What type of microscopy do I want?
In choosing what type of microscopy you need, there
are several questions to ask yourself which will help
in making this decision:
- What is the
sample and how small is the detail you wish to
observe?
- Further considerations ?
- Still interested in
Microscopy ?
1. What is
the sample and what do you want to see?
This question relates specifically to the sample and
may dictate which type of microscope to use; whether
it be a light microscope (LM) or an electron microscope
(EM). Do you just want to look at differences between cell types within
a tissue e.g. to study the organisation of the cell layers within a leaf, or
specifically at detail within an organelle e.g. to study the membranes within
a chloroplast, or find out where a particular protein is located within a cell
or to identify what type of virus you are looking at? This
information will guide you to know what type of microscope
should be used, the level of resolution required and
may also dictate the technique for preparing your sample.
This comes down to some very basic questions one would
always need to answer before proceeding. You need to know how big the sample is and how big is
the bit you’re interested in? Is it a whole plant, a tissue, a cell
or organelle? Are you going to work at the mm,
mm or nm level? Then there are some more specific questions
to be answered to further refine what technique you should
use, as described below.
a) Do you have a sense of scale?
A sense of scale between living
cells and atoms. Each diagram shows an image magnified
by a factor of 10 in an imaginary progression from a
thumb, through skin cells, to a ribosome, to a cluster
of atoms forming part of one of the many protein molecules
in our bodies. Details of molecular structure, as shown
in the last two panels, are beyond the power of the electron
microscope. This image has been taken
from Molecular Biology of the Cell, fourth edition, courtesy
of Garland science publishing
The resolving power of a microscope can
be defined as the smallest distance apart at which two
points on a specimen can still be seen separately and depends
on several factors, including the wavelength of light
(or electrons in an EM), the quality of the lenses and
the quality of your sample preparation. This is not the
same as magnifying power. Although magnification alone
helps, to be able to distinguish further detail the microscope
must provide sufficient resolution otherwise the image
will just look blurred. There is a limit in all
microscopes, beyond which any further magnification becomes "empty" rather
than "useful" since it is merely enlarging
unresolvable blurs in the image rather than introducing
further detail.
As a rule of thumb, the resolution of the human eye
is about 0.2mm. The resolution of the LM is 200nm in
X or Y and 700nm in Z. For our SEM, it is about 3nm and
in our TEM, 2nm.
Note: you can still "see" something without resolving it, especially
if it emits light e.g. using fluorescence microscopy.
b) Will your specimen be living or dead (can it be chemically or cryo-fixed)?
Do you want to perform dynamic studies in living cells?
This is the case if you are using GFP (green
fluorescent protein), following tagged proteins or expression
patterns, using micro-injection (e.g. of tubulin to study
it’s incorporation into living
microtubules within the cytoskeleton of a cell), using vital stains or performing
time lapse experiments.
If you do not need to look at living cells, then the
material can be chemically or cryo-fixed. This is usually
a requirement for all specimens which will be put in
an electron microscope and is also usually the case for
studies of general morphology, immuno-localisation, nucleic
acid in situ hybridisation, and
the use of general stains (e.g. calcofluor, toluidine
blue) and reporter genes (e.g. GUS).
c) Where in the sample do you want to look?
One must think about how to handle the specimen; if it
is large and you wish to see inside it, then you are
likely to want to cut it (section it)!
This can be done to a certain extent by optical sectioning
in the confocal microscope. In this case, the thickness
limit depends on the opacity of the sample, but is generally
50mm.
For other microscopes you will need to physically section
the material. For the TEM, the specimen will initially
need to be trimmed to about 1mm3 pieces prior to fixation.
After embedding in resin, it will be sectioned into 100nm
slices.
For light microscopy, the sample should preferably
be fairly flat, or at least within the working distance
of the objective lens, if it is to be mounted whole.
Otherwise it can also be embedded (e.g. in wax or resin)
and/or sectioned, by hand or by using a microtome or
vibratome to generate thin slices.
If you will only be observing single cells in a light
microscope, then they simply need to be immobilised on
a glass microscope slide, normally using poly-L-lysine,
Aptes, low gelling-point agarose or by air-drying to
ensure they adhere successfully.
d) Will it need to be labelled?
