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Few branches of science are as aesthetically satisfying as the study
of plant anatomy. The internal architecture of plant tissues is invariably
pleasing to the eye and often breathtakingly beautiful. Yet until relatively
recently, access to this world was anything but straightforward. The fluorescence
microscope has changed all that. Plant anatomy is now open to anyone who
can cut a piece of tissue in half with a razor.

Conventional microscopy demands a high degree of manual dexterity. Tissues
must first be carved into thin, translucent slices if they are to reveal
their cellular structure. Students often find themselves cutting thin sections
by hand, armed with a cutthroat razor. It is a technique that is almost
guaranteed to put them off the subject for life. Expecting students to master
this craft before they can study plant anatomy is rather like insisting
that authors should master copperplate script before they can use a word
processor.

The fluorescence microscope does away with the need to cut thin sections.
Instead of shining light through a translucent specimen from below, the
microscope illuminates the specimen from above with the help of its own
objective lens. The thickness of the specimen is unimportant, because only
the light emitted from its top surface forms the image. These optical arrangements
bring some notable advantages. Because the same lens both illuminates the
object and collects light from it, the microscope is always in perfect adjustment.
If the observer decides to use a more powerful objective lens, the intensity
of illumination increases automatically.

The origins of the fluorescence microscope can be traced back to the
early years of the century, when researchers discovered that certain biological
molecules would fluoresce if illuminated with an intense beam of light of
a particular wavelength. Since then the technique has generated a wide range
of applications, many of them in the field of medical microscopy. One example
is the use of fluorescent antibodies to bind to, and to reveal the whereabouts
of, specific chemicals inside cells.

In recent years the technique has revolutionised the use of the microscope
in botany. Many plant tissues, ranging from roots of Berberis to the food
stores in grains of barley, contain molecules that can be made to fluoresce.
Umbelliferones in the stems of parsley, for example, appear brilliant yellow
when irradiated with blue-violet light.

The range of plant tissues that contains fluorescent molecules is limited,
but researchers have extended the technique by the use of fluorochromes.
These are molecules that fluoresce when they are attached to certain chemicals
found in plant tissues. Many fluorochromes bind only to one variety of cellular
molecule. They first found favour with plant breeders, who discovered that
a fluorochrome called decolourised aniline blue had a particular affinity
for callose – a polymer of glucose found in germinating pollen grains. When
pollen grains find themselves on a flower with which they are sexually incompatible,
plugs of callose appear in the tubes that grow out from the grains. Tubes
treated with fluorochrome give off a bright yellow-green fluorescence when
illuminated with ultraviolet light. The technique provides breeders with
quick a check on the success or failure of pollinations.

Calcofluor – the blue whitener in washing powders – is arguably the
most useful fluorochrome of all. It binds to cellulose fibrils in cotton
fabrics and fluoresces at a barely visible blue wavelength when irradiated
with ultraviolet light such as that in sunlight. Calcofluor is the universal
fluorochrome for plant tissues. Almost all plant tissues appear ice blue
if stained with calcofluor and illuminated with ultraviolet light from a
mercury vapour lamp.

Auramine O is almost as valuable as calcofluor. It binds to two important
chemical constituents of plants – lignin and cutin – and fluoresces with
a yellow hue in blue light. A few year ago, Nick Harris and I discovered
that we could use it to stain two varieties of organelle inside living plant
cells, the endoplasmic reticulum and the Golgi bodies. In conjunction with
electron microscopy, the technique allows researchers to correlate relatively
easily the structure of these organelles with their function and behaviour
in the living cell.

Many fluorochromes are effective at very low concentrations, so they
do not poison plant tissues. Researchers can use them to study the movements
of specific substances around the growing plant. The palette of available
fluorochromes is expanding rapidly. Acridine orange, for example, stains
both RNA and DNA, while 4′,6-diamidino-2-phenyl indole (DAPI) is a very
sensitive detector of DNA. Another fluorochrome, 8-anilino-1-naphthalenesulphonic
acid (ANSA), is particularly useful for locating lipids and proteins in
cells and tissues. Several fluorochromes can be used on the same tissue.

Fluorescence microscopy is both fast and straightforward. A deft cut
with a sharp razor blade and immersion of the specimen in a solution of
fluorochrome are all that is needed to produce a detailed image of cellular
structure. The results can be as good as, or better than, those obtained
by embedding tissues in wax and cutting thin sections on a microtome. The
work of days can be compressed into a couple of minutes. Because there is
no need to fix the specimen, dehydrate it, or embed it in wax, the risk
of introducing artefacts is minimal. This combination of speed and accuracy
makes the fluorescence microscope an ideal tool for screening large amounts
of biological material.

The microscope can be made yet more powerful by linking it to a computerised
device that analyses colour images. Such devices capture the image via a
television camera and then employ sophisticated software to generate quantitative
data. With the aid of this equipment, researchers can gather information
about certain aspects of plant anatomy – the amount of lignin in a plant
stem, say – almost instantaneously. The technique is fast enough to be used
as a way of monitoring production processes, where conventional microscopy
would be completely impracticable.

At Durham we have used it to monitor the development of plant tissues
that we are culturing in the laboratory. Rather than wait until visible
symptoms of some treatment appear – a process that can take weeks – we sample
the tissues every day and use fluorochromes to search for evidence of cell
division. Using this technique, we can discern signs of rooting in tissues
between four and seven days after the start of the experiment. By the time
external signs of growth appear, we have moved on to the next experiment.
The technique has also been adopted by at least one commercial company specialising
in the micropropagation of plants.

Yet the real joy of the technique lies in the ease with which the beauty
and intricacies of plant tissues can be revealed. Having been brought up
in the Sweeney Todd school of plant anatomy, I take great pleasure in showing
students that they do not need the skill of a watchmaker to explore the
internal architecture of a plant. The fluorescence microscope has quite
literally shed new light on plant anatomy.

Dr Phil Gates lectures in the department of biological sciences at the
University of Durham.