Technological Advances in Microscopes
Researchers wanted to study living cells in the tiniest molecular detail.
In 1873, Ernst Abbe showed a physical limit for the maximum resolution of traditional
optical microscopes.
The resolution it could never be better than 0.2 micrometers.
Compound Light Microscopes
Uses light
Has two lenses
Magnification limited
Electron Microscope
Developed in the 1930s
the electron microscope allowed for higher magnification
used electron beams (instead of light) and focused with an electromagnet (no lenses)
the light microscope produces magnifications up to 2000X
the electron microscope produces images that are magnified up to 50 000X or higher
The electron microscope allowed scientists to see better quality images at higher
Magnification
Scanning Electron Microscope (SEM)
Electrons are reflected from the surface of the specimen
Produces a 3-D image
Good for the thicker specimens
Lacks the magnification and resolution of the transmission electron microscope
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Transmission Electron Microscope (TEM)
Uses beams of electrons
Magnification of 2 000 000x
Has two limitations:
Good only for thin specimens
Only dead cells can be observed
Fluorescence
The development of dyes was very useful to study the parts of cells.
But these required the absorption of light which provide problems with measurement.
Fluorescent dyes overcome this problem because we just measure the intensity of the
emitted light. The intensity is directly proportional to the amount of excited dye.
Epiflourescence is useful because most of the excitation light is transmitted through the
specimen.
Only reflected excitatory light reaches the objective together with the emitted light and the
epifluorescence method therefore gives a high signal-to-noise ratio.
Schematic of a fluorescent microscope.
https://en.wikipedia.org/wiki/Fluorescence_microscope#/media/File:FluorescenceFilters_2008-09-28.svg CC BY-SA 3.0
Source of fluorescence
There are several methods of creating a fluorescent sample; the main techniques are
labeling with fluorescent stains or, in the case of biological samples, expression of
a fluorescent protein. Alternatively the natural fluorescence of a sample
(i.e.,autofluorescence) can be used.
In the life sciences fluorescence microscopy is a powerful tool which allows the specific and
sensitive staining of a specimen in order to detect the distribution of proteins or other
molecules of interest. As a result there is a diverse range of techniques for fluorescent
staining of biological samples.
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Biological fluorescent stains
Many fluorescent stains have been designed for a range of biological molecules. Some of
these are small molecules which are fluorescent and bind a biological molecule of interest.
Major examples of these are nucleic acid stains which bind the minor groove of DNA, thus
labeling the nuclei of cells.
Other fluorescent stains are drugs or toxins which bind specific cellular structures and have
been made with a fluorescent reporter. A major example of this class of fluorescent stain
is phalloidin which is used to stain actin fibres in mammalian cells.
There are many fluorescent molecules called fluorophores or fluorochromes which can be
chemically linked to a different molecule which binds the target of interest within the
sample.
Eg. Fluorescently labeled antibodies.
Immunofluorescence
This technique uses the highly specific binding of an antibody to its antigen in order to label
specific proteins or other molecules within the cell. A sample is treated with a primary
antibody specific for the molecule of interest. A fluorophore can be directly conjugated to
the primary antibody.
Alternatively a secondary antibody, conjugated to a fluorophore, which binds specifically to
the first antibody can be used.
Indirect immuno-cytochemistry
This detection method is very sensitive because the primary antibody is recognized by many
molecules of the secondary antibody. The secondary antibody is linked to a marker
molecule that makes it detectable.
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Molecular structure of green fluorescent protein GFP
Stimulated emission depletion (STED) microscopy, developed by Stefan Hell in 2000. Two
laser beams are used; one stimulates fluorescent molecules to glow, another cancels out all
fluorescence except for that in a nanometre-sized volume. Scanning over the sample,
nanometre for nanometre, gives an image with a resolution better than Abbe's limit.
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Eric Betzig and William Moerner worked on single-molecule microscopy.
The fluorescence of individual molecules are turned on and off.
Scientists image the same area multiple times, letting just a few molecules glow each time.
Adding these images gives a dense super-image resolved at the nanolevel.
In 2006 Betzig used this method for the first time.
The following image is a 3-D version of PALM showing microtubules in a cell from a fruit fly.
The tubules are labeled for depth, with red lower and blue and violet higher. A conventional
optical microscopy image of the same cell (left) is shown for comparison.
Credit: Jim and Cathy Galbraith, Gleb Shtengel, Harald Hess, HHMI/Janelia Research Campus.
There are still some problems, such as capturing good images of cellular parts in motion and
removing optical errors caused by the deep three-dimensional environment of the cell.
But super-resolution techniques are revolutionizing our understanding of cell biology.
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