The Birth of Light Microscopy
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| From "The Microscope and Its Revelations" Carpenter,
1891:
image of Hooke's compound microscope 1665 |
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| From "The Microscope and Its Revelations" Carpenter,
1891:
images of Leeuwenhoek's simple microscope |
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| From "Anthony Van Leeuwenhoek and his 'little
animals'",
edited by Clifford Dobel:
plate showing the drawings of bacteria
from the human mouth |
The English scientist, Robert Hooke (1635-1703), was
the first to publish work based upon the use of an optical
microscope, in 1665. His publication was entitled "Micrographia" or "Some
physiological descriptions of minute bodies made by magnifying
glasses with observations and inquiries thereupon".
This
book contained a description and illustration of the microscope
that he used for his observations and several illustrations
of parts of plants and animals. He was the first to use
the word “cell” in the biological context.
Hooke
used a compound microscope, having two lenses, whereas Antoni
van Leeuwenhoek, a Dutch cloth merchant and amateur scientist,
used only a single glass lens in his simple microscope, some
10 years or so later.
Leeuwenhoek made observations on bacteria,
plants, blood cells and minerals and is generally considered
to have made a greater contribution to our understanding
of the microscopical world than Hooke.
The understanding of how to solve the problems of spherical
and chromatic aberrations, two types of distortion related
to the shape and type of the glass used in lens production,
led to a major leap forward in the quality and resolving
power of optical microscopes and hence, by about 1830, we
have compound microscopes capable of relatively high magnification
and good resolution.
Around 1870, Ernst Abbe formulated his famous sine theory
for the resolving power of the light microscope, which demonstrates
the importance of the numerical aperture of the lenses used
and the wavelength of light. The higher the numerical
aperture and the shorter the wavelength, the better the resolving
power. Thus, the theoretically possible resolution in
light microscopy is approximately 0.2 mm. The resolving
power of an objective lens is the ability to show two object
details separately from each other in the microscope image.
Resolution: the resolving power of the microscope depends
on the width of the cone of illumination and therefore on
both the condenser and the objective lens. It is calculated
using the formula
Resolution = 0.61l / n sinq
Where n = the refractive index of the medium (usually
air or oil) separating the specimen from the objective
and condenser lenses.
l = the wavelength of light
used (for white light the figure of 0.53 microns is commonly
assumed)
q = half the angular width
of the cone of rays collected by the objective lens from
a typical point in the specimen (since the maximum width
is 180 degrees, sinq has a
maximum value of 1).
Numerical aperture: n sinq in
the equation above is called the numerical aperture (NA)
of the lens and is a function of its light-collecting ability. For dry
lenses this cannot be more than 1, but for oil-immersion
lenses it can be as a high as 1.4. The higher the
numerical aperture, the greater the resolution and the
brighter the image (brightness is important in fluorescence
microscopy). However, this advantage is obtained at the
expense of very short working distances and a very small
depth of field.
By 1900 the theoretical principles of the microscope were
well understood and the microscope had become a well-established
research tool for professional scientists. Further
clever refinements have improved the performance of the instrument
and these, together with the development of photographic
and now digital imaging techniques, have led us to the modern
research microscope used as an essential tool in laboratories
all over the world.
Techniques:
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| "Using the microscope in an upright position" from "The
Microscope and Its Revelations" Carpenter, 1891 |
Transmitted Light Microscopy
Transmitted light microscopy is the general term used for
any type of microscopy where the light is transmitted from
a source on the opposite side of the specimen to the objective
lens. Usually, the light is passed through a condenser to
focus it on the specimen to get maximum illumination. After
the light passes through the specimen it goes through the
objective lens to magnify the image of the sample and then
to the oculars, where the enlarged image is viewed.
In order to get a usable image in the microscope, the specimen
must be properly illuminated. The light path of the microscope
must be correctly set up for each optical method and the
components used for image generation. The condenser was invented
to concentrate the light on the specimen in order to obtain
a bright enough image to be useful. The optimum set-up for
specimen illumination and image generation is known as Köhler
illumination after the man who invented it. It is used for
most of the optical configurations listed below. The microscope
techniques requiring a transmitted light path include bright
field, dark field, phase contrast, polarisation and differential
interference contrast optics.
Bright Field (Köhler illumination) Microscopy
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| From "Micrographia", by Robert Hooke, 1665:
plate showing the drawing of a flea |
This is "normal" microscopy when no optical contrast
technique is employed. It uses transmitted light to
view a specimen that contains inherent contrast/colour or
is stained. In order to get the best image possible from
any transmitted light form of microscopy, and it is crucial
that the light path be set up properly. The method for doing
this is called Köhler illumination after August Köhler;
the man who invented it. It is also know as double diaphragm
illumination because it employs both a field and an aperture
iris diaphragm to set up the illumination. The condenser
is used to focus parallel rays of light on the specimen,
as if coming from infinity, thereby giving you the advantages
of an evenly illuminated field, a bright image without glare
and minimum heating of the specimen. As most cells and tissues
have insufficient contrast in themselves, staining techniques
are generally used. Common stains include Papanicolaou’s
stain, which is used for cervical smears, and toluidine blue,
a general stain used for semi-thin sections of all tissue
types.
