Microscope

A microscope (from Ancient Greek μικρός (mikrós) 'small', and σκοπέω (skopéō) 'to
look (at); examine, inspect') is a laboratory instrument used to examine objects that
are too small to be seen by the naked eye. Microscopy is the science of investigating
small objects and structures using a microscope. Microscopic means being invisible
to the eye unless aided by a microscope.
 Microscope

Uses Small sample

observation

Notable Discovery of cells

experiments

Related items Optical

microscope,
electron
microscope

There are many types of microscopes, and they may be grouped in different ways.
One way is to describe the method an instrument uses to interact with a sample and
produce images, either by sending a beam of light or electrons through a sample in its
optical path, by detecting photon emissions from a sample, or by scanning across
and a short distance from the surface of a sample using a probe The most common
microscope (and the first to be invented) is the optical microscope, which uses lenses
to refract visible light that passed through a thinly sectioned sample to produce an
observable image. Other major types of microscopes are the fluorescence
microscope, electron microscope (both the transmission electron microscope and the
scanning electron microscope) and various types of scanning probe microscopes.[1]

History

18th-century microscopes from

the Musée des Arts et Métiers,
Paris

Although objects resembling lenses date back 4,000 years and there are Greek
accounts of the optical properties of water-filled spheres (5th century BC) followed by
many centuries of writings on optics, the earliest known use of simple microscopes
(magnifying glasses) dates back to the widespread use of lenses in eyeglasses in the
13th century.[2][3][4] The earliest known examples of compound microscopes, which
combine an objective lens near the specimen with an eyepiece to view a real image,
appeared in Europe around 1620.[5] The inventor is unknown, even though many
claims have been made over the years. Several revolve around the spectacle-making
centers in the Netherlands, including claims it was invented in 1590 by Zacharias
Janssen (claim made by his son) or Zacharias' father, Hans Martens, or both,[6][7]
claims it was invented by their neighbor and rival spectacle maker, Hans Lippershey
(who applied for the first telescope patent in 1608),[8] and claims it was invented by
expatriate Cornelis Drebbel, who was noted to have a version in London in 1619.[9][10]
Galileo Galilei (also sometimes cited as compound microscope inventor) seems to
have found after 1610 that he could close focus his telescope to view small objects
and, after seeing a compound microscope built by Drebbel exhibited in Rome in 1624,
[11][12][13]
for the compound microscope Galileo submitted to the Accademia dei Lincei in
1625[14] (Galileo had called it the occhiolino 'little eye'). René Descartes (Dioptrique,
1637) describes microscopes wherein a concave mirror, with its concavity towards
the object, is used, in conjunction with a lens, for illuminating the object, which is
mounted on a point fixing it at the focus of the mirror.[15]

Rise of modern light microscopes

Carl Zeiss binocular

compound microscope, 1914

The first detailed account of the microscopic anatomy of organic tissue based on the
use of a microscope did not appear until 1644, in Giambattista Odierna's L'occhio
della mosca, or The Fly's Eye.[16]

The microscope was still largely a novelty until the 1660s and 1670s when naturalists
in Italy, the Netherlands and England began using them to study biology. Italian
scientist Marcello Malpighi, called the father of histology by some historians of
biology, began his analysis of biological structures with the lungs. The publication in
1665 of Robert Hooke's Micrographia had a huge impact, largely because of its
impressive illustrations. Hooke created tiny lenses of small glass globules made by
fusing the ends of threads of spun glass.[15] A significant contribution came from
Antonie van Leeuwenhoek who achieved up to 300 times magnification using a
simple single lens microscope He sandwiched a very small glass ball lens between
the holes in two metal plates riveted together, and with an adjustable-by-screws
needle attached to mount the specimen.[17] Then, Van Leeuwenhoek re-discovered
red blood cells (after Jan Swammerdam) and spermatozoa, and helped popularise
the use of microscopes to view biological ultrastructure. On 9 October 1676, van
Leeuwenhoek reported the discovery of micro-organisms.[16]

The performance of a compound light microscope depends on the quality and correct
use of the condensor lens system to focus light on the specimen and the objective
lens to capture the light from the specimen and form an image.[5] Early instruments
were limited until this principle was fully appreciated and developed from the late
19th to very early 20th century, and until electric lamps were available as light
sources. In 1893 August Köhler developed a key principle of sample illumination,
Köhler illumination, which is central to achieving the theoretical limits of resolution for
the light microscope. This method of sample illumination produces even lighting and
overcomes the limited contrast and resolution imposed by early techniques of sample
illumination. Further developments in sample illumination came from the discovery of
phase contrast by Frits Zernike in 1953, and differential interference contrast
illumination by Georges Nomarski in 1955; both of which allow imaging of unstained,
transparent samples.

