Mapping molecules in scanning far-field fluorescence nanoscopy

Abstract

In fluorescence microscopy, the distribution of the emitting molecule number in space is usually obtained by dividing the measured fluorescence by that of a single emitter. However, the brightness of individual emitters may vary strongly in the sample or be inaccessible. Moreover, with increasing (super-) resolution, fewer molecules are found per pixel, making this approach unreliable. Here we map the distribution of molecules by exploiting the fact that a single molecule emits only a single photon at a time. Thus, by analysing the simultaneous arrival of multiple photons during confocal imaging, we can establish the number and local brightness of typically up to 20 molecules per confocal (diffraction sized) recording volume. Subsequent recording by stimulated emission depletion microscopy provides the distribution of the number of molecules with subdiffraction resolution. The method is applied to mapping the three-dimensional nanoscale organization of internalized transferrin receptors on human HEK293 cells.

Figure 1. Mapping molecule distributions in far-field fluorescence microscopy through analysis of coincident photon detection.

(a) Experimental confocal/STED setup equipped with four independent detection channels. BS, 1:1 beam splitter; DM, dichroic mirror; D_i, i^th detector, i=1, 2, 3 and 4; FB, fibre; L, lens; M, mirror; OB, objective lens; PP, phase plate; QW, quarter wave plate. (b-f) Double-stranded DNAs (dsDNA) labelled with up to four ATTO 647N was sparsely immobilized on a glass surface. The numbers of dye molecules in each dsDNA were established by analysing the distribution of coincident photon detection, which was recorded by confocal microscopy. (b) Fluorescence bleaching steps of single dsDNAs. The corresponding dsDNAs are indicated in d by triangles with the same colours. (c) Comparison of number of dye molecules (mean and s.d.) derived from photon coincidence recordings with the number of bleaching steps of the same single dsDNAs. Red line: y=x. The number of molecules from photon statistics is slightly higher than that from bleaching steps due to the bleaching during scanning. The statistics of each point were based on 39–108 dsDNAs. (d,e) An example image pair of one- and two- photon detection events of immobilized dsDNA labelled with up to four ATTO 647N molecules. The positions where single dye molecules were located are indicated by open triangles in e. (f) The established map of the number of ATTO 647N molecules on each dsDNA from d and e. The numbers indicate the numbers of dye molecules in each dsDNA. H is the maximum value of the pseudocolour intensity scale, meaning counts in d and e and the number of molecules in f at each pixel. Original pixel size in d and e is 20 nm and is binned to 40 nm for better visualization. Scale bars, 1 μm.

Figure 2. Mapping the number of molecules in a controlled biological sample.

DNA origami labelled with ATTO 647N molecules were immobilized on the surface and measured with confocal and STED microscopy. (a) Sketch of one DNA origami. Red dots represent the locations where ATTO 647N molecules can be conjugated. Each DNA origami can accommodates up to 24 fluorophores (12 in each line). (b) Confocal one-photon (left) and two-photon (right) detection images. (c) Map of the number of ATTO 647N molecules on single DNA origami calculated based on b. (d) STED one-photon (left) and two-photon (right) detection images. (e) Map of the number of ATTO 647N molecules on single DNA origami based on b and d. (f) The histogram of the distance between the two fluorescent lines in one DNA origami from the reconstructed images. (g,h) Histograms of the numbers of ATTO 647N molecules in one DNA origami (g) and one line of DNA origami (h) from the extracted number maps. H is the maximum value of the pseudocolor intensity scale and specified on the top-right corner of the corresponding images, meaning counts in b, d and the number of molecules at each pixel in c and e. The numbers in the histograms (f,g,h) are the mean values and s.d. Scale bars, 200 nm.

Figure 3. Mapping the number of transferrin receptors (TfR) in HEK293 cells.

Living cells were incubated with ATTO 647N-conjugated anti-TfR aptamer c2 for 60 min. After incubation, excess aptamer molecules were washed off and cells were chemically fixed. Stained receptors were imaged by confocal and STED microscopy (see Methods for details). (a) Confocal and STED images (raw data): summation projection along the axial dimension (0.9 μm). H is the maximum value of the pseudocolour intensity, meaning counts. (b) 3D molecular map generated by photon statistics of both confocal and STED recordings. Colours represent the axial (z) position. (c) Isosurfaces of the molecular map (corresponding to the box region in a and b). The isosurfaces embrace 70% of the overall molecules in the corresponding regions. The grey surfaces are the summation projections of the number of molecules to the corresponding dimensions. Colours represent the number of molecules in the corresponding region. The numbers of molecules in the corresponding segmentations are indicated on the z-projection plane. (d) The histogram of the number of molecules in the recognized separated clusters in the generated molecule map. Identification of the clustering is performed with built-in watershed algorithm provided in MATLAB. The red line is the exponential distribution fit to the occurrences of up to 24 molecules in each spot (blue circle). Clusters of TfRs with more than 24 molecules are not considered due to potential overlapping of multiple clusters under the given resolution of the STED microscope. Inset: the residual of the fit from the experimental observation. Scale bars, 1 μm.