Figure 2.

Localization of promyelocytic leukemia (PML) bodies in the nucleus of human lymphocytes using hybrid light and x-ray imaging. (A) Transmitted light microscope image of a lymphocyte showing localization of PML bodies in the nucleus, labeled using gold-enhanced FluoroNanogold® secondary antibodies. (B) Localization of PML bodies in the nucleus of a lymphocyte from the same population of cells seen using cryo x-ray tomography. This is a maximum intensity projection image of all orthoslices from the tomographic reconstruction showing only the segmented nucleus. (C) The same image seen in ‘B’ with distribution of PML bodies highlighted by gold dots. (D) One orthoslice from the tomographic reconstruction shown in ‘B, C’ showing two PML bodies (red arrows). (E) One orthoslice from control nucleus processed in parallel, omitting only the primary antibody labeling step. (F) One orthoslice from the tomographic reconstruction of a live, unlabeled, rapidly frozen lymphocyte for comparison. The nucleus is outlined in red and contains well-defined heterochromatin and a nucleolus (yellow arrow) that are not seen in ‘B – E’, presumably due to extensive processing for antibody labeling. (G) Labeled antibodies are easily identified using the Linear Absorption Coefficient (LAC), since only the gold enhanced particles they have a LAC > 0.6 μm-1, as seen in the plot of all voxels in the nucleus after aldehyde fixation (black dashed line), aldehyde fixation plus secondary antibodies (red solid line), and gold enhancement of antibodies (light blue solid line). Scale bars = 2 μm.