Electron microscopes
 Electron microscope constructed by
Ernst Ruska in 1933

In the early 20th century a significant alternative to the light microscope was
developed, an instrument that uses a beam of electrons rather than light to generate
an image. The German physicist, Ernst Ruska, working with electrical engineer Max
Knoll, developed the first prototype electron microscope in 1931, a transmission
electron microscope (TEM). The transmission electron microscope works on similar
principles to an optical microscope but uses electrons in the place of light and
electromagnets in the place of glass lenses. Use of electrons, instead of light, allows
for much higher resolution.

Development of the transmission electron microscope was quickly followed in 1935

by the development of the scanning electron microscope by Max Knoll.[18] Although
TEMs were being used for research before WWII, and became popular afterwards, the
SEM was not commercially available until 1965.

Transmission electron microscopes became popular following the Second World War.
Ernst Ruska, working at Siemens, developed the first commercial transmission
electron microscope and, in the 1950s, major scientific conferences on electron
microscopy started being held. In 1965, the first commercial scanning electron
microscope was developed by Professor Sir Charles Oatley and his postgraduate
student Gary Stewart, and marketed by the Cambridge Instrument Company as the
One of the latest discoveries made about using an electron microscope is the ability
to identify a virus.[19] Since this microscope produces a visible, clear image of small
organelles, in an electron microscope there is no need for reagents to see the virus or
harmful cells, resulting in a more efficient way to detect pathogens.

Scanning probe microscopes

First atomic force microscope

From 1981 to 1983 Gerd Binnig and Heinrich Rohrer worked at IBM in Zürich,
Switzerland to study the quantum tunnelling phenomenon. They created a practical
instrument, a scanning probe microscope from quantum tunnelling theory, that read
very small forces exchanged between a probe and the surface of a sample. The probe
approaches the surface so closely that electrons can flow continuously between
probe and sample, making a current from surface to probe. The microscope was not
initially well received due to the complex nature of the underlying theoretical
explanations. In 1984 Jerry Tersoff and D.R. Hamann, while at AT&T's Bell
Laboratories in Murray Hill, New Jersey began publishing articles that tied theory to
the experimental results obtained by the instrument. This was closely followed in
1985 with functioning commercial instruments, and in 1986 with Gerd Binnig, Quate,
and Gerber's invention of the atomic force microscope, then Binnig's and Rohrer's
Nobel Prize in Physics for the SPM.[20]

New types of scanning probe microscope have continued to be developed as the

ability to machine ultra-fine probes and tips has advanced.
Fluorescence microscopes

Fluorescence microscope
with the filter cube turret
above the objective lenses,
coupled with a camera

The most recent developments in light microscope largely centre on the rise of
fluorescence microscopy in biology.[21] During the last decades of the 20th century,
particularly in the post-genomic era, many techniques for fluorescent staining of
cellular structures were developed.[21] The main groups of techniques involve
targeted chemical staining of particular cell structures, for example, the chemical
compound DAPI to label DNA, use of antibodies conjugated to fluorescent reporters,
see immunofluorescence, and fluorescent proteins, such as green fluorescent
protein.[22] These techniques use these different fluorophores for analysis of cell
structure at a molecular level in both live and fixed samples.

The rise of fluorescence microscopy drove the development of a major modern

microscope design, the confocal microscope. The principle was patented in 1957 by
Marvin Minsky, although laser technology limited practical application of the
technique. It was not until 1978 when Thomas and Christoph Cremer developed the
first practical confocal laser scanning microscope and the technique rapidly gained
popularity through the 1980s.
Super resolution microscopes
Much current research (in the early 21st century) on optical microscope techniques is
focused on development of superresolution analysis of fluorescently labelled
samples. Structured illumination can improve resolution by around two to four times
and techniques like stimulated emission depletion (STED) microscopy are
approaching the resolution of electron microscopes.[23] This occurs because the
diffraction limit is occurred from light or excitation, which makes the resolution must
be doubled to become super saturated. Stefan Hell was awarded the 2014 Nobel
Prize in Chemistry for the development of the STED technique, along with Eric Betzig
and William Moerner who adapted fluorescence microscopy for single-molecule
visualization.[24]