There are a huge number of probes available ranging from;
histochemical and fluor-histochemical markers e.g. Ruthenium
Red and DAPI (which stains the nucleus), antibodies to
specific targets (such as cytoplasmic proteins or components
of the cell wall) and tagged proteins/DNA e.g. GFP chimeras.
There are also dynamic probes, which change their fluorescent
properties depending on the environment such as Ca2+
and pH. If you are using immuno-localisation methods, then a suitable secondary
antibody must be used with the appropriate conjugated marker (eg gold particles
which can be seen in the TEM or fluorescent markers which are suitable only for
optical microscopes).
Bright-field microscopy using transmitted white light
can be used for many biological samples without the need
for a probe, as long as the specimen contains some inherent
contrast or colour. However, most biological material
is transparent therefore we to use either specialist
contrast enhancing techniques e.g. phase / DIC or
histochemical markers which leave an insoluble colour
stain. Fluorescent and confocal microscopy
can also be used to visualise biological material, using
primary fluorescence (auto-fluorescence) such as that
found in chlorophyll. However, most biological molecules
or structures do not fluorescence of their own accord;
so they must be linked with fluorescent molecules (fluorochromes)
in order to create specific fluorescent probes. In both
light and electron microscopy the most common technique
used is immuno-localisation which uses commercially available
secondary antibodies conjugated to the appropriate marker
e.g. fluorochromes or enzymes for light microscopy, or
different sizes of gold particles
for the TEM. Silver-enhancement of gold can be used for
both EM and LM.
2. Further considerations
Now you know all about your sample and what type of
microscope would be best to use, we’d like to throw
in some extra things to consider!
a) Do you really need to use an electron microscope?
Sometimes, knowing that the resolving power of a light
microscope overlaps with that of an electron microscope,
there can be a tendency to think that one would be better
to use the electron microscope; that it somehow might
give more impressive results. However, preparation
of samples for the light microscope is far easier and
quicker and generally involves fewer health and safety
risks (because many chemicals used for fixation of specimens
for the TEM are known to be toxic). The operation
of a light microscope is considered relatively routine
compared to the operation of an electron microscope and
there are generally more light microscopes available. Unless
one really requires the resolution or special capabilities
of an electron microscope, such as the increased depth
of field in the SEM, it is usually better to stick to
a light microscope.
b) Collection of data
How many samples will be required to get a sufficient
sampling number and what volume of data will be generated?
There is a maximum number of samples that you can easily
handle at any one time for each type of microscopy. For
example, this may mean that you have to do several embedding
runs, which can drastically increase the time taken to
complete a project.
If you generate 100 embedded blocks of material, think
about how long this is going to take you to section them
all! The labour-intensiveness of electron microscopy,
together with the sheer volume of data that can be created
by taking images of every microscopic detail, can often
dictate how far one decides to take a project involving
microscopy. Nevertheless, it is difficult to resist
the temptation of looking for that elusive "perfect
picture" of what you are interested in, when the
microscopic world is so alluring.
c) Interpretation of results
Have you prepared enough samples to give your data statistical
value? Getting one good picture from one cell in one
100nm section from one leaf may take you a long time,
but it doesn’t mean anything unless it is repeatable
and you can show that the same result can be found from
a larger sampling area.
Is the quality of data sufficient? Microscopists must
learn to be critical of their images and look for artefacts
of fixation. When using probes, they must be careful
of using the appropriate controls to compare labelling
with background levels and to be aware of non-specific
labelling or cross-reaction of antibodies. When working
with sections, they must learn to put the two-dimensional
images together in their mind and think of things in
three dimensions in the sample.
If you want to quantify the data from a labelling experiment,
you can (if you have sufficient images) count the number
of gold particles on labelled sections or the level of
fluorescent intensity on many samples, but be prepared
for this to require a lot of your time (and patience)!
3. Still interested
in Microscopy?
Microscopy is typically a very time-consuming and often
labour-intensive technique. It involves the use
of expensive, specialist equipment which can be used
as a tool to further our understanding of the world around
us. A good microscopist will always allow enough
time for the preparation of the sample, the actual imaging
and thorough analysis of results when planning a piece
of work, as well as knowing how to tinker and modify
conditions to get the best out of any microscope. It
is not unusual for people to be shocked when they find
out how long it can take to get that one elusive picture
they want for a publication!
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