Supporting Information
Tip Induced Fluorescence Quenching for Nanometer Optical and
Topographical Resolution
Olaf Schulz1,&, Zhao Zhao 2,3, Alex Ward1, Marcelle Koenig4, Felix Koberling4,
Yan Liu2,3, Jörg Enderlein5, Hao Yan2,3, and Robert Ros1,*
1
Department of Physics, 2Biodesign Institute, 3Department of Chemistry and
Biochemistry, Arizona State University, Tempe, AZ 85287, USA
4
PicoQuant GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
5
III. Institute of Physics, Georg August University, 37077 Göttingen, Germany
&
Present address: 5III. Institute of Physics, Georg August University, 37077 Göttingen,
Germany
*Robert.Ros@asu.edu, phone 480 727 9280, fax 480 965 7954
1. Orientation of the molecule on the sample surface
Defocused wide-field imaging of single molecules is an established technique to probe
the orientation of a dipole emitter in 3D (Patra et al, 2004). Recently, it has been adapted
to confocal microscopy (Marshall et al, 2011). In confocal microscopy, the laser beam is
focused to a small spot on the sample surface. The polarization in this spot is not uniform,
S1
which can be used to find the orientation of the molecular dipole. The excitation of the
molecule depends on the angle between the dipole moment and the polarization of the
laser and is greatest when the incident light is polarized parallel to its transition dipole.
When the laser is slightly defocused, we get unique patterns in the fluorescence image
that allow us to infer the orientation of the molecule by comparing the image to
calculated patterns (Patra et al, 2004).
Patterns for different orientations of the molecule are calculated using Matlab
(2011b 64bit), and compared to the experiment (Fig. S1a). As a first step, patterns are
calculated for a wide range of angles and values of defocussing to be compared visually
to the experimental pattern (Fig. S1b). From this starting point, a search algorithm is
performed for angles +/- 10 degrees in steps of 1 degree, and defocussing 100 nm,
200 nm, and 300 nm. For the algorithm, invalid pixels are removed from the
experimental data (i.e. emission from other molecules or blinking events). To find the
best fit, the calculated pattern is subtracted from the data and the standard deviation over
the whole image is calculated. For the best fit of all angles and values of defocussing
(within the pre-selected range), the offset between calculated pattern and experimental
data is adjusted with the same fitting procedure. The search for best angle and focus, and
adjusting the offset is run again until the result does not change any more. The best match
for the data in Fig. S1b is found to be at an angle of 13 degrees with respect to the
vertical axis of the image and a defocussing of 200 nm (Fig. S1c). This result can be
compared with the data from a combined scan to evaluate the effect that the AFM tip has
onto the fluorescence regarding the respective orientation of the molecule to the tip
surface. (Fig S1d, and Suppl. Information ‘2. Modelling of the tip induced quenching’).
S2
For the samples prepared for the single molecule quenching experiments, we
found that intensity patterns are consistent with orientations of the molecules parallel to
the sample surface.
a
0°
5°
10°
15°
20°
b
d
5.3ns
300 nm
200 nm
c
100 nm
2.1ns
Figure S1:
Determination of the orientation of the dipole emitter. From a defocussed image of the
fluorescence emission of a molecule, its orientation can be determined. (a) calculated pattern for different
focus positions and angle values. For a fixed molecule, unique patterns arise from the shape and
polarization inside the laser beam. From the defocussed image of a molecule with the tip withdrawn from
the surface (b), the molecule’s orientation can be extracted by fitting a simulated pattern (c) to the image.
Panel d (same data as Fig. 2f) shows the fluorescence intensity (top) and fluorescence lifetime (bottom) of
the same molecule in close proximity to a silicon AFM probe. The orientation of the molecular dipole,
which is determined by fitting the intensity patterns from (b) is shown as a red arrow in the fluorescence
lifetime image. The scale bar for (b) and (c) is 500 nm, and for (d) 50 nm. In the intensity image (b) of the
defocussed pattern, the emission from neighboring molecules is visible and was omitted for the fit.
