Figure 1. Simultaneously acquired 3D... | Rockefeller University Press

Figure 1.

$Figure 1. Simultaneously acquired 3D image of the nuclear envelope. (A) Schematic drawing of the multifocus relay optic enabling simultaneous 3D image acquisition. The lens L1 forms a secondary Fourier plane (an image of the objective pupil) in which the MFG is placed to split the beam into zero- and first-order diffraction beams. Lens L2 forms the secondary image plane on the camera. The first-order diffraction forms eight beams around the zero order with only the above (+3) and below (−3) beams depicted. The zero-order beam travels straight through an empty panel in the CCG, the central, flat facet of the prism, and is focused on the camera by L2. The first-order beams are separated and refocused by the MFG, corrected for chromatic dispersion by the CCG, redirected to proper positions on the camera by the prism, and focused onto the camera by L2. An example of the nine image planes (NPC image recorded in the red channel) projected in a 3 × 3 array and simultaneously recorded by a CCD camera is shown. Numbers on the subfields of the image correspond to the relative position of the plane in the stack. (B) A magnified series of a region of interest from A (red box) shown over seven planes (planes +3 and +4 not shown as out of focus). Arrowheads (plane −3) point to nuclear pores and the circle marks unexpected invagination. The region of the nuclear envelope that is out of focus near the bottom of the cell comes into focus near the middle of the cell. Image is shown with black and white values inverted.$

Simultaneously acquired 3D image of the nuclear envelope. (A) Schematic drawing of the multifocus relay optic enabling simultaneous 3D image acquisition. The lens L1 forms a secondary Fourier plane (an image of the objective pupil) in which the MFG is placed to split the beam into zero- and first-order diffraction beams. Lens L2 forms the secondary image plane on the camera. The first-order diffraction forms eight beams around the zero order with only the above (+3) and below (−3) beams depicted. The zero-order beam travels straight through an empty panel in the CCG, the central, flat facet of the prism, and is focused on the camera by L2. The first-order beams are separated and refocused by the MFG, corrected for chromatic dispersion by the CCG, redirected to proper positions on the camera by the prism, and focused onto the camera by L2. An example of the nine image planes (NPC image recorded in the red channel) projected in a 3 × 3 array and simultaneously recorded by a CCD camera is shown. Numbers on the subfields of the image correspond to the relative position of the plane in the stack. (B) A magnified series of a region of interest from A (red box) shown over seven planes (planes +3 and +4 not shown as out of focus). Arrowheads (plane −3) point to nuclear pores and the circle marks unexpected invagination. The region of the nuclear envelope that is out of focus near the bottom of the cell comes into focus near the middle of the cell. Image is shown with black and white values inverted.