Optical microscope - AbsoluteAstronomy.com

The optical microscope, often referred to as the "light microscope", is a type of microscope

Microscope

A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

which uses visible light

Visible spectrum

The visible spectrum is the portion of the electromagnetic spectrum that is visible to the human eye. Electromagnetic radiation in this range of wavelengths is called visible light or simply light. A typical human eye will respond to wavelengths from about 390 to 750 nm. In terms of...

and a system of lenses

Lens (optics)

A lens is an optical device with perfect or approximate axial symmetry which transmits and refracts light, converging or diverging the beam. A simple lens consists of a single optical element...

to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the 17th century. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution

Optical resolution

Optical resolution describes the ability of an imaging system to resolve detail in the object that is being imaged.An imaging system may have many individual components including a lens and recording and display components...

and sample contrast

Contrast (vision)

Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background. In visual perception of the real world, contrast is determined by the difference in the color and brightness of the object and other objects within the same field of view...

. Historically optical microscopes were easy to develop and are popular because they use visible light so the sample can be directly observed by eye

Human eye

The human eye is an organ which reacts to light for several purposes. As a conscious sense organ, the eye allows vision. Rod and cone cells in the retina allow conscious light perception and vision including color differentiation and the perception of depth...

.

The image from an optical microscope can be captured by normal light-sensitive cameras to generate a micrograph

Micrograph

A micrograph or photomicrograph is a photograph or digital image taken through a microscope or similar device to show a magnified image of an item.Micrographs are widely used in all fields of microscopy.-Photomicrograph:...

. Originally images were captured by photographic film

Photographic film

Photographic film is a sheet of plastic coated with an emulsion containing light-sensitive silver halide salts with variable crystal sizes that determine the sensitivity, contrast and resolution of the film...

but modern developments in CMOS

CMOS

Complementary metal–oxide–semiconductor is a technology for constructing integrated circuits. CMOS technology is used in microprocessors, microcontrollers, static RAM, and other digital logic circuits...

and charge-coupled device

Charge-coupled device

A charge-coupled device is a device for the movement of electrical charge, usually from within the device to an area where the charge can be manipulated, for example conversion into a digital value. This is achieved by "shifting" the signals between stages within the device one at a time...

(CCD) cameras allow the capture of digital image

Digital image

A digital image is a numeric representation of a two-dimensional image. Depending on whether or not the image resolution is fixed, it may be of vector or raster type...

s. Purely digital microscope

Digital microscope

A digital microscope is a variation of a traditional optical microscope that uses optics and a charge-coupled device camera to output a digital image to a monitor, sometimes by means of software running on a computer. A digital microscope differs from an optical microscope in that there is no...

s are now available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eyepieces.

Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy

Transmission electron microscopy

Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...

Optical configurations

There are two basic configurations of the conventional optical microscope, the simple (one lens) and compound (many lenses). The vast majority of modern research

Research

Research can be defined as the scientific search for knowledge, or as any systematic investigation, to establish novel facts, solve new or existing problems, prove new ideas, or develop new theories, usually using a scientific method...

microscopes are compound microscopes while some cheaper commercial digital microscope

Digital microscope

s are simple single lens microscopes. A magnifying glass

Magnifying glass

A magnifying glass is a convex lens that is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle ....

is, in essence, a basic single lens microscope. In general microscope optics are static; to focus at different focal depths the lens to sample distance is adjusted and to get a wider or narrower field of view a different magnification objective lens must be used. Most modern research microscopes also have a separate set of optics for illuminating the sample.

Single lens (simple) microscope

A simple microscope is a microscope that uses only one lens for magnification, and is the original design of light microscope. Van Leeuwenhoek's microscopes consisted of a small, single converging lens mounted on a brass plate, with a screw mechanism to hold the sample or specimen to be examined. Demonstrations by British microscopist have images from such basic instruments.
Though now considered primitive, the use of a single, convex lens for viewing is still found in simple magnification devices, such as the magnifying glass

Magnifying glass

A magnifying glass is a convex lens that is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle ....

, and the loupe

Loupe

A loupe is a simple, small magnification device used to see small details more closely. Unlike a magnifying glass, a loupe does not have an attached handle, and its focusing lens are contained in an opaque cylinder or cone. Loupes are also called hand lenses .- Optics :Three basic types of loupes...

Compound microscope

A compound microscope is a microscope which uses multiple lenses to collect light from the sample and then a separate set of lenses to focus the light into the eye or camera. Compound microscopes are heavier, larger and more expensive than simple microscopes due to the increased number of lenses used in construction. The main advantages of multiple lenses are improved numerical aperture (see resolution limit below), reduced chromatic aberration and exchangeable objective lenses to adjust the magnification. A compound microscope also makes more advanced illumination setups, such as phase contrast.