2. Modeling of the tip induced quenching
We are using a classical electrodynamical model and geometrical considerations to
calculate the fluorescence of an organic dye on a glass surface in close proximity to a
S3
sharp silicon AFM tip. The presence of the silicon AFM tip alters the observed
fluorescence from the molecule in two ways. Both the excitation and the emission of the
fluorophore are modified by the tip. The incident light and the emission from the
molecule both polarize the tip material locally, thus creating a feedback onto the electric
field at the position of the molecule, which can enhance or inhibit the excitation and
emission of the fluorophore, depending on its distance from the tip and its orientation to
the surface. This classical electrodynamic phenomenon can be studied analytically for a
system of layered materials, in this case glass, which is the substrate for the molecule, air,
which is the imaging medium and silicon as the tip material. The observed fluorescence
depends on the distance of the fluorophore to the silicon layer and its angle to it. The
angle of the molecule refers to the orientation of its dipole moment with respect to the
silicon surface.
We have modified this model to include the geometry of the AFM tip. This allows
us to reproduce the experimental data qualitatively. We model the AFM tip as a silicon
sphere with the radius r, which corresponds roughly to the very end of an AFM tip (Fig.
S2a). The calculations were performed using the following parameters. n(glass)=1.51,
n(air)=1, n(Si)=3.84+0.15i (Green & Keevers, 1995) wavelength=640 nm, NA=1.45.
Danos et al. have found no significant difference in the influence of silicon of different
crystal directions onto the fluorescence of DiO molecules(Danos et al, 2008), which
justifies that we do not take into account the change in crystal direction around the tip.
The fluorescent molecule is represented by an oscillating point dipole with its dipole
moment oriented parallel to the glass sample surface. This is justified by the analysis of
our samples using defocused imaging (see Suppl. Information ‘1. Orientation of the
S4
molecule on the sample surface’). For the calculations, we employ two parallel half
spaces of glass and silicon separated by air as indicated in Fig. S2b. The silicon half
space is created tangential to the AFM tip at the point where the distance d between the
point dipole and the AFM tip is shortest. The glass half space is positioned parallel to the
silicon at the distance d. In this configuration the point dipole has an angle of inclination
θ to the surface normal. Both excitation in a confocal scheme and emission of the
fluorescent molecule are calculated using the parameters d and θ solving Maxwell’s
equations by using Sommerfeld integrals (Chizhik et al, 2009; Enderlein, 2000; Enderlein
& Ruckstuhl, 2005).
Since the geometry of the model is symmetric, only one quadrant of the sphere
was used for the calculations. This quadrant was segmented into 129 equidistant values
for θ and 17 equidistant values for the angle Φ in the x-y-plane. For each value of θ the
distance of the point dipole from the point of contact between tip and surface (x-position)
and the shortest distance between fluorophore and tip (d) were calculated. The calculation
was limited to values of the x-position smaller than 128 nm. With the values for θ and
distance, the fluorescence intensity was calculated as follows. The excitation is calculated
by the same procedure as used for the calculation of the defocussed patterns and is
averaged over the entire focal spot. The emission of the excited molecule that is collected
by the microscope objective is summed up. These two values are multiplied to give the
observed fluorescence intensity. The results are plotted versus x-position and Φ to give a
2D intensity distribution that corresponds to a confocal scan (Fig. S4).
