Microscopy
Oat root

A sense of scale

 

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:

  1. What is the sample and how small is the detail you wish to observe?
  2. Further considerations ?
  3. 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?

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

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

Recommended link: http://learn.genetics.utah.edu/content/begin/cells/scale/

This is a great interactive demonstration of the relative sizes of different biological features (from a coffee bean to a carbon atom)

 

 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 2nm and in our TEM, 0.1nm.

Note: you can still "see" something without resolving it, especially if it emits light e.g. using fluorescence microscopy.

What can be seen at different magnification

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!