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Hafi, N. et al. Nat. Methods 11, 579–584 (2014).
We thank A. Albrecht for help with generating some figures. Parts of this work were supported by the German Science Foundation (CRC 803).
The University of Braunschweig, with which N.H., L.S.v.d.H., M.K. and P.J.W. are affiliated, has filed patents (US patent 14/362,346 and German patent 102011087770.3) for parts of this work.
Supplementary Figure 1, Supplementary Methods and Supplementary Note (PDF 3236 kb)
Diffraction limited images, movies of the modulated part of the emission, images of the constant part of the emission, movies of the total signal and color coded phase information from fluorescence modulation for various samples. (a) Single fluorescence molecules on coverslip. (b) Liposomes in liquid environment. (c) Membrane-labelled neurons. (d) Immunolabeled microtubules. (e) Molecule pair simulations of varying distances and relative orientation differences. (f) Labeled actin filaments on cover slide. The diffraction limited images are the average of the total signals in the raw data movies. The raw data movies are composed of a modulated part and a constant part. The modulated part is the time-dependent, modulated fraction in the raw data (constant offset in each pixel subtracted). The remaining non-modulated part is the constant, time-independent offset in the total signal. The phase information is derived from Fourier analysis in each pixel and is color coded. As described in Hafi et al. the relative modulation amplitude depends approximately on 1/√Nmol and is thus smaller in the total signal for more densely labelled samples. The modulated raw data are averages from (a) 70, (b) 10, (c) 90, 80, (d) 70, (e) 1 and (f) 20 periods and are repeated ten times in the Video. Set-up dependent periodical deviations in the overall excitation intensity (typically ~5%) were corrected for. (MOV 7664 kb)
(a-d) Diffraction limited images, (e-h) modulated parts and (i-l) SPEED images as well as profile plots of single molecule pairs (a,e,i), membrane labeled neurons (b,f,j) and immunolabeled microtubules (c,d,g,h,k,l). The profile plots for the modulation movies (e-h) show the modulation amplitudes (black). Colored profile plots: individual time frames from the modulated data with varying contributions from the spine apparatus and the closest membrane (f) and from the two fibers (g). The modulated raw data are averages from 6, 90 and 70 periods for the single molecule, neuron and microtubule data, respectively, and are repeated ten times in the Video. A drift correction was applied for samples that moved more than one pixel during that time. (MOV 11866 kb)
(a) Total signal modulation movie. (b) Corresponding movie of the modulated part of the emission (constant offset in each pixel subtracted). (c) Total signal modulation movie from inset i. (d) Corresponding color coded phase image. The phase was derived from Fourier analysis in each pixel and is a measure for the label polarization. (e) Corresponding movie of the modulated part of the emission. (f,g) Two different image frames from raw modulating data from inset ii. Blue lines: linear fits of the fiber positions. (h) Total signal modulation movie from inset ii differing by 40° in label polarization along with profile plot lines used for the data shown in i-k. (i) Profile plots from the total signal modulation movie shown in h. Blue vertical lines indicate the true positions of the fibers. (j) Corresponding profile plots series of the modulated part of the emission. (k) Corresponding profile plots of the raw modulating data in two selected image frames. (l-n) Results of fitting two modulating Gaussian functions to the data shown in i, j and k, respectively. For both Gaussians the position, the modulation amplitude, the modulation phase and a constant offset were optimized. Blue vertical lines indicate the true positions of the fibers, black vertical lines indicate fitted positions. The fitted positions are also indicated by white dots in (h). To improve fit quality, the agreement with the pure raw modulating data (compare j and m) was weighed twice as high as the agreement with the total signal (compare i and l). (o) Corresponding fit to time-averaged data (no modulation). Blue vertical lines indicate the true positions of the fibers, red vertical lines indicate fitted positions. (p) Deviation of the fitted fiber positions from the true fiber positions for the 40° data. Black dots denote difference between fitted and true positions in the modulated data (l). Red dots denote the difference between fitted and true positions in the unmodulated data (o). (q) Data observed for two fibers differing by 70° in label polarization (inset iii). (r) Data observed for two fibers differing by 5° in label polarization (inset iv). The modulated data are averages from 20 periods and are repeated ten times in the Video. (MOV 7864 kb)
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Hafi, N., Grunwald, M., van den Heuvel, L. et al. Reply to "Polarization modulation adds little additional information to super-resolution fluorescence microscopy". Nat Methods 13, 8–9 (2016). https://doi.org/10.1038/nmeth.3721
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