X-ray microscopes
X-ray microscopes are instruments that use electromagnetic radiation usually in the
soft X-ray band to image objects. Technological advances in X-ray lens optics in the
early 1970s made the instrument a viable imaging choice.[25] They are often used in
tomography (see micro-computed tomography) to produce three dimensional images
of objects, including biological materials that have not been chemically fixed.
Currently research is being done to improve optics for hard X-rays which have greater
penetrating power.[25]

Types
 Types of microscopes illustrated by the principles
of their beam paths

Evolution of spatial resolution achieved with optical,

transmission (TEM) and aberration-corrected
electron microscopes (ACTEM)[26]

Microscopes can be separated into several different classes. One grouping is based
on what interacts with the sample to generate the image, i.e., light or photons (optical
microscopes), electrons (electron microscopes) or a probe (scanning probe
microscopes). Alternatively, microscopes can be classified based on whether they
analyze the sample via a scanning point (confocal optical microscopes, scanning
electron microscopes and scanning probe microscopes) or analyze the sample all at
once (wide field optical microscopes and transmission electron microscopes).

Wide field optical microscopes and transmission electron microscopes both use the
theory of lenses (optics for light microscopes and electromagnet lenses for electron
microscopes) in order to magnify the image generated by the passage of a wave
transmitted through the sample, or reflected by the sample. The waves used are
electromagnetic (in optical microscopes) or electron beams (in electron
microscopes). Resolution in these microscopes is limited by the wavelength of the
radiation used to image the sample, where shorter wavelengths allow for a higher
resolution.[21]

Scanning optical and electron microscopes, like the confocal microscope and
scanning electron microscope, use lenses to focus a spot of light or electrons onto
the sample then analyze the signals generated by the beam interacting with the
sample. The point is then scanned over the sample to analyze a rectangular region.
Magnification of the image is achieved by displaying the data from scanning a
physically small sample area on a relatively large screen. These microscopes have
the same resolution limit as wide field optical, probe, and electron microscopes.

Scanning probe microscopes also analyze a single point in the sample and then scan
the probe over a rectangular sample region to build up an image. As these
microscopes do not use electromagnetic or electron radiation for imaging they are
not subject to the same resolution limit as the optical and electron microscopes
described above.

Optical microscope
The most common type of microscope (and the first invented) is the optical
microscope. This is an optical instrument containing one or more lenses producing
an enlarged image of a sample placed in the focal plane. Optical microscopes have
refractive glass (occasionally plastic or quartz), to focus light on the eye or on to
another light detector. Mirror-based optical microscopes operate in the same manner.
Typical magnification of a light microscope, assuming visible range light, is up to
1,250× with a theoretical resolution limit of around 0.250 micrometres or
250 nanometres.[21] This limits practical magnification to ~1,500×. Specialized
techniques (e.g., scanning confocal microscopy, Vertico SMI) may exceed this
magnification but the resolution is diffraction limited. The use of shorter wavelengths
of light, such as ultraviolet, is one way to improve the spatial resolution of the optical
i d i h th fi ld i ti l i
Sarfus is a recent optical technique that increases the sensitivity of a standard optical
microscope to a point where it is possible to directly visualize nanometric films (down
to 0.3 nanometre) and isolated nano-objects (down to 2 nm-diameter). The technique
is based on the use of non-reflecting substrates for cross-polarized reflected light
microscopy.

Ultraviolet light enables the resolution of microscopic features as well as the imaging
of samples that are transparent to the eye. Near infrared light can be used to visualize
circuitry embedded in bonded silicon devices, since silicon is transparent in this
region of wavelengths.

In fluorescence microscopy many wavelengths of light ranging from the ultraviolet to

the visible can be used to cause samples to fluoresce, which allows viewing by eye or
with specifically sensitive cameras.

Unstained cells viewed by typical

brightfield (left) compared to phase-
contrast microscopy (right)

Phase-contrast microscopy is an optical microscopic illumination technique in which

small phase shifts in the light passing through a transparent specimen are converted
into amplitude or contrast changes in the image.[21] The use of phase contrast does
not require staining to view the slide. This microscope technique made it possible to
study the cell cycle in live cells.

The traditional optical microscope has more recently evolved into the digital
microscope. In addition to, or instead of, directly viewing the object through the
eyepieces, a type of sensor similar to those used in a digital camera is used to obtain
an image, which is then displayed on a computer monitor. These sensors may use
CMOS or charge-coupled device (CCD) technology, depending on the application.

