Biomedical Engineering Reference
In-Depth Information
double thickness of virtual section. Here, we describe the simplest
procedure for the examination of living cells containing quickly
moving organelles.
Initially, CVLEM was demanding, and to master its various
steps requires a whole array of skills. However, microscopy is devel-
oping fast both in the fi eld of live-cell imaging and in the fi eld of
EM, and new powerful technologies are rapidly becoming avail-
able in user-friendly versions.
This should make the use of CLEM appealing to a number of
cell biologists. Through the use of fl uorescent proteins of different
colors, two or more marker proteins can be observed simultane-
ously. This will allow the analysis of the interactions between dif-
ferent organelles and organelle sub-domains. Combining these
and other methods with electron microscope tomography increases
the subtlety and range of the questions that can be answered by the
CLEM approach.
Taken together, all the steps of CLEM represent quite a long
procedure (
see
below) and require signifi cant effort of a researcher
or technician. Hence, it would be especially disappointing to lose
such tour-de-force experiments due to small troubles in specimen
handling. The entire CVLEM procedure requires quite a long time
to be completed (4-5 days). During the fi rst day, the cells have to
be transfected with cDNA. The second day, it is possible to make
observations in living cells, fi x them, and start the immune label-
ing. The third day is required to complete the immune labeling
and resin embedding of the cells. During the fourth day, the cell of
interest has to be identifi ed in the resin block and be cut in serial
sections. However, it is possible to store already embedded speci-
men for a long time and to cut them later.
Our experience suggests that people often ask us whether
treatment of sample with 70 % ethanol could be longer than
5-10 min or whether it is possible to leave samples in glutaralde-
hyde for more than one day before Epon embedding. Here, we
give the minimal times, and in the brackets, one could fi nd the
time during which it is possible to leave samples without signifi cant
alteration of the structure.
Before deciding what method of CLEM could be used, it is
necessary to consult with the established specialist in EM. The sci-
entist planning to perform correlative microscopy should decide
which combination he will apply: CLLM, CLEM, or CLLM and
CLEM. It is necessary to decide whether one needs to examine the
rare event/structure using simple 3D reconstruction describing
the shape and connections or it is necessary to combine this with
immune labeling. For example, the strategy of CLEM will be dif-
ferent if the identifi cation of the organelle will be in the fl at cell or
in the tall cell. It is necessary to select what kind of sections he will
be applying for TEM or STEM tomography and so on: tangential
or vertical sections, simple serial section, or thick serial sections.
