Light Microscope Related terms:

Using a binocular light microscope with 4–100× objective lenses preferably with
Chloroplast, Genus, Tissues,
an integrated digital photography and computer-linked editing software is
Electron Microscopy,
standard equipment nowadays for the fish pathologist.
Light Microscopy,
From: Aquaculture Pathophysiology, 2022
Chromosomes

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MICROSCOPY | Overview MICROSCOPY | Light Microscopy

P.J. Evennett, C. Hammond, in and Histochemical Methods
Encyclopedia of Analytical Science (Second Edition), 2005
J.H. Holgate, J. Webb, in
Advantages and Limitations of Light Encyclopedia of Food Sciences and Nutrition (Second
Edition)
Microscopy
, 2003
The light microscope has many advantages over other
forms of microscope. Light microscopes are extremely Principles
versatile instruments. They can be used to examine a The light microscope is an instrument for visualizing fine
wide variety of types of specimen, frequently with detail of an object. It does this by creating a magnified
minimal preparation. Light microscopes can be adapted image through the use of a series of glass lenses, which
to examine specimens of any size, whole or sectioned, first focus a beam of light onto or through an object, and
living or dead, wet or dry, hot or cold, and static or fast- convex objective lenses to enlarge the image formed. In
moving. They offer a wide range of contrast techniques, the majority of light microscopes, the image is viewed
providing information on the physical, chemical, and directly through binocular eyepieces that act as a
biological attributes of specimens. The image from a secondary lens in the form of a magnifying glass to
light microscope is presented in color. It can be observed observe the projected image. Such instruments are
with the eye directly, recorded by photographic, video or termed ‘compound microscopes,’ and the total
computer techniques, and image components can be magnification is the sum of the objective magnification
analyzed. And the instruments themselves are (relatively) and the eyepiece magnification. The magnification range
inexpensive, small, require no vacuum, and are extends from ×10 to ×1000, with a resolving power of the
undemanding of operating conditions, services and order of 0.2 μm, depending on the type and numerical
maintenance. aperture (area available for passage of light) of the
The principal limitation of the light microscope is its objective lenses. A number of books are available,
resolving power. Using an objective of NA 1.4, and green providing comprehensive details on the theory of the
light of wavelength 500 nm, the resolution limit is light microscope and guidance to the practical use of the
∼0.2 μm. This value may be approximately halved, with instrument, including methods of image enhancement
some inconvenience, using ultraviolet radiation of and instrument care. The reader is referred to these, in
shorter wavelengths. By good fortune, however, the particular an extensive series of handbooks published by
resolving power of the light microscope is adequate for the Royal Microscopical Society, for further information.
many areas of work in the biological and physical
sciences.
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Microbiology laboratory techniques

Soldier Determination in Ants of Tim Sandle, in Pharmaceutical Microbiology, 2016

the Genus Pheidole 6.5 Microscopy

The light microscope is an important tool in the study of
L. PASSERA, in Caste Differentiation in Social Insects,
microorganisms, particularly for identification purposes.
1985
The compound light microscope uses visible light to
23.2 LARVAL DIFFERENTIATION INTO directly illuminate specimens in a two-lens system,
SOLDIERS resulting in the illuminated specimen appearing dark
Light microscope examination of larvae taken from against a bright background. The two lenses present in a
developing colonies of P. pallidula societies allowed three compound microscope are the ocular lens in the
larval instars to be recognized in the minor worker line. eyepiece and the objective lens located in the revolving
Instar 1: length of larvae: 0.4 mm. The mandible with nosepiece. Compound light microscopes typically have
one-pointed tooth looks like a very large paddle; low the following components (as outlined below and set out
pilosity; on dorsal abdomen two lines of five or six fairly in Figure 6.1):
thick short hairs characteristic of the first instar. Ocularlens
(eyepiece)
Instar 2: length of larvae: 0.40 to 0.75 mm. The mandible Bodytube
with three-pointed teeth is fairly elongated; thick pilosity
with single hairs and two lines of five or six thick forked Nosepiece
Arm
hairs on the dorsal abdomen, characteristic of the second Objectives