Two of the flaws of this simple model, the fact that the tip is a finite object as
opposed to an infinite half-space and the fact that the orientation of the molecule with
S5
respect to the glass layer does not change in the actual experiment, can be addressed by a
simple rescaling procedure. For every position of the molecule, the fluorescence is
calculated without the tip material. This value is subtracted from the fluorescence with
the silicon half space present. Fig. S3 illustrates this procedure. The resulting value
contains the corrected influence from the AFM tip onto the fluorescence and is negative
in the case of quenching and positive in the case of fluorescence enhancement. To take
into account the fact that the AFM tip is not infinitely extended, we scale this value
according to
æ r ö
(Fluorescence - Fluorescenceno tip )ç
÷
èr+d ø
3
(1)
where d-3 stems from the magnitude of an induced dipole in a sphere as opposed to an
infinite plane. Finally, the fluorescence of a molecule parallel to the glass surface without
silicon is added to yield the total fluorescence. Fig. S4 illustrates that the calculated
values reproduce the trends in the experimental data nicely.
S6
Figure S2: Illustration of the model system. The sketch shows a cut through the x-z-plane. The point dipole
lies parallel on the sample surface (x-y-plane). The origin is located at the contact point AFM tip – surface.
S7
Figure S3: Illustration of the normalization procedure. The fluorescence intensity (arbitrary units) in the
presence of the silicon half-space (fluorescence) and the fluorescence without silicon present are calculated
for each angle θ, here translated into distance along the x-axis. The correction is subtracted from the
fluorescence data to yield the enhancement and quenching due to the silicon. These values are scaled
according to equation 1, after which the normal fluorescence for a dipole parallel on the surface is added
(scaled fluorescence). The calculations were performed with a tip radius of 10 nm.
S8
Figure S4: Comparison of calculated fluorescence and experimental data. (a) 2D fluorescence intensity
distributions of the inner part of the spots. The molecular dipole as well as the laser polarization is oriented
along the horizontal axis. The calculations are performed with a tip radius of 2 nm, 5 nm, 10 nm, and 15
nm. (b) The graph shows horizontal sections of the fluorescence intensity from the quenched spot outward.
The FWHMs for the different tip sizes are 2.2 nm, 5.6 nm, 11.4, and 15.6 nm.
S9
3. Application of different organic dye molecules
A
B
C
D
Figure S5: Tip-induced quenching microscopy with different organic dyes. Fluorescence intensity images
from combined AFM/confocal scans on samples with single (a) Atto647N, (b) Alexa647, (c) Atto655, and
(d) Alexa 488 dye molecules on glass. Scale bars are 500nm.
For most of the single molecule experiments, and also for the DNA origami, we chose
Atto655 as fluorophore, since it has proven to be the most stable under our measurement
conditions in very dry air (about 5% rel. humidity at 24 degrees Celsius). The superior
stability of Atto655 under these conditions becomes obvious when one compares
fluorescence intensity time traces of different fluorophores. For Atto647N, we find an
average time before bleaching of 21.5 seconds for 12 molecules. For Atto655 this value is
70.8 seconds averaged over 10 molecules.
Fig. S6: Fluorescence emission of a single Atto655 molecule under ambient conditions on glass.
S10
4. Preparation of the DNA origami nanostructures
Triangular
shaped
origami
template
was
formed
according
to
Rothemund
paper(Rothemund, 2006). A molar ratio of 1:5 between the long viral ssDNA M13 (New
England Biolabs, Inc.) and the short helper strands, including the unmodified helpers
(Integrated Technologies, Inc.) and ATTO655 modified helper strands (IBA GmbH) was
used. DNA origami was assembled in 1× TAE-Mg2+ buffer (Tris, 40 mM; Acetic acid, 20
mM; EDTA, 2 mM; and Magnesium acetate, 12.5 mM; pH 8.0) by cooling slowly from
90 °C to room temperature. DNA origami was then filtered with 100 kDa MWCO
(Amicon) centrifuge filters to remove extra DNA helper strands.
