In Search for the Best Environment for Single Molecule Studies ...
In Search for the Best Environment for Single Molecule Studies: Photostability of Single Terrylenediimide Molecules in Various Polymer Matrices
Hubert Piwoński, Adam Sokołowski, Jacek Waluk
Supporting information
1
Microscopy Setup. Single molecule fluorescence spectra were obtained with a home-built scanning confocal optical microscope based on the instrument developed in the laboratory of Prof. A. Meixner (see, e.g., Phys. Chem. Chem. Phys., 2011, 13, 1722-1733). Light from a He-Ne gas laser (05-LYP-173-152, Melles Griot) provides continuous wave excitation at 594 nm. In order to eliminate laser plasma lines, the laser beam passes through a band-pass excitation filter (XL10 594NB3, Omega Optical) followed by a beam expander with a spatial filter (25 µm pinhole) that increases the diameter of the laser beam to fill the back aperture of the objective, and by λ/4 plate to ensure that all in-plane molecular absorption dipoles are excited with the same probability. The collimated laser beam is then reflected by a dichroic mirror (Z594RDC, Chroma) towards the back aperture of an oil immersion microscope objective (CP-ACHROMAT 100x/1.25 Oil Objective, Zeiss), which focuses the beam to a diffraction-limited spot into the sample plane providing light intensities in the range of 0.6–92 kW/cm2. The emission from the specimen is collected by the same objective as used for excitation, and passed through a dichroic beamsplitter. The backscattered laser light in the fluorescence is blocked by a dielectric long-pass filter (ET605LP, Chroma). The fluorescence light is focused onto a single photon avalanche photodiode SPAD (SPCM-AQ 14, Perkin– Elmer). The active region of the SPAD has a diameter of 175 µm, so that it acts as a pinhole. A piezo-driven x–y scanning stage (P-517.3CD, Physikinstrumente) allows for raster imaging the sample in a range of 100 x 100 µm2. Alternatively, selected molecules can be positioned in the focus of the objective to record variation of their fluorescence intensities with time (“timetraces”) within a selectable integration time window (typically, 15 ms interval, minimum 10k intervals). The microscope is operated by a high-speed multifunction data acquisition card (PCI-6251, National Instruments) controlled by a home-written software (LabView, National Instruments). In order to record spectrally resolved emission spectra, the fluorescence light is redirected to a spectrograph (Shamrock SR-303i-B, Andor) equipped with a Peltier/liquid cooled EMCCD detector (Newton EM, Andor).
Sample Preparation. TDI (terrylene diimide, N-(2,6-diisopropylphenyl)-N′-(n-octyl)terrylene-3,4:11,12-tetracarboxidiimide, see Figure 1 for the formula) was kindly provided by Prof. Jerzy Sepioł. The samples were prepared from co-solutions of a selected polymer (10 mg/g) and TDI (5x10-10M) in an appropriate solvent, by spin coating onto a cleaned microscope coverslip, The following polymers were used: poly(vinyl chloride) (PVC, Sigma2
Aldrich); polyvinyl butyral (PVB , POCH); polyvinyl alcohol (PVA, Sigma-Aldrich); polystyrene (PS, Acros Organic); polyethylene (PE,Merck UVASOL, for spectroscopy); poly(methyl methacrylate) (PMMA, Acros Organic); polycarbonate (PC, ultra-pure LEXAN, portion from blank uncoated virgin Digital Versatile Discs, Sonopress GmbH). The solvents used were: toluene (Merck), tetrahydrofuran (Sigma-Aldrich), xylene (Carl-Roth), and MilliQ millipore water. All the measurements were done under ambient conditions. Measurements of photostability in deoxygenated PMMA were performed in a gas flow chamber under continuous flow of nitrogen. Polymer film samples were prepared by spin coating of 1% (w/w) co-solution of a chosen polymer and TDI (0.5 nM) in an appropriate solvent directly onto a clean borosilicate glass slide. This procedure lead to 20-30 nm thick films (measurement performed using AFM). In case of PE, the co-solution of polymer and TDI in xylene were heated up to 140 degree of Celsius before spun on a hot borosilicate slide and later baked at 70 oC for at least 10 minutes. In case of PVA the solutions of TDI in methanol were spin-coated first on a borosilicate glass and later coated by hot (70 oC, 2% (w/w) milli-Q water solution of PVA.
Figure 1S. Consecutive wide field images of photobleaching observed upon irradiation of TDI in PVB.
3
Figure 2S. Percentage of molecules surviving the first scan for different laser powers.
