Figure 5: Microscope alignment. | Nature Protocols

Figure 5: Microscope alignment.

From: 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy

Figure 5

(a) An unaligned Bessel beam tilted in the xy plane. (b) An unaligned Bessel beam tilted in xy plane. The right side of the beam is in focus, but the left side is not. (c) A Bessel beam that, when swept in the x direction, does not remain in focus, so that the virtual light sheet and the detection focal plane are not coincident. (d) An aligned Bessel beam that is in focus at all x positions across the scan plane. (e) The cross-sectional intensity profile of a properly aligned Bessel beam. (f) The cross-sectional intensity profile of a Bessel beam caused by nonuniform illumination across the annular ring and mis-conjugation of the the annular ring to the rear pupil of excitation objective EO. (g) The detection wide-field xz PSF with strong spherical aberration. (h) The detection wide-field xz PSF after aberration correction with the correction collar of DO. (i) xz MIP view of a field of fluorescent beads imaged in the sheet-scan mode showing symmetric PSFs in the axial direction across the entire field of view. (j) xz MIP view of a field of fluorescent beads imaged in the sheet-scan mode, with a 1 μm offset between the center of the light sheet and the focal plane of DO, showing an identical axial asymmetry in the PSF across the entire field of view. (k) Same as in i, except acquired in the OS-SIM mode with N = 7 phases. Note the substantially tighter PSF in the axial direction. (l) Same as in j, except acquired in the OS-SIM mode with N = 7 phases, and a 0.3 μm offset between the excitation and detection planes. (mp) Zoomed-in view of the same fluorescent bead marked in il. In general, the SIM modes require an even tighter axial alignment than the swept-sheet modes.

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