instar. Stage
Instar 3: length of larvae: 0.75 to 1.50 mm. The three- Stageclips Coarseadjustment
Diaphragm
pointed mandible has asperities. Pilosity is less than in
Fineadjustment
Light
the preceding instar but single hairs and forked thin
Base
hairs are present but scarce on the entire surface of the
body. On the dorsal face of the abdomen, two lines of Sign in to download full-size image
five to eight long flexuous (springlike) forked hairs Figure 6.1. Microscope design.
replace the second instar's forked hairs.
A histogram of the diameters of the first thoracic spiracle ● Illuminator: the light source in the base of the
clearly shows three peaks corresponding to the three microscope;
instars (Fig. 23.2). ● Abbe Condensor: a two lens system that collects and
concentrates light from the illuminator and directs it
to the iris diaphragm;
● Iris diaphragm: regulates the amount of light
entering the lens system;
● Mechanical stage: a platform used to place the slide
on which has a hole in the center to let light from the
illuminator pass through. Often contains stage clips
to hold the slide in place;
● Body tube: houses the lens system that magnifies the
specimens;
● Upper end of body tube—oculars/eye pieces: what
you view through;
● Lower end of body tube—nose-piece: revolves and
contains the objectives.
Essentially, a light microscope magnifies small objects
and makes them visible. The science of microscopy is
Sign in to download full-size image based on the following concepts and principles:
FIG. 23.2. Histogram of the diameter (μm) of the first thoracic spiracle of the three larval instars in ● Magnification is simply the enlargement of the
Pheidole pallidula. Hatched columns show worker-biased larvae. Open columns show soldier-biased
specimen. In a compound lens system, each lens
larvae. Numbers above the columns designate instars.
sequentially enlarges or magnifies the specimen;
Soldier larvae are larger, up to 3.5 mm long. Their ● The objective lens magnifies the specimen,
teguments have the same characteristics as the third producing a real image that is then magnified by the
instar worker larvae, and the same spring-shaped hairs ocular lens resulting in the final image;
are present. ● The total magnification can be calculated by
The histograms of the diameter of the first thoracic multiplying the objective lens value by the ocular lens
spiracle are the same for the soldier and third instar value.
worker. Larvae, i.e., the spiracle diameters of the oldest
worker larvae and those of soldier larvae of all ages View chapter Purchase book
belong to the same population (Fig. 23.2). Thus the
soldier larvae are of the third instar: the increased growth
of soldier larvae is accomplished without a
supplementary moult, but by cuticle extension. This
phenomenon is well-known in many arthropods Polarization Microscopy
(Bordereau, 1982).
The time at which soldier larvae become visibly different Randy Wayne, in
from worker larvae was identified in a detailed study of Light and Video Microscopy (Third Edition), 2019
the third instar larvae (Passera, 1973, 1974; Suzzoni et al.,
Reflected Light Polarization Microscopy
1982). When the worker larvae reach 1.30 mm they are
Polarized light microscopes can be used in the reflected
orange, because of the colour of the gut contents. Their
light or epi-illumination mode. In epipolarization
development temporarily ceases on emptying the
microscopy, the light passes through the objective before
hindgut, resulting in the acquisition of a grey colour
it strikes the specimen and then the reflected light is
before pupation (Fig. 23.3), which occurs when the larvae
captured by the same objective lens. Epipolarization
are 1.5 mm long and 0.4 to 0.5 mg in weight. The
microscopes are used for metallurgical work and have
resulting worker pupae develop through the following
also been used in biological work to localize antibodies
stages: first, white pupae with white eyes; second, white
that are conjugated to colloidal gold (Hughes, 1987;
pupae with coloured eyes; then yellow pupae. However,
Hughes et al., 1991; Gao and Cardell, 1994; Gao et al.,
the larvae may continue to grow, and reach 3.5 mm in
1995; Stephenson et al., 1998; Ermert et al., 1998, 2000,
length and 2.5 to 3 mg in weight before emptying the
2001).
hindgut, and pupating as before, but to form soldier
pupae (Fig. 23.3). Thus orientation towards soldiers takes
place when the coloured orange third instar larvae reach View chapter Purchase book
a length slightly over 1.5 mm.