S11
Figure S7: DNA Origami design and helper sequence
A01, CGGGGTTTCCTCAAGAGAAGGATTTTGAATTA,
A02, AGCGTCATGTCTCTGAATTTACCGACTACCTT,
A03, TTCATAATCCCCTTATTAGCGTTTTTCTTACC,
A04, ATGGTTTATGTCACAATCAATAGATATTAAAC,
A05, TTTGATGATTAAGAGGCTGAGACTTGCTCAGTACCAGGCG,
A06, CCGGAACCCAGAATGGAAAGCGCAACATGGCT,
A07, AAAGACAACATTTTCGGTCATAGCCAAAATCA,
S12
A08, GACGGGAGAATTAACTCGGAATAAGTTTATTTCCAGCGCC,
A09, GATAAGTGCCGTCGAGCTGAAACATGAAAGTATACAGGAG,
A10, TGTACTGGAAATCCTCATTAAAGCAGAGCCAC,
A11, CACCGGAAAGCGCGTTTTCATCGGAAGGGCGA,
A12, CATTCAACAAACGCAAAGACACCAGAACACCCTGAACAAA,
A13, TTTAACGGTTCGGAACCTATTATTAGGGTTGATATAAGTA,
A14, CTCAGAGCATATTCACAAACAAATTAATAAGT,
A15, GGAGGGAATTTAGCGTCAGACTGTCCGCCTCC,
A16, GTCAGAGGGTAATTGATGGCAACATATAAAAGCGATTGAG,
A17, TAGCCCGGAATAGGTGAATGCCCCCTGCCTATGGTCAGTG,
A18, CCTTGAGTCAGACGATTGGCCTTGCGCCACCC,
A19, TCAGAACCCAGAATCAAGTTTGCCGGTAAATA,
A20, TTGACGGAAATACATACATAAAGGGCGCTAATATCAGAGA,
A21, CAGAGCCAGGAGGTTGAGGCAGGTAACAGTGCCCG,
A22, ATTAAAGGCCGTAATCAGTAGCGAGCCACCCT,
A23, GATAACCCACAAGAATGTTAGCAAACGTAGAAAATTATTC,
A24, GCCGCCAGCATTGACACCACCCTC,
A25, AGAGCCGCACCATCGATAGCAGCATGAATTAT,
A26, CACCGTCACCTTATTACGCAGTATTGAGTTAAGCCCAATA,
A27, AGCCATTTAAACGTCACCAATGAACACCAGAACCA,
A28, ATAAGAGCAAGAAACATGGCATGATTAAGACTCCGACTTG,
A29, CCATTAGCAAGGCCGGGGGAATTA,
A30, GAGCCAGCGAATACCCAAAAGAACATGAAATAGCAATAGC,
A31, TATCTTACCGAAGCCCAAACGCAATAATAACGAAAATCACCAG,
A32, CAGAAGGAAACCGAGGTTTTTAAGAAAAGTAAGCAGATAGCCG,
A33, CCTTTTTTCATTTAACAATTTCATAGGATTAG,
A34, TTTAACCTATCATAGGTCTGAGAGTTCCAGTA,
A35, AGTATAAAATATGCGTTATACAAAGCCATCTT,
S13
A36, CAAGTACCTCATTCCAAGAACGGGAAATTCAT,
A37, AGAGAATAACATAAAAACAGGGAAGCGCATTA,
A38, AAAACAAAATTAATTAAATGGAAACAGTACATTAGTGAAT,
A39, TTATCAAACCGGCTTAGGTTGGGTAAGCCTGT,
A40, TTAGTATCGCCAACGCTCAACAGTCGGCTGTC,
A41, TTTCCTTAGCACTCATCGAGAACAATAGCAGCCTTTACAG,
A42, AGAGTCAAAAATCAATATATGTGATGAAACAAACATCAAG,
A43, ACTAGAAATATATAACTATATGTACGCTGAGA,
A44, TCAATAATAGGGCTTAATTGAGAATCATAATT,
A45, AACGTCAAAAATGAAAAGCAAGCCGTTTTTATGAAACCAA,
