(
A) The emission point spread function (ePSF) measured from fluorescence emitted from an imaged bead as previously described (
DiGregorio et al., 2007). Left:
xz plane (
y = 0; 4.0 × 9.6 µm) of a 3D wide-field ePSF created from axial images (
xy plane; 1 pixel = 0.05 µm) of a 110 nm fluorescent bead (Molecular Probes yellow-green FluoSpheres: 505 nm excitation, 515 nm emission) taken in multiple planes in 0.4 µm steps (
dz). The resolution in the axial plane was increased by a factor of 8 (i.e.
dz = 0.05 µm) by cubic spline interpolation. Vertical symmetry along
x-axis was created by averaging. Values were normalized between 0 and 1, displayed with a colour scale from blue to yellow. Middle: 2D fit of ePSF to a diffraction integral representation of a high-NA objective (
Sheppard and Torok, 1997) that includes a sin
2θ series function to account for spherical aberrations (
DiGregorio et al., 2007). Right: difference between ePSF and the 2D fit displayed with a colour scale from green (−0.14) to red (+0.14). (
B) 1D profiles of ePSF and the fit in
A along
x-axis (left;
y = 0,
z = 0) and
z-axis (right;
x = 0,
z = 0). (
C) Left: 1D profile of the average confocal PSF (cPSF) in the
x-axis (black line; FWHM
xy = 255 nm) and range (gray; FWHM
xy = 218–336 nm) computed from fluorescence measured from 110 nm beads as a focused laser spot (488 nm) was stepped across their lateral dimensions. Plots of average fluorescence versus spot location were fit with a Gaussian function and the resulting Gaussian widths were corrected for bead size using deconvolution (
Chaigneau et al., 2011): FWHM
actual = [(FWHM
measured)
2 – (FWHM
bead)
2]
1/2. Red line is derived from a theoretical cPSF (
Wilson and Carlini, 1987). Green dashed line is a Gaussian fit to the theoretical cPSF (FWHM
xy = 238 nm). Right: 1D profile of the average cPSF in the
z-axis (black line; FWHM
z = 916 nm, range 780–1047 nm), with theoretical cPSF (red line) and its Gaussian fit (green dashed line; FWHM
z = 975 nm).