Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

LOOKING AT CELLS IN THE LIGHT MICROSCOPE
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100 photons 1000 photons 10,000 photons
(A)
100 nm
successive cycles of activation and bleaching allow well-separated single fluorescent molecules
to be detected
the exact center of each fluorescent molecule is determined and its position added to the map
a super-resolution image of the fluorescent structure is built up as the positions of successive small
groups of molecules are added to the map
(B)
Figure 9–38 Single fluorescent molecules can be located with great accuracy. (A) Determining the exact mathematical
center of the blurred image of a single fluorescent molecule becomes more accurate the more photons contribute to the final
image. The point spread function described in the text dictates that the size of the molecular image is about 200 nm across,
but in very bright specimens, the position of its center can be pinpointed to within a nanometer. (B) In this imaginary specimen,
sparse subsets of fluorescent molecules are individually switched on briefly and then bleached. The exact positions of all these
well-spaced molecules MBoC6 can be n9.236/9.38 gradually built up into an image at superresolution. (C) In this portion of a cell, the microtubules
have been fluorescently labeled and imaged at the top in a TIRF microscope (see Figure 9–32) and below, at superresolution,
in a PALM microscope. The diameter of the microtubules in the lower panel now resembles their true size, about 25 nm,
rather than the 250 nm in the blurred image at the top. (A, from A.L. McEvoy et al., BMC Biol. 8:106, 2010; C, courtesy of
Carl Zeiss Ltd.)
(C)
1 µm
development of novel fluorescent probes that exhibit the appropriate switching
behavior. All these methods are now being extended to incorporate multicolor
imaging, three-dimensional imaging (Figure 9–39), and live-cell imaging in real
time. Ending the long reign of the diffraction limit has certainly reinvigorated light
microscopy and its place in cell biology research.
Figure 9–39 Small fluorescent structures can be imaged in three
dimensions with superresolution. (A) The image of two touching
180-nm-diameter clathrin-coated pits on the plasma membrane of a cultured
cell is diffraction-limited, and the individual pits cannot be distinguished in
this conventional fluorescence image. (B) Using STORM superresolution
microscopy, however, the pits are clearly resolvable. Not only can such pits
be imaged using probes of different colors, but additional three-dimensional
information can also be obtained. (C) and (D) Shown are two different
orthogonal views of one single coated pit. The clathrin is labeled red and
transferrin—the cargo within the pit—is labeled green. Images of this sort can
be acquired in less than one second, making possible dynamic observations
on living cells. These techniques depend heavily on the development of new,
very fast-switching, and extremely bright fluorescent probes. (A and
B, from M. Bates et al., Science 317:1749–1753, 2007; C and D, from
S.A. Jones et al., Nat. Methods 8:499–508, 2011. With permission from
Macmillan Publishers Ltd.)
z
(A)
x
(C)
transferrin
cargo
(green)
y
clathrincoated
pit
(red)
(B)
(D)
x
200 nm
200 nm