RESOLVING THE HIGH-RESOLUTION ARCHITECTURE, ASSEMBLY AND FUNCTIONAL REPERTOIRE OF BACTERIAL SYSTEMS BY IN VITRO ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 89

through the formation and slow expansion of ~25 nm deep lat-bottomed
apertures ( Fig. 4.12a-f ) . The cortex was fully lysed by the time spore coat
layers dissolved. Hence, the lat-bottomed apertures in this undercoat layer
show the underlying cell wall of the emerging vegetative cell,

which, based on its lighter AFM phase contrast ( Fig. 4.12f ) , has different
physicochemical properties or/and hence composition than the surrounding
coat remnants. The nascent surface of the emerging germ cell appears to be
formed by a porous network ( Fig. 4.12e-f ) of peptidoglycan ibres, similar to
one described earlier for

C. novyi-NT

B. atrophaeus vegetative cells.

(a) (b) (c)

(d) (e) (f)

Figure 4.12. (a-e) AFM height images of the inal outgrowth stage. (a) After the ~6
spore coat layers were largely dissolved, the underlying structural layer was exposed.
(b-e) In this layer, 25 nm deep apertures appeared and grew laterally. (f ) Phase image
zoom-in of the largest aperture depicted in (c-e), showing the pronounced phase
contrast, indicating the different material properties of the emerging cell wall (light)
and remaining spore layer (dark). Inset in (f ) is the concurrent height image, showing
the 25 nm deeper position of the cell wall with respect to the surrounding spore layer.
Time in germination medium in hr:min was 1:40 (a), 2:15 (b), 2:50 (c), 3:35 (d), 3:50
(e), 3:55 (f ). Images reproduced, with permission from Ref. 8. ©(2007) American
Society for Microbiology.

Note that the spore coat degradation process presented in Figs. 4.8 - 4.12
appears not to be affected by the scanning AFM tip. 7,8 The shapes of issures
and apertures remained unaltered after repeated scanning. Furthermore,
when we zoomed out to a larger previously non-scanned area after prolonged