Journal: ACS Photonics

Article Title: Background Reduction in STED-FCS Using a Bivortex Phase Mask

doi: 10.1021/acsphotonics.0c00388

Figure Lengend Snippet: Influence of the bivortex parameter ρ on CH-STED-FCS measurements, as experimentally determined from measurements of Abberior Star Red in water/glycerol solution. (a) The inner radius ρ of the central π phase step of the STED phase mask (top) determines the concavity of the effective depletion pattern. Bottom: pictures of the xz cross sections of the depletion pattern imaged using scattering gold beads. Scale bar: 300 nm. (b) Representative normalized FCS curves in confocal and CH-STED modes with different bivortex parameters ρ (STED laser power: 32 mW). (c, d) Average size of the effective observation volume (c) and average number of molecules in the observation volume (d) determined from STED-FCS recordings for different values of the parameter ρ and at two different STED laser power, as indicated in the legend in panel (e), and normalized with the confocal value. (e, f) Estimation of background contributions in STED-FCS curves. (e) Variation of the observed number of molecules in the observation volume with size of the observation volume for different values of the parameter ρ within the range 0.7–1.0 in steps of 0.05. If no undepleted background is present, the normalized observation volume and number of molecules are equal (gray line). (f) ρ-Dependent variation of the nRMSD (indicator of noise in FCS curves). Error bars: standard deviation, n = 6. Excitation power: 17 μW.

Article Snippet: For each aberration value, we acquired a set of STED-FCS measurements of freely diffusing Abberior Star Red dye in a water/glycerol solution.

Techniques: Standard Deviation

Journal: ACS Photonics

Article Title: Background Reduction in STED-FCS Using a Bivortex Phase Mask

doi: 10.1021/acsphotonics.0c00388

Figure Lengend Snippet: STED-FCS with different confinement modes in an aqueous solution of freely diffusing Abberior Star Red dyes. (a) Representative FCS curves at a STED laser power of 55 mW, for different confinement modes, as indicated in the legend. (b, c) STED laser power-dependent effective observation volume (b) and average number of molecules in the observation volume (c), normalized with values determined from confocal recordings, and (d) their pairwise scatterplot. STED-FCS recordings without any background would follow the dotted gray line (proportionality between number of molecules in the observation volume and size of the observation volume). (e) Variation of nRMSD with STED laser power, for different STED confinement modes as indicated in the legend. Error bars: s.d., n = 6. Excitation power: 8 μW.

Article Snippet: For each aberration value, we acquired a set of STED-FCS measurements of freely diffusing Abberior Star Red dye in a water/glycerol solution.

Techniques:

Journal: ACS Photonics

Article Title: Background Reduction in STED-FCS Using a Bivortex Phase Mask

doi: 10.1021/acsphotonics.0c00388

Figure Lengend Snippet: Effect of common aberrations on the depletion pattern on STED-FCS experiments. (a–c) Spherical aberration. (a, b) FCS curves of Abberior Star Red in water/glycerol solution, obtained with different confinement modes, without (a) and with (b) 1 rad rms of spherical aberration introduced in the depletion beam. Insets: images ( xz ) of the corresponding depletion patterns obtained with a sample of scattering gold beads (scale bar: 1 μm). (c) Relative variation of size of the observation volume ( x -axis) and average number of molecules in the observation volume ( y -axis) upon various amounts of introduced spherical aberration (indicated by the size of the marker) for different STED-FCS confinement modes, as indicated in the legend in panel (f). Aberrations either reduce the resolution or increase the strength of background contributions, as indicated by the arrows. The inset represents the phase distribution of 1 rad rms of spherical aberration. (d–i) Corresponding effects of (d, g) astigmatism (e, h) coma, and (f, i) tilt on STED-FCS parameters (d–f) and gold bead images (g–i) in xy (top) and xz (bottom) planes for the depletion patterns with 1 rad rms of the corresponding aberrations. In panel (i), the white dotted line represents the position of the excitation focus. STED laser power: 16 mW.

Article Snippet: For each aberration value, we acquired a set of STED-FCS measurements of freely diffusing Abberior Star Red dye in a water/glycerol solution.

Techniques: Marker