Polarization Microscopy
Randy Wayne, in
Light and Video Microscopy (Second Edition), 2014

Reflected Light Polarization Microscopy

Polarized light microscopes can be used in the reflected
light or epi-illumination mode. In epipolarization
microscopy, the light passes through the objective before
it strikes the specimen and then the reflected light is
captured by the same objective lens. Epipolarization
microscopes are used for metallurgical work, and have
also been used in biological work to localize antibodies
that are conjugated to colloidal gold (Hughes, 1987;
Hughes et al., 1991; Gao and Cardell, 1994; Gao et al.,
1995; Stephenson et al., 1998; Ermert et al., 1998, 2000,
2001Ermert et al., 1998Ermert et al., 2000Ermert et al.,
2001).

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FIG. 23.3. Relationship between weight and length of worker and soldier larvae in Pheidole pallidula.
Abscissa: length in mm; ordinate: weight in mg. ○ = orange worker larvae; • = grey worker larvae; ▪ =
orange soldier larvae; □ = grey soldier larvae; 1 = regression line for worker larvae; 2 = regression line for
soldier larvae; 3 = regression line for orange soldier larvae; 4 = regression line for grey soldier larvae.
(From Suzzoni et al., 1982.)Copyright © 1982

Quantitative Imaging in Cell

The American species, P. bicarinata, shows similar
development: three larval instars with soldier Biology
differentiation at the end of the last instar (Wheeler &
Paul C. Goodwin, in Methods in Cell Biology, 2014
Nijhout, 1981a).
Larval development into soldier is characterized by the Abstract
existence of a pair of mesothoracic wing discs. This The light microscope is an essential tool for the study of
phenomenon was discovered by Wheeler & Nijhout cells, organelles, biomolecules, and subcellular
(1981a) in Pheidole bicarinata and later confirmed in P. dynamics. A paradox exists in microscopy whereby the
pallidula (Suzzoni et al., 1982). higher the needed lateral resolution, the more the image
Imaginal wing discs in Pheidole pallidula do not become is degraded by out-of-focus information. This creates a
apparent until the orange-coloured larvae begin to significant need to generate axial contrast whenever high
differentiate into soldiers (Fig. 23.4). The discs are dense, lateral resolution is required. One strategy for generating
compact masses which have a spherical shape in larvae contrast is to measure or model the optical properties of
and become ellipsoid in the pupae. Their external cells the microscope and to use that model to algorithmically
are rather elongated and tall and the central cells are reverse some of the consequences of high-resolution
more rounded; the nucleus is 3 μ by 5 μm. The imaging. Deconvolution microscopy implements model-
development of the discs is shown in Figure 23.5. From based methods to enable the full diffraction-limited
the orange larvae stage to the grey larvae stage they grow resolution of the microscope to be exploited even in
slightly, the surface area increasing from 0.01 to 0.0135 complex and living specimens.
mm2. During pupation they emerge from the body in the
form of a minute bud behind and above each
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mesothoracic spiracle (Fig. 23.4). Their size then
decreases and by emergence they have disappeared.

Equipment
In Microbiological Analysis of Food and Water, 1998

5.19 Microscopes
Ordinary light microscopes are equipped with three
objective lenses (5 ×/10 ×, 40 ×, and 90/100 ×), and two
ocular (5 ×, 10 ×) lenses. Many other types of microscope
are used for particular microbiological examinations and
may be equipped with a special type of illumination, e.g.
phase contrast, ultraviolet or epifluorescence. Calibration
of fields can be done by using specially calibrated
microslides for counting/estimation of microbes fixed on
slides. A check on the power of UV illumination
produced is important in fluorescent microscopy. For all
Sign in to download full-size image types of microscope, cleaning after use and regular
FIG. 23.4. Wing discs in Pheidole pallidula. A: orange soldier larva; ventral view (whole mount); B: orange
maintenance service is a task for which designated
worker larva; lateral view (whole mount); C: mesothoracic wing disc of an orange soldier larva; D: members of staff are responsible. Maintenance service
Scanning electron micrograph of the thorax of a white soldier pupa; E: Scanning electron micrograph of
the mesothoracic region of the same pupa (soldier); l = leg imaginal disc; w = wing imaginal disc; s =
should be recorded and signed for in a special book.
mesothoracic spiracle.