Dark Field Microscopy
The specimen is illuminated obliquely, with no direct
light entering the objective. Features in the specimen plane
which scatter light can clearly be seen against a dark background. Darkfield illumination is provided by either a simple patch stop, a
darkfield element in a phase contrast condenser or purpose-built
darkfield condenser. The latter is required for high-resolution
objectives to prevent the oblique rays entering the wide
aperture of the objective. Applications include detection
of micro-organisms in unstained smear preparations and classical
diatom studies.
Phase Contrast
Devised by Zernicka, this technique exploits the fact
that light slows slightly when passing through biological
specimens. The
specimen is illuminated by a hollow cone of light coming
through a phase annulus in the condenser. Phase
contrast objectives must be used, which have a corresponding phase
plate. Light rays passing through the specimen are
slightly retarded, and further retardation takes place in
the phase plate. When these rays combine with rays
which have not taken this path, degrees of constructive and
destructive interference occur which produce the characteristic
light and dark features in the image.
Polarised Light Microscopy
Polarised light microscopy uses plane-polarised light
to analyse structures that are birefringent; structures
that have two different refractive indices at right angles
to one another (e.g. cellulose microfibrils). Normal,
un-polarised, light can be thought of as many sine waves,
each oscillating at any one of an infinite number of
orientations (planes) around the central axis. Plane-polarised
light, produced by a polar, only oscillates in one plane
because the polar only transmits light in that plane.
The polarised light microscope must be equipped with
both a polarizer, positioned in the light path somewhere
before the specimen, and an analyser (a second polarizer),
placed in the optical pathway after the objective rear
aperture. Image contrast arises from the interaction
of plane-polarized light with a birefringent (or doubly-refracting)
specimen to produce two individual wave components that
are each polarized in mutually perpendicular planes.
The velocities of these components are different and
vary with the propagation direction through the specimen.
After exiting the specimen, the light components become out
of phase, but are recombined with constructive and destructive
interference when they pass through the analyzer. Polarised
light microscopy can be used to measure the amount of retardation
that occurs in each direction and so give information about
the molecular structure of the birefringent object (e.g.
orientation).
Differential Interference Contrast
In this complex form of polarised light microscopy two
slightly separate, plane polarised beams of light are
used to create a 3D-like image with shades of grey. Wollaston prisms
situated in the condenser and above the objective produce
the effect, and additional elements add colour to the image. Care
must be taken to interpreting DIC images as the apparent
hills and valleys in the specimen can be misleading. The
height of a "hill" (e.g. the nucleus) is a product
of both the actual thickness of the feature (i.e. ray path
length) and its refractive index. Variations of the
DIC system are named after their originators, Nomarski and
de Senarmont. Options can be selected to maximise
either resolution or contrast.
Fluorescence Microscopy
Fluorescence can be used as a label or tag when preparing
specific biological probes. Some biological substances, such
as chlorophyll and some oils and waxes, have primary fluorescence
(auto-fluorescence). However, most biological molecules do
not fluoresce on their own, so they must be linked with
fluorescent molecules fluorochromes)
in order to create specific fluorescent probes. The fluorochromes
emit light of a given wavelength when excited by incident
light of a different (shorter) wavelength. Specimens labelled
with a fluorochrome such as fluorescein or green fluorescent
protein (GFP) are illuminated with the relevant wavelength
of light (blue in these examples) and emit the energy as
a longer wavelength (green).
The key feature of fluorescence microscopy is that it employs
reflected rather than transmitted light, which means transmitted
light techniques such as phase contrast and DIC can be combined
with fluorescence microscopy. To view the fluorescence
in the microscope, several light filtering components are
needed. At the heart of the fluorescence microscope
is the dichroic mirror cube which comprises three components:
a dichroic beam splitter (partial mirror), an excitation
filter and a barrier filter. Specific filters are used
to isolate the excitation and emission wavelengths for each
fluorochrome. The dichroic mirror reflects shorter
wavelengths of light and allows longer wavelengths to pass
and is required because the objective acts as both the condenser
lens (excitation light) and objective lens (emission light);
therefore the beam splitter isolates the emitted light from
the excitation wavelength. This epi-illumination type of
light path is required to create a dark background so that
the fluorescence can be easily seen. The wavelength at which
a beam splitter allows the longer wavelengths to pass must
be set between the excitation and emission wavelengths of
any given fluorochrome so that excitation light is reflected
and emission light is allowed to pass through it. A bright
light source producing the correct wavelengths for excitation
is also required for fluorescence microscopy, normally a
mercury arc lamp. When using confocal
microscopy to view fluorescence, where up to 95% of
the emission light is filtered out, specific wavelength
lasers are used, as these are extremely bright and monochromatic.