Table 1S. Permeability coefficients, diffusion coefficients, and solubility coefficients of polymers used in this work. P(x1013) 25st C
D(x106)
S(x106)
Poly(ethylene)
2.2 a
0.46 a
0.472 a
Poly(styrene)
1.9 a
0.22 b
0.19f
Poly(carbonate) Lexan
1.4 d
0.119 c
0.21 c
Poly(vinyl butyral)
0.30 a
0.17 a
0.18 a
Poly(methyl methacrylate)
0.0653 a
0.014 b
0.21 g
Poly(vinyl chloride)
0.034 a
0.012 a
0.29 a
Poly(vinyl alcohol)
0.00665 a
0.00054 - 0.014 e
0.045 e
P in cm3 (273.15K; 1.013x105 Pa) x cm/(cm2 x s x Pa); D in cm2/s; S in cm3 (273.15K; 1.013x105 Pa)/(cm3 x Pa) a
Polymer Handbook 4th Edition, John Wiley & Sons, Inc. (1999)
b
Charlesworth, J. M.; Gan, T. H., Kinetics of quenching of ketone phosphorescence by oxygen in a glassy
matrix. J. Phys. Chem. 1996, 100, 14922-14927. c
López-González, M.; Saiz, E.; Guzmán, J.; Riande, E., Experimental and simulation studies on the transport of
gaseous diatomic molecules in polycarbonate membranes. J. Chem. Phys. 2001, 115, 6728-6736. d
Klinger, M.; Tolbod, L. P.; Gothelf, K. V.; Ogilby, P. R., Effect of Polymer Cross-Links on Oxygen Diffusion
in Glassy PMMA Films. ACS Appl. Mater. Inter. 2009, 1, 661-667. e
Marek, P.; Velasco-Veléz, J. J.; Doll, T.; Sadowski, G., Compensation for the influence of temperature and
humidity on oxygen diffusion in a reactive polymer matrix. J. Sens. Sens. Syst. 2014, 3, 291-303. f
Korolev, V. V.; Mamaev, A. L.; Bol'shakov, B. V.; Bazhin, N. M., Oxygen diffusion in poly(methyl
methacrylate) at low temperatures. J.Pol. Sci. B: Polymer Physics 1998, 36, 127-131. g
Peterson, C. M., Permeability studies on heterogeneous polymer films. J. Appl. Polym. Sci. 1968, 12, 2649-
2667.
4
Hubert Piwoński, Adam Sokołowski, Jacek Waluk
Supporting information
1
Microscopy Setup. Single molecule fluorescence spectra were obtained with a home-built scanning confocal optical microscope based on the instrument developed in the laboratory of Prof. A. Meixner (see, e.g., Phys. Chem. Chem. Phys., 2011, 13, 1722-1733). Light from a He-Ne gas laser (05-LYP-173-152, Melles Griot) provides continuous wave excitation at 594 nm. In order to eliminate laser plasma lines, the laser beam passes through a band-pass excitation filter (XL10 594NB3, Omega Optical) followed by a beam expander with a spatial filter (25 µm pinhole) that increases the diameter of the laser beam to fill the back aperture of the objective, and by λ/4 plate to ensure that all in-plane molecular absorption dipoles are excited with the same probability. The collimated laser beam is then reflected by a dichroic mirror (Z594RDC, Chroma) towards the back aperture of an oil immersion microscope objective (CP-ACHROMAT 100x/1.25 Oil Objective, Zeiss), which focuses the beam to a diffraction-limited spot into the sample plane providing light intensities in the range of 0.6–92 kW/cm2. The emission from the specimen is collected by the same objective as used for excitation, and passed through a dichroic beamsplitter. The backscattered laser light in the fluorescence is blocked by a dielectric long-pass filter (ET605LP, Chroma). The fluorescence light is focused onto a single photon avalanche photodiode SPAD (SPCM-AQ 14, Perkin– Elmer). The active region of the SPAD has a diameter of 175 µm, so that it acts as a pinhole. A piezo-driven x–y scanning stage (P-517.3CD, Physikinstrumente) allows for raster imaging the sample in a range of 100 x 100 µm2. Alternatively, selected molecules can be positioned in the focus of the objective to record variation of their fluorescence intensities with time (“timetraces”) within a selectable integration time window (typically, 15 ms interval, minimum 10k intervals). The microscope is operated by a high-speed multifunction data acquisition card (PCI-6251, National Instruments) controlled by a home-written software (LabView, National Instruments). In order to record spectrally resolved emission spectra, the fluorescence light is redirected to a spectrograph (Shamrock SR-303i-B, Andor) equipped with a Peltier/liquid cooled EMCCD detector (Newton EM, Andor).