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Correlative Light and Electron

Microscopy IV
Korbinian Buerger, ... Ralph Witzgall, in
Methods in Cell Biology, 2021

3.5 Fluorescence microscopy of resin sections

Instrumentation:
Inverted light microscope (Zeiss Axiovert 200M) with a
HXP 120 V Compact Light Source (Leistungselektronik
JENA GmbH, Jena, Germany); Fluar 40 ×/1.30 oil
immersion objective (Carl Zeiss AG, Oberkochen,
Germany); cooled pco.edge 4.2 sCMOS camera (PCO AG,
Sign in to download full-size image Kelheim, Germany); VisiView software package (version
4.1.0.4); filter D 350/50 excitation and ET 460/50
FIG. 23.5. Relationship between surface area of mesothoracic discs to development of larvae and pupae
during soldier differentiation in Pheisole pallidula. OL = orange larvae; GL = grey larvae; PP = prepupae;
emission; filter ET 470/40 excitation and ET 525/50
P1 = white pupae with white eyes; P2 = White pupae with black eyes; P3 = yellow pupae; S = queen emission; filter ET 560/40 excitation and ET 630/75
prepupae.
emission.
(From Suzzoni et al. 1982.)Copyright © 1982
Materials:
Metathoracic imaginal wing discs have not been Rhodamine B-labeled gold nanoparticles (Fokkema et al.,
observed in larvae of either species. However, we did 2018); coverslips (cat. no. 0107032, high precision, No.
once find two pairs of wing buds in a pupa of Pheidole 1.5H, Paul Marienfeld GmbH & Co. KG, Lauda-
pallidula infected by Mermis larvae (Nematoda). It has Königshofen, Germany).
already been shown that mermithization causes
important morphological alterations in P. pallidula View chapter Purchase book
(Passera, 1976); when the infecting larvule enters an
orange larva it disturbs the worker/soldier differentiation.
The result is an ant with morphology intermediate
between that of a worker and of a soldier, i.e., an
intercaste. Moreover some intercastes develop a queen- Organic disorders
like character in certain morphological features, such as
the presence of ocelli, gynecoidy of the thorax, and Alan J Carson, ... Tom Brown, in
indeed the presence of two pairs of wing buds. During Companion to Psychiatric Studies (Eighth Edition), 2010
the imaginal ecdysis these buds disappear, as they do in
Pathology and aetiology
healthy soldiers. However, Tinaut & Millan (1981) have
The light microscope reveals ‘spongiform change’ in the
reported the presence of wing stumps in some adult
brain associated with neuronal loss, gliosis and
mermithized intercastes of P. pallidula collected in Spain.
deposition of ‘amyloid’. Immunocytochemistry and direct
Workers of neither of the species possess wing imaginal
biochemical analysis indicate that the amyloid is
discs at any time (Wheeler & Nijhout, 1981a; Suzzoni et
composed of a protease-resistant form of prion protein
al., 1982). This is surprising because, as a general rule,
(PrP). PrP is a membrane-associated neuronal protein
workers of ants have two pairs of wing imaginal discs
coded on the short arm of chromosome 20. In TSEs, PrP
where development is synchronized with leg discs.
is thought to undergo a conformational change to a
However, these wing discs are always smaller than those
disease-associated form, PrPsc, which both renders PrP
of the soldiers and disappear before pupation (Wheeler &
resistant to normal degradation, and confers upon it the
Nijhout, 1981a).
capacity to convert other molecules of PrP to PrPsc. This
process of catalysis gradually results in the toxic
View chapter Purchase book accumulation of PrPsc. In spCJD the initial
conformational change occurs ‘spontaneously’; in
transmitted cases it is catalysed by exogenous PrPsc; and
in inherited cases, a mutation in the PrP gene renders
the molecule more than usually vulnerable to
Recommended publications
spontaneous transformation to PrPsc. This radical ‘prion
hypothesis’ explains how one and the same disorder can
Aquaculture occur in sporadic, infective and inherited forms (Aguzzi
Journal et al 2000).

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