Biology Reference
In-Depth Information
Abstract
Electron microscopy (EM) has dominated high-resolution cellular imaging for over
50 years, thanks to its ability to resolve on nanometer-scale intracellular structures
such as the microtubules of the mitotic spindle. It is advantageous to view the cell of
interest prior to processing the sample for EM. Correlative light-electron microscopy
(CLEM) is a technique that allows one to visualize cells of interest by light micros-
copy (LM) before being transferred to EM for ultrastructural examination. Here, we
describe how CLEM can be applied as an effective tool to study the spindle apparatus
of mitotic cells. This approach allows transfected cells of interest, in desirable stages
of mitosis, to be followed from LM to EM. CLEM has often been considered as a
technically challenging and laborious technique. In this chapter, we provide step-
by-step pictorial guides that allow successful CLEM to be achieved. In addition,
we explain how it is possible to vary the sectioning plane, allowing spindles and mi-
crotubules to be analyzed from different angles, and the outputs that can be obtained
from these methods when applied to the study of kinetochore fiber ultrastructure.
INTRODUCTION
The mitotic spindle is a complex machine consisting of microtubules, motor proteins,
and nonmotor proteins which, together, generate the forces needed to separate the
sister chromatids between the two daughter cells (
Scholey, Brust-Mascher, &
Mogilner, 2003
). A better visualization of its ultrastructure is necessary to under-
stand the mechanisms underlying its functions.
Light microscopy (LM) and the discovery of the green fluorescent protein (GFP)
led to many important discoveries due to the possibility of tracking protein dynamics
in live cells. However, LM has a relatively low resolution, which does not allow one
to visualize structures smaller than
200 nm. This diffraction limit has been a major
imaging weakness, and electron microscopy (EM) has been one of the few techniques
to overcome it. Another disadvantage of LM is the restricted number of separate
wavelength channels which can be used on a single sample without overlap, while
the rest of the cell remains unobservable.
EM also possesses its share of drawbacks, other than the tricky and time-
consuming nature of sample preparation. Only static samples can be observed,
making the analysis of dynamic changes impossible. Also, routine EM does not
allow one to easily locate cells of interest, such as cells expressing a fluorescent
protein or in a particular stage of the cell cycle. It is possible to overcome these
limitations by combining the ease and dynamic nature of LM with the
subnanometer resolving power of EM in the form of correlative light-electron mi-
croscopy (CLEM).
CLEM techniques are useful for studying the mitotic spindle. The complexity
of spindle microtubules means that they cannot be viewed individually by
LM. Also, mitosis is a very dynamic process; each of its stages lasts less than