A46, GAGCAAAAGAAGATGAGTGAATAACCTTGCTTATAGCTTA,
A47, GATTAAGAAATGCTGATGCAAATCAGAATAAA,
A48, CACCGGAATCGCCATATTTAACAAAATTTACG,
A49, AGCATGTATTTCATCGTAGGAATCAAACGATTTTTTGTTT,
A50, ACATAGCGCTGTAAATCGTCGCTATTCATTTCAATTACCT,
A51, GTTAAATACAATCGCAAGACAAAGCCTTGAAA,
A52, CCCATCCTCGCCAACATGTAATTTAATAAGGC,
A53, TCCCAATCCAAATAAGATTACCGCGCCCAATAAATAATAT,
A54, TCCCTTAGAATAACGCGAGAAAACTTTTACCGACC,
A55, GTGTGATAAGGCAGAGGCATTTTCAGTCCTGA,
A56, ACAAGAAAGCAAGCAAATCAGATAACAGCCATATTATTTA,
A57, GTTTGAAATTCAAATATATTTTAG,
A58, AATAGATAGAGCCAGTAATAAGAGATTTAATG,
A59, GCCAGTTACAAAATAATAGAAGGCTTATCCGGTTATCAAC,
A60, TTCTGACCTAAAATATAAAGTACCGACTGCAGAAC,
A61, GCGCCTGTTATTCTAAGAACGCGATTCCAGAGCCTAATTT,
A62, TCAGCTAAAAAAGGTAAAGTAATT,
A63, ACGCTAACGAGCGTCTGGCGTTTTAGCGAACCCAACATGT,
S14
A64, ACGACAATAAATCCCGACTTGCGGGAGATCCTGAATCTTACCA,
A65, TGCTATTTTGCACCCAGCTACAATTTTGTTTTGAAGCCTTAAA,
B01, TCATATGTGTAATCGTAAAACTAGTCATTTTC,
B02, GTGAGAAAATGTGTAGGTAAAGATACAACTTT,
B03, GGCATCAAATTTGGGGCGCGAGCTAGTTAAAG,
B04, TTCGAGCTAAGACTTCAAATATCGGGAACGAG,
B05, ACAGTCAAAGAGAATCGATGAACGACCCCGGTTGATAATC,
B06, ATAGTAGTATGCAATGCCTGAGTAGGCCGGAG,
B07, AACCAGACGTTTAGCTATATTTTCTTCTACTA,
B08, GAATACCACATTCAACTTAAGAGGAAGCCCGATCAAAGCG,
B09, AGAAAAGCCCCAAAAAGAGTCTGGAGCAAACAATCACCAT,
B10, CAATATGACCCTCATATATTTTAAAGCATTAA,
B11, CATCCAATAAATGGTCAATAACCTCGGAAGCA,
B12, AACTCCAAGATTGCATCAAAAAGATAATGCAGATACATAA,
B13, CGTTCTAGTCAGGTCATTGCCTGACAGGAAGATTGTATAA,
B14, CAGGCAAGATAAAAATTTTTAGAATATTCAAC,
B15, GATTAGAGATTAGATACATTTCGCAAATCATA,
B16, CGCCAAAAGGAATTACAGTCAGAAGCAAAGCGCAGGTCAG,
B17, GCAAATATTTAAATTGAGATCTACAAAGGCTACTGATAAA,
B18, TTAATGCCTTATTTCAACGCAAGGGCAAAGAA,
B19, TTAGCAAATAGATTTAGTTTGACCAGTACCTT,
B20, TAATTGCTTTACCCTGACTATTATGAGGCATAGTAAGAGC,
B21, ATAAAGCCTTTGCGGGAGAAGCCTGGAGAGGGTAG,
B22, TAAGAGGTCAATTCTGCGAACGAGATTAAGCA,
B23, AACACTATCATAACCCATCAAAAATCAGGTCTCCTTTTGA,
B24, ATGACCCTGTAATACTTCAGAGCA,
B25, TAAAGCTATATAACAGTTGATTCCCATTTTTG,
S15
B26, CGGATGGCACGAGAATGACCATAATCGTTTACCAGACGAC,