Digital microscopy with very low light levels to avoid damage to vulnerable biological
samples is available using sensitive photon-counting digital cameras. It has been
demonstrated that a light source providing pairs of entangled photons may minimize
the risk of damage to the most light-sensitive samples In this application of ghost
imaging to photon-sparse microscopy, the sample is illuminated with infrared
photons, each of which is spatially correlated with an entangled partner in the visible
band for efficient imaging by a photon-counting camera.[27]

Modern transmission
electron microscope

Electron microscope

Transmission electron micrograph of

a dividing cell undergoing cytokinesis

The two major types of electron microscopes are transmission electron microscopes
(TEMs) and scanning electron microscopes (SEMs).[21][22] They both have series of
electromagnetic and electrostatic lenses to focus a high energy beam of electrons on
a sample. In a TEM the electrons pass through the sample, analogous to basic optical
microscopy.[21] This requires careful sample preparation, since electrons are
scattered strongly by most materials.[22] The samples must also be very thin (below
100 nm) in order for the electrons to pass through it.[21][22] Cross-sections of cells
stained with osmium and heavy metals reveal clear organelle membranes and
proteins such as ribosomes.[22] With a 0.1 nm level of resolution, detailed views of
viruses (20 – 300 nm) and a strand of DNA (2 nm in width) can be obtained.[22] In
contrast, the SEM has raster coils to scan the surface of bulk objects with a fine
electron beam. Therefore, the specimen do not necessarily need to be sectioned, but
coating with a nanometric metal or carbon layer may be needed for nonconductive
samples.[21] SEM allows fast surface imaging of samples, possibly in thin water vapor
to prevent drying.[21][22]

Scanning probe
The different types of scanning probe microscopes arise from the many different
types of interactions that occur when a small probe is scanned over and interacts
with a specimen. These interactions or modes can be recorded or mapped as
function of location on the surface to form a characterization map. The three most
common types of scanning probe microscopes are atomic force microscopes (AFM),
near-field scanning optical microscopes (NSOM or SNOM, scanning near-field optical
microscopy), and scanning tunneling microscopes (STM).[28] An atomic force
microscope has a fine probe, usually of silicon or silicon nitride, attached to a
cantilever; the probe is scanned over the surface of the sample, and the forces that
cause an interaction between the probe and the surface of the sample are measured
and mapped. A near-field scanning optical microscope is similar to an AFM but its
probe consists of a light source in an optical fiber covered with a tip that has usually
an aperture for the light to pass through. The microscope can capture either
transmitted or reflected light to measure very localized optical properties of the
surface, commonly of a biological specimen. Scanning tunneling microscopes have a
metal tip with a single apical atom; the tip is attached to a tube through which a
current flows.[29] The tip is scanned over the surface of a conductive sample until a
tunneling current flows; the current is kept constant by computer movement of the tip
and an image is formed by the recorded movements of the tip.[28]
 Leaf surface viewed by a scanning
electron microscope

Other types
Scanning acoustic microscopes use sound waves to measure variations in acoustic
impedance. Similar to Sonar in principle, they are used for such jobs as detecting
defects in the subsurfaces of materials including those found in integrated circuits.
On February 4, 2013, Australian engineers built a "quantum microscope" which
provides unparalleled precision.[30]

Mobile apps
Mobile app microscopes can optionally be used as optical microscope when the
flashlight is activated. However, mobile app microscopes are harder to use due to
visual noise, are often limited to 40x, and the resolution limits of the camera lens
itself.

See also

Fluorescence interference contrast

 microscopy
Laser capture microdissection
Microscope image processing
Microscope slide
Multifocal plane microscopy
Royal Microscopical Society

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External links

Wikimedia Commons has media

related to Microscopes.
Milestones in Light Microscopy (htt
p://www.nature.com/milestones/mi
lelight/index.html) , Nature
Publishing

FAQ on Optical Microscopes (http

s://web.archive.org/web/20090404
024608/http://www.micro.magnet.f
su.edu/primer/faq.html) (archived
4 April 2009)
Nikon MicroscopyU, tutorials from
Nikon (http://www.microscopyu.co
m)
 Molecular Expressions : Exploring
the World of Optics and Microscopy,
Florida State University. (http://micr
o.magnet.fsu.edu/index.html)

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