectroscopy. An avalanche photodiode (APD) was used to collect photons from the bottom microscope port. Excitation entering the back port of the microscope was redirected by an appropriate filter set into the back aperture of the objective. Emission light was collected through the same objective and directed to the side or bottom port of the microscope.