History

Invention

It is difficult to say who invented the compound microscope. Dutch

Dutch people

The Dutch people are an ethnic group native to the Netherlands. They share a common culture and speak the Dutch language. Dutch people and their descendants are found in migrant communities worldwide, notably in Suriname, Chile, Brazil, Canada, Australia, South Africa, New Zealand, and the United...

spectacle-makers Hans Janssen and his son Zacharias Janssen

Zacharias Janssen

Zacharias Jansen was a Dutch spectacle-maker from Middelburg associated with the invention of the first optical telescope. Jansen is sometimes also credited for inventing the first truly compound microscope...

are often said to have invented the first compound microscope in 1590, but this was a declaration made by Zacharias Janssen himself during the mid 17th century. The date is unlikely, as it has been shown that Zacharias Janssen actually was born around 1590. Another favorite for the title of 'inventor of the microscope' was Galileo Galilei

Galileo Galilei

Galileo Galilei , was an Italian physicist, mathematician, astronomer, and philosopher who played a major role in the Scientific Revolution. His achievements include improvements to the telescope and consequent astronomical observations and support for Copernicanism...

. He developed an occhiolino or compound microscope with a convex and a concave lens in 1609. Galileo's microscope was celebrated in the Accademia dei Lincei

Accademia dei Lincei

The Accademia dei Lincei, , is an Italian science academy, located at the Palazzo Corsini on the Via della Lungara in Rome, Italy....

in 1624 and was the first such device to be given the name "microscope" a year later by fellow Lincean Giovanni Faber

Giovanni Faber

Giovanni Faber was a German papal doctor, botanist and art collector, originally from Bamberg in Bavaria, who lived in Rome from 1598. He was curator of the Vatican botanical garden, a member and the secretary of the Accademia dei Lincei. He acted throughout his career as a political broker...

. Faber coined the name from the Greek

Greek language

Greek is an independent branch of the Indo-European family of languages. Native to the southern Balkans, it has the longest documented history of any Indo-European language, spanning 34 centuries of written records. Its writing system has been the Greek alphabet for the majority of its history;...

words μικρόν (micron) meaning "small", and σκοπεῖν (skopein) meaning "to look at", a name meant to be analogous with "telescope

Telescope

A telescope is an instrument that aids in the observation of remote objects by collecting electromagnetic radiation . The first known practical telescopes were invented in the Netherlands at the beginning of the 1600s , using glass lenses...

", another word coined by the Linceans.

Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular system in the late 17th century that was achromatically

Achromatic lens

An achromatic lens or achromat is a lens that is designed to limit the effects of chromatic and spherical aberration. Achromatic lenses are corrected to bring two wavelengths into focus in the same plane....

corrected, and therefore a huge step forward in microscope development. The Huygens ocular is still being produced to this day, but suffers from a small field size, and other minor problems.

Popularisation

Anton van Leeuwenhoek

Antonie Philips van Leeuwenhoek was a Dutch tradesman and scientist from Delft, Netherlands. He is commonly known as "the Father of Microbiology", and considered to be the first microbiologist...

(1632–1723) is credited with bringing the microscope to the attention of biologists, even though simple magnifying lenses were already being produced in the 16th century. Van Leeuwenhoek's home-made microscopes were very small simple instruments, with a single, yet strong lens. They were awkward in use, but enabled van Leeuwenhoek to see detailed images. It took about 150 years of optical development before the compound microscope was able to provide the same quality image as van Leeuwenhoek's simple microscopes, due to difficulties in configuring multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the inventor of the microscope.

Lighting techniques

While basic microscope technology and optics have been available for over 400 years it is much more recently that techniques in sample illumination were developed to generate the high quality images seen today.

In August 1893 August Köhler developed Köhler illumination

Köhler illumination

Köhler illumination is a method of specimen illumination used for transmitted and reflected light optical microscopy. Köhler illumination acts to generate an extremely even illumination of the sample and ensures that an image of the illumination source is not visible in the resulting image...

. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Before development of Köhler illumination the image of the light source, for example a lightbulb filament, was always visible in the image of the sample.

The Nobel Prize

Nobel Prize

The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...

in physics was awarded to Fritz Zernike in 1953 for his development of phase contrast illumination which allows imaging of transparent samples. By using interference

Interference

In physics, interference is a phenomenon in which two waves superpose to form a resultant wave of greater or lower amplitude. Interference usually refers to the interaction of waves that are correlated or coherent with each other, either because they come from the same source or because they have...

rather than absorption

Absorption (electromagnetic radiation)

In physics, absorption of electromagnetic radiation is the way by which the energy of a photon is taken up by matter, typically the electrons of an atom. Thus, the electromagnetic energy is transformed to other forms of energy for example, to heat. The absorption of light during wave propagation is...

of light, extremely transparent samples, such as live mammalian cells, can be imaged without having to use staining techniques. Just two years later, in 1955, George Nomarski published the theory for differential interference contrast microscopy, another interference

Interference

-based technique for imaging transparent samples.

Fluorescence microscopy

Modern biological microscopy depends heavily on the development of fluorescent probe

Probe

- Science and technology :* Medical equipment** Anal probe** Endoscope** Proctoscope* Atom probe, an atomic-resolution microscope* Hybridization probe or chemical probe, used in molecular biology...

s for specific structures within a cell. In contrast to normal transilluminated light microscopy in fluorescence microscopy the sample is illuminated through the objective lens with a narrow set of wavelengths of light. This light interacts with fluorophores in the sample which then emit light of a longer wavelength

Wavelength

In physics, the wavelength of a sinusoidal wave is the spatial period of the wave—the distance over which the wave's shape repeats.It is usually determined by considering the distance between consecutive corresponding points of the same phase, such as crests, troughs, or zero crossings, and is a...

