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

550 Chapter 9: Visualizing Cells
(A) (B) (C)
The first of these so-called superresolution approaches, structured illumination
microscopy (SIM), is a fluorescence imaging method with a resolution of
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about 100 nm, or twice the resolution of conventional bright-field and confocal
microscopy. SIM overcomes the diffraction limit by using a grated or structured
pattern of light to illuminate the sample. The microscope’s physical set-up and
operation is quite complex, but the general principle can be thought of as similar
to creating a moiré pattern, an interference pattern created by overlaying two
grids with different angles or mesh sizes (Figure 9–34). In a similar way to creating
a moiré pattern, the illuminating grid and the sample features combine into
an interference pattern, from which the original high-resolution contributions
to the image of features beyond the classical resolution limit can be calculated.
Illumination by a grid means that the parts of the sample in the dark stripes of
the grid are not illuminated and therefore not imaged, so the imaging is repeated
several times (usually three) after translating the grid through a fraction of the
grid spacing between each image. As the interference effect is strongest for image
components close to the direction of the grid bars, the whole process is repeated
with the grid pattern rotated through a series of angles to obtain an equivalent
enhancement in all directions. Finally, mathematically combining all these separate
images by computer creates an enhanced superresolution image. SIM is versatile
because it can be used with any fluorescent dye or protein, and combining
SIM images captured at consecutive focal planes can create three-dimensional
data sets (Figure 9–35).
Figure 9–34 Structured illumination
microscopy. The principle, illustrated here,
is to illuminate a sample with patterned
light and measure the moiré pattern.
Shown are (A) the pattern from an unknown
structure and (B) a known pattern.
(C) When these are combined, the resulting
moiré pattern contains more information
than is easily seen in (A), the original
pattern. If the known pattern (B) has higher
spatial frequencies, then better resolution
will result. However, because the spatial
patterns that can be created optically
are also diffraction-limited, SIM can only
improve the resolution by about a factor
of two. (From B.O. Leung and K.C. Chou,
Appl. Spectrosc. 65:967–980, 2011.)
(A) (B) (C)
2 µm
Figure 9–35 Structured illumination microscopy can be used to create three-dimensional data. These three-dimensional projections of the
meiotic chromosomes at pachytene in a maize cell show the paired lateral elements of the synaptonemal complexes. (A) The chromosome set has
been stained with a fluorescent antibody to cohesin and is viewed here by conventional fluorescence microscopy. Because the distance between
the two lateral elements is about 200 nm, the diffraction limit, the two lateral elements that make up each complex are not resolved. (B) In the
three-dimensional SIM image, the improved resolution enables each lateral element, about 100 nm across, to be clearly resolved, and the two
chromosomes can clearly be seen to coil around each other. (C) Because
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the complete three-dimensional data set for the whole nucleus is available,
the path of each separate pair of chromosomes can be traced and artificially assigned a different color. (Courtesy of C.J. Rachel Wang, Peter Carlton
and Zacheus Cande.)