B27, TAATTGCTTGGAAGTTTCATTCCAAATCGGTTGTA,
B28, GATAAAAACCAAAATATTAAACAGTTCAGAAATTAGAGCT,
B29, ACTAAAGTACGGTGTCGAATATAA,
B30, TGCTGTAGATCCCCCTCAAATGCTGCGAGAGGCTTTTGCA,
B31, AAAGAAGTTTTGCCAGCATAAATATTCATTGACTCAACATGTT,
B32, AATACTGCGGAATCGTAGGGGGTAATAGTAAAATGTTTAGACT,
B33, AGGGATAGCTCAGAGCCACCACCCCATGTCAA,
B34, CAACAGTTTATGGGATTTTGCTAATCAAAAGG,
B35, GCCGCTTTGCTGAGGCTTGCAGGGGAAAAGGT,
B36, GCGCAGACTCCATGTTACTTAGCCCGTTTTAA,
B37, ACAGGTAGAAAGATTCATCAGTTGAGATTTAG,
B38, CCTCAGAACCGCCACCCAAGCCCAATAGGAACGTAAATGA,
B39, ATTTTCTGTCAGCGGAGTGAGAATACCGATAT,
B40, ATTCGGTCTGCGGGATCGTCACCCGAAATCCG,
B41, CGACCTGCGGTCAATCATAAGGGAACGGAACAACATTATT,
B42, AGACGTTACCATGTACCGTAACACCCCTCAGAACCGCCAC,
B43, CACGCATAAGAAAGGAACAACTAAGTCTTTCC,
B44, ATTGTGTCTCAGCAGCGAAAGACACCATCGCC,
B45, TTAATAAAACGAACTAACCGAACTGACCAACTCCTGATAA,
B46, AGGTTTAGTACCGCCATGAGTTTCGTCACCAGGATCTAAA,
B47, GTTTTGTCAGGAATTGCGAATAATCCGACAAT,
B48, GACAACAAGCATCGGAACGAGGGTGAGATTTG,
B49, TATCATCGTTGAAAGAGGACAGATGGAAGAAAAATCTACG,
B50, AGCGTAACTACAAACTACAACGCCTATCACCGTACTCAGG,
B51, TAGTTGCGAATTTTTTCACGTTGATCATAGTT,
B52, GTACAACGAGCAACGGCTACAGAGGATACCGA,
B53, ACCAGTCAGGACGTTGGAACGGTGTACAGACCGAAACAAA,
S16
B54, ACAGACAGCCCAAATCTCCAAAAAAAAATTTCTTA,
B55, AACAGCTTGCTTTGAGGACTAAAGCGATTATA,
B56, CCAAGCGCAGGCGCATAGGCTGGCAGAACTGGCTCATTAT,
B57, CGAGGTGAGGCTCCAAAAGGAGCC,
B58, ACCCCCAGACTTTTTCATGAGGAACTTGCTTT,
B59, ACCTTATGCGATTTTATGACCTTCATCAAGAGCATCTTTG,
B60, CGGTTTATCAGGTTTCCATTAAACGGGAATACACT,
B61, AAAACACTTAATCTTGACAAGAACTTAATCATTGTGAATT,
B62, GGCAAAAGTAAAATACGTAATGCC,
B63, TGGTTTAATTTCAACTCGGATATTCATTACCCACGAAAGA,
B64, ACCAACCTAAAAAATCAACGTAACAAATAAATTGGGCTTGAGA,
B65, CCTGACGAGAAACACCAGAACGAGTAGGCTGCTCATTCAGTGA,
Link-A1C, TTAATTAATTTTTTACCATATCAAA,
Link-A2C, TTAATTTCATCTTAGACTTTACAA,
Link-A3C, CTGTCCAGACGTATACCGAACGA,
Link-A4C, TCAAGATTAGTGTAGCAATACT,
Link-B1A, TGTAGCATTCCTTTTATAAACAGTT,
Link-B2A, TTTAATTGTATTTCCACCAGAGCC,
Link-B3A, ACTACGAAGGCTTAGCACCATTA,
Link-B4A, ATAAGGCTTGCAACAAAGTTAC,
Link-C1B, GTGGGAACAAATTTCTATTTTTGAG,