Confocal Microscopy
Although conventional light and fluorescence microscopy
allow the examination of both living and fixed specimens,
certain problems exist with these techniques. One of
the main problems is out-of-focus blur degrading the
image by obscuring important structures of interest,
particularly in thick specimens. In conventional microscopy,
not only is the plane of focus illuminated, but much
of the specimen above and below this point is also illuminated
resulting in out-of-focus blur from these areas. This
out-of-focus light leads to a reduction in image contrast
and a decrease in resolution. In the confocal microscope,
all out-of-focus structures are suppressed at image formation.
This is obtained by an arrangement of diaphragms, which,
at optically conjugated points of the path of rays, act
as a point source and as a point detector respectively.
The detection pinhole does not permit rays of light from
out-of-focus points to pass through it. The wavelength of
light, the numerical aperture of the objective and the diameter
of the diaphragm (wider detection pinhole reduces the confocal
effect) affect the depth of the focal plane. To obtain a
full image, the point of light is moved across the specimen
by scanning mirrors. The emitted/reflected light passing
through the detector pinhole is transformed into electrical
signals by a photomultiplier and displayed on a computer
monitor.
What is Green Fluorescent Protein (GFP)?
Green Fluorescent Protein (GFP) is a protein naturally produced
by the jellyfish Aequorea victoria; which produces
glowing points of light around the margin of it’s umbrella.
The light arises from yellow tissue masses that each consist
of about 6000-7000 photogenic cells. These cells generate
light by a process of bioluminescence, whose components include
a calcium-activated photoprotein (aequorin) that emits blue-green
light and an accessory green fluorescent protein (GFP), which
accepts energy from aequorin and re-emits it as green light.
GFP is a 238 amino acid long protein which is very stable
in neutral buffers up to 65oC, and displays a broad range
of pH stability from 5.5 to 12. The protein is intensely
fluorescent, with a quantum efficiency of approximately 80%
and molar extinction coefficient of 2.2 x 104 cm-1 M-1. GFP
fluoresces maximally when excited at 400nm with a lesser
peak at 475nm, and fluorescence emission peaks at 509nm.
The intrinsic fluorescence of the protein is due to a unique
covalently attached chromophore, which is formed post-translationally
within the protein upon cyclisation and oxidation of residues
65-67, Ser-Tyr-Gly. The gene for GFP has been isolated and
has become a useful tool for making expressed proteins fluorescent
by creating chimeric genes composed of those of GFP and its
different colour variants linked to genes for proteins of
interest. This makes it possible to have an in vivo fluorescent
protein, which may be followed in a living system.
There have been several recent developments for the use
of GFP and its colour variants. Wild type GFP has two excitation
peaks, a major one at 395nm (long wave UV; causes rapid quenching
of the fluorescence) and a smaller one at 475nm (blue) and
an emission peak at 509nm (green). For general fluorescence
microscopy purposes, investigators have been using
normal FITC filter sets for viewing GFP. These are inadequate
for wild type GFP both in excitation 475-495nm, and emission
520-560nm. To alleviate this problem, several modified versions
of GFP were constructed which have increased fluorescence
(serine to threonine substitution at position 65 increased
fluorescence 5-6 times), but perhaps more important, the
major excitation peak has been red-shifted to 490nm with
the emission staying at 509nm. This is better for use of
FITC filter sets as this modified GFP has the same excitation
range as FITC. Furthermore, in confocal
microscopy the main laser line used for GFP excitation
is from the argon laser at 488nm, there is no good commonly
used laser line near 395nm. In Arabidopsis plants and cells,
which we regularly use for our research at The John Innes
Centre, poor or no fluorescence was seen when transformed
with GFP-cDNA because the expression of GFP was curtailed
by aberrant mRNA splicing. Therefore, modified forms of GFP
were created to restore and improve expression of the fluorescent
protein. The modified gene now contains an altered codon
to remove a cryptic plant intron. Since then, other modifications
have given further improvements in the brightness of the
emission and different colour variants of GFP have been produced
e.g. in order from shortest to longest emission spectra:
blue (FP or BFP), cyan (CFP), green (GFP), yellow (YFP) and
red (RFP). This makes it possible to prepare double-labelled
specimens expressing two or more fluorescently labelled proteins.
Added peptide sequences also allow targeting of GFP intracellular
organelles like the lumen of the endoplasmic reticulum.
Other useful information about GFP in plants can be found
on Jim
Haseloff’s web-site, of Cambridge University.
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