Figure 2.

Correlative microscopy. A 150-micron square area has been imaged by HeIM (A), phase-contrast microscopy (B) and fluorescence microscopy (C). Our usual sequence of acquisition is to take a phase-contrast image first, then a fluorescence image, than a HeIM image, all on the same area. HeIM modality, by avoiding electroconductive coating, can allow changes in this sequence when needed. HeIM (A) and phase-contrast (B) modalities show the morphology of the cells. Fluorescence microscopy (C) highlights their infectious status, because only infected cells fluoresce in green. In this area, HeIM (A) shows that two cells (1 and 2) are connected by a TNT about 100 microns long. There is also a rounded bipolar cell (3) emitting TNT, and correlative microscopy shows that it is infected; this cell (3) did not change its position regarding the prior acquired phase-contrast photomicrograph. Rounded infected cells, however, tend to detach from the floor; correlation between Phase Contrast (B) and HeIM (A) shows that cell 4 changed its position slightly and cell 5 moved out of the field. Only HeIM was able to pick-up the presence of vesicles in the area; the tiny one indicated by “v” in (A) will be shown at higher magnification in Fig. 8D (HeIM (A) × 1800; Optical Microscopy (B,C) × 400).