Link-C2B, CGGTGCGGGCCTTCCAAAAACATT,
Link-C3B, ATGAGTGAGCTTTTAAATATGCA,
Link-C4B, ACTATTAAAGAGGATAGCGTCC,
Loop, GCGCTTAATGCGCCGCTACAGGGC,
C01, TCGGGAGATATACAGTAACAGTACAAATAATT,
S17
C02, CCTGATTAAAGGAGCGGAATTATCTCGGCCTC,
C03, GCAAATCACCTCAATCAATATCTGCAGGTCGA,
C04, CGACCAGTACATTGGCAGATTCACCTGATTGC,
C05, TGGCAATTTTTAACGTCAGATGAAAACAATAACGGATTCG,
C06, AAGGAATTACAAAGAAACCACCAGTCAGATGA,
C07, GGACATTCACCTCAAATATCAAACACAGTTGA,
C08, TTGACGAGCACGTATACTGAAATGGATTATTTAATAAAAG,
C09, CCTGATTGCTTTGAATTGCGTAGATTTTCAGGCATCAATA,
C10, TAATCCTGATTATCATTTTGCGGAGAGGAAGG,
C11, TTATCTAAAGCATCACCTTGCTGATGGCCAAC,
C12, AGAGATAGTTTGACGCTCAATCGTACGTGCTTTCCTCGTT,
C13, GATTATACACAGAAATAAAGAAATACCAAGTTACAAAATC,
C14, TAGGAGCATAAAAGTTTGAGTAACATTGTTTG,
C15, TGACCTGACAAATGAAAAATCTAAAATATCTT,
C16, AGAATCAGAGCGGGAGATGGAAATACCTACATAACCCTTC,
C17, GCGCAGAGGCGAATTAATTATTTGCACGTAAATTCTGAAT,
C18, AATGGAAGCGAACGTTATTAATTTCTAACAAC,
C19, TAATAGATCGCTGAGAGCCAGCAGAAGCGTAA,
C20, GAATACGTAACAGGAAAAACGCTCCTAAACAGGAGGCCGA,
C21, TCAATAGATATTAAATCCTTTGCCGGTTAGAACCT,
C22, CAATATTTGCCTGCAACAGTGCCATAGAGCCG,
C23, TTAAAGGGATTTTAGATACCGCCAGCCATTGCGGCACAGA,
C24, ACAATTCGACAACTCGTAATACAT,
C25, TTGAGGATGGTCAGTATTAACACCTTGAATGG,
C26, CTATTAGTATATCCAGAACAATATCAGGAACGGTACGCCA,
C27, CGCGAACTAAAACAGAGGTGAGGCTTAGAAGTATT,
C28, GAATCCTGAGAAGTGTATCGGCCTTGCTGGTACTTTAATG,
C29, ACCACCAGCAGAAGATGATAGCCC,
S18
C30, TAAAACATTAGAAGAACTCAAACTTTTTATAATCAGTGAG,
C31, GCCACCGAGTAAAAGAACATCACTTGCCTGAGCGCCATTAAAA,
C32, TCTTTGATTAGTAATAGTCTGTCCATCACGCAAATTAACCGTT,
C33, CGCGTCTGATAGGAACGCCATCAACTTTTACA,
C34, AGGAAGATGGGGACGACGACAGTAATCATATT,
C35, CTCTAGAGCAAGCTTGCATGCCTGGTCAGTTG,
C36, CCTTCACCGTGAGACGGGCAACAGCAGTCACA,
C37, CGAGAAAGGAAGGGAAGCGTACTATGGTTGCT,
C38, GCTCATTTTTTAACCAGCCTTCCTGTAGCCAGGCATCTGC,
C39, CAGTTTGACGCACTCCAGCCAGCTAAACGACG,
C40, GCCAGTGCGATCCCCGGGTACCGAGTTTTTCT,
C41, TTTCACCAGCCTGGCCCTGAGAGAAAGCCGGCGAACGTGG,
C42, GTAACCGTCTTTCATCAACATTAAAATTTTTGTTAAATCA,
C43, ACGTTGTATTCCGGCACCGCTTCTGGCGCATC,