Sample Preparation. TDI (terrylene diimide, N-(2,6-diisopropylphenyl)-N′-(n-octyl)terrylene-3,4:11,12-tetracarboxidiimide, see Figure 1 for the formula) was kindly provided by Prof. Jerzy Sepioł. The samples were prepared from co-solutions of a selected polymer (10 mg/g) and TDI (5x10-10M) in an appropriate solvent, by spin coating onto a cleaned microscope coverslip, The following polymers were used: poly(vinyl chloride) (PVC, Sigma2
Aldrich); polyvinyl butyral (PVB , POCH); polyvinyl alcohol (PVA, Sigma-Aldrich); polystyrene (PS, Acros Organic); polyethylene (PE,Merck UVASOL, for spectroscopy); poly(methyl methacrylate) (PMMA, Acros Organic); polycarbonate (PC, ultra-pure LEXAN, portion from blank uncoated virgin Digital Versatile Discs, Sonopress GmbH). The solvents used were: toluene (Merck), tetrahydrofuran (Sigma-Aldrich), xylene (Carl-Roth), and MilliQ millipore water. All the measurements were done under ambient conditions. Measurements of photostability in deoxygenated PMMA were performed in a gas flow chamber under continuous flow of nitrogen. Polymer film samples were prepared by spin coating of 1% (w/w) co-solution of a chosen polymer and TDI (0.5 nM) in an appropriate solvent directly onto a clean borosilicate glass slide. This procedure lead to 20-30 nm thick films (measurement performed using AFM). In case of PE, the co-solution of polymer and TDI in xylene were heated up to 140 degree of Celsius before spun on a hot borosilicate slide and later baked at 70 oC for at least 10 minutes. In case of PVA the solutions of TDI in methanol were spin-coated first on a borosilicate glass and later coated by hot (70 oC, 2% (w/w) milli-Q water solution of PVA.
Figure 1S. Consecutive wide field images of photobleaching observed upon irradiation of TDI in PVB.
3
Figure 2S. Percentage of molecules surviving the first scan for different laser powers.
Table 1S. Permeability coefficients, diffusion coefficients, and solubility coefficients of polymers used in this work. P(x1013) 25st C
D(x106)
S(x106)
Poly(ethylene)
2.2 a
0.46 a
0.472 a
Poly(styrene)
1.9 a
0.22 b
0.19f
Poly(carbonate) Lexan
1.4 d
0.119 c
0.21 c
Poly(vinyl butyral)
0.30 a
0.17 a
0.18 a
Poly(methyl methacrylate)
0.0653 a
0.014 b
0.21 g
Poly(vinyl chloride)
0.034 a
0.012 a
0.29 a
Poly(vinyl alcohol)
0.00665 a
0.00054 - 0.014 e
0.045 e
P in cm3 (273.15K; 1.013x105 Pa) x cm/(cm2 x s x Pa); D in cm2/s; S in cm3 (273.15K; 1.013x105 Pa)/(cm3 x Pa) a
Polymer Handbook 4th Edition, John Wiley & Sons, Inc. (1999)
b
Charlesworth, J. M.; Gan, T. H., Kinetics of quenching of ketone phosphorescence by oxygen in a glassy
matrix. J. Phys. Chem. 1996, 100, 14922-14927. c
López-González, M.; Saiz, E.; Guzmán, J.; Riande, E., Experimental and simulation studies on the transport of
gaseous diatomic molecules in polycarbonate membranes. J. Chem. Phys. 2001, 115, 6728-6736. d
Klinger, M.; Tolbod, L. P.; Gothelf, K. V.; Ogilby, P. R., Effect of Polymer Cross-Links on Oxygen Diffusion
in Glassy PMMA Films. ACS Appl. Mater. Inter. 2009, 1, 661-667. e
Marek, P.; Velasco-Veléz, J. J.; Doll, T.; Sadowski, G., Compensation for the influence of temperature and
humidity on oxygen diffusion in a reactive polymer matrix. J. Sens. Sens. Syst. 2014, 3, 291-303. f
Korolev, V. V.; Mamaev, A. L.; Bol'shakov, B. V.; Bazhin, N. M., Oxygen diffusion in poly(methyl
methacrylate) at low temperatures. J.Pol. Sci. B: Polymer Physics 1998, 36, 127-131. g
Peterson, C. M., Permeability studies on heterogeneous polymer films. J. Appl. Polym. Sci. 1968, 12, 2649-
2667.
4