C44, CCAGGGTGGCTCGAATTCGTAATCCAGTCACG,
C45, TAGAGCTTGACGGGGAGTTGCAGCAAGCGGTCATTGGGCG,
C46, GTTAAAATTCGCATTAATGTGAGCGAGTAACACACGTTGG,
C47, TGTAGATGGGTGCCGGAAACCAGGAACGCCAG,
C48, GGTTTTCCATGGTCATAGCTGTTTGAGAGGCG,
C49, GTTTGCGTCACGCTGGTTTGCCCCAAGGGAGCCCCCGATT,
C50, GGATAGGTACCCGTCGGATTCTCCTAAACGTTAATATTTT,
C51, AGTTGGGTCAAAGCGCCATTCGCCCCGTAATG,
C52, CGCGCGGGCCTGTGTGAAATTGTTGGCGATTA,
C53, CTAAATCGGAACCCTAAGCAGGCGAAAATCCTTCGGCCAA,
C54, CGGCGGATTGAATTCAGGCTGCGCAACGGGGGATG,
C55, TGCTGCAAATCCGCTCACAATTCCCAGCTGCA,
C56, TTAATGAAGTTTGATGGTGGTTCCGAGGTGCCGTAAAGCA,
C57, TGGCGAAATGTTGGGAAGGGCGAT,
S19
C58, TGTCGTGCACACAACATACGAGCCACGCCAGC,
C59, CAAGTTTTTTGGGGTCGAAATCGGCAAAATCCGGGAAACC,
C60, TCTTCGCTATTGGAAGCATAAAGTGTATGCCCGCT,
C61, TTCCAGTCCTTATAAATCAAAAGAGAACCATCACCCAAAT,
C62, GCGCTCACAAGCCTGGGGTGCCTA,
C63, CGATGGCCCACTACGTATAGCCCGAGATAGGGATTGCGTT,
C64, AACTCACATTATTGAGTGTTGTTCCAGAAACCGTCTATCAGGG,
C65, ACGTGGACTCCAACGTCAAAGGGCGAATTTGGAACAAGAGTCC,
Atto modified helpers
Ab16,(ATTO655)TTTTCGCCAAAAGGAATTACAGTCAGAAGCAAAGCGCAGGTCAG
Ab28,(ATTO655)TTTTGATAAAAACCAAAATATTAAACAGTTCAGAAATTAGAGCT
Supporting References
Chizhik A, Schleifenbaum F, Gutbrod R, Khoptyar D, Meixner AJ, Enderlein J (2009)
Tuning the Fluorescence Emission Spectra of a Single Molecule with a Variable Optical
Subwavelength Metal Microcavity. Phys Rev Lett 102: 073002-073006
Danos L, Greef R, Markvart T (2008) Efficient fluorescence quenching near crystalline
silicon from Langmuir-Blodgett dye films. Thin Solid Films 516: 7251-7255
Enderlein J (2000) A theoretical investigation of single-molecule fluorescence detection
on thin metallic layers. Biophys J 78: 2151-2158
Enderlein J, Ruckstuhl T (2005) The efficiency of surface-plasmon coupled emission for
sensitive fluorescence detection. Optics Express 13: 8855-8865
Green MA, Keevers MJ (1995) OPTICAL-PROPERTIES OF INTRINSIC SILICON AT
300 K. Progress in Photovoltaics 3: 189-192
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