. It is this emitted light which makes up the image.

Since the mid 20th century chemical fluorescent stains, such as DAPI

DAPI

DAPI or 4',6-diamidino-2-phenylindole is a fluorescent stain that binds strongly to A-T rich regions in DNA. It is used extensively in fluorescence microscopy...

which binds to DNA

DNA

Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...

, have been used to label specific structures within the cell. More recent developments include immunofluorescence

Immunofluorescence

Immunofluorescence is a technique used for light microscopy with a fluorescence microscope and is used primarily on biological samples. This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows...

, which uses fluorescently labelled antibodies to recognise specific proteins within a sample, and fluorescent proteins like GFP

Green fluorescent protein

The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...

which a live cell can express

Protein expression

Protein expression is a subcomponent of gene expression. It consists of the stages after DNA has been translated into polypeptide chains, which are ultimately folded into proteins...

making it fluorescent.

Components

All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path, listed here in the order the light travels through them:
In addition the vast majority of microscopes have the same 'structural' components:

Ocular lens (eyepiece) (1)
Objective turret or Revolver or Revolving nose piece (to hold multiple objective lenses) (2)
Objective
Objective (optics)
In an optical instrument, the objective is the optical element that gathers light from the object being observed and focuses the light rays to produce a real image. Objectives can be single lenses or mirrors, or combinations of several optical elements. They are used in microscopes, telescopes,...

(3)
Focus wheel to move the stage (4 – coarse adjustment, 5 – fine adjustment)
Frame (6)
Light source, a light
Light
Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...

or a mirror
Mirror
A mirror is an object that reflects light or sound in a way that preserves much of its original quality prior to its contact with the mirror. Some mirrors also filter out some wavelengths, while preserving other wavelengths in the reflection...

(7)
Diaphragm and condenser lens (8)
Stage (to hold the sample) (9)

These entries are numbered according to the image on the right.

Eyepiece (ocular)

The eyepiece, or ocular

Eyepiece

An eyepiece, or ocular lens, is a type of lens that is attached to a variety of optical devices such as telescopes and microscopes. It is so named because it is usually the lens that is closest to the eye when someone looks through the device. The objective lens or mirror collects light and brings...

, is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 2×, 5× and 10×. In some high performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance. This occurs most commonly with apochromat

Apochromat

An apochromat, or apochromatic lens , is a photographic or other lens that has better correction of chromatic and spherical aberration than the much more common achromat lenses.-Explanation:...

ic objectives.

Objective turret or Revolver or Revolving nose piece

Objective turret or Revolver is the part that holds the set of objective lenses, it allows to change them.

Objective

At the lower end of a typical compound optical microscope there are one or more objective lenses that collect light from the sample. The objective is usually in a cylinder housing containing a glass single or multi-element compound lens. Typically there will be around three objective lenses screwed into a circular nose piece which may be rotated to select the required objective lens. These arrangements are designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in focus

Focus (optics)

In geometrical optics, a focus, also called an image point, is the point where light rays originating from a point on the object converge. Although the focus is conceptually a point, physically the focus has a spatial extent, called the blur circle. This non-ideal focusing may be caused by...

. Microscope objectives are characterized by two parameters, namely, magnification

Magnification

Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called "magnification"...

and numerical aperture

Numerical aperture

In optics, the numerical aperture of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the property that it is constant for a beam as it goes from one...

. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal length

Focal length

The focal length of an optical system is a measure of how strongly the system converges or diverges light. For an optical system in air, it is the distance over which initially collimated rays are brought to a focus...

s of about 40 to 2 mm, respectively. Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field

Depth of field

In optics, particularly as it relates to film and photography, depth of field is the distance between the nearest and farthest objects in a scene that appear acceptably sharp in an image...

in the resulting image. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance.

Oil immersion objective

Some microscopes make use of oil-immersion objectives or water-immersion objectives for greater resolution at high magnification. These are used with index-matching material

Index-matching material

In optics and fiber optics, an index-matching material is a substance, usually a liquid, cement , or gel, which has an index of refraction that closely approximates that of an optical element or fiber, and is used to reduce Fresnel reflection at the surface of the element.In fiber optics and...

such as immersion oil or water and a matched cover slip between the objective lens and the sample. The refractive index of the index-matching material is higher than air allowing the objective lens to have a larger numerical aperture (greater than 1) so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. Numerical apertures as high as 1.6 can be achieved. The larger numerical aperture allows collection of more light making detailed observation of smaller details possible. An oil immersion lens usually has a magnification of 40 to 100×.

Focus wheels

Adjustment wheels move the stage up and down with separate adjustment for coarse and fine focussing. The same controls enable the microscope to adjust to specimens of different thickness. In older designs of microscopes, the focus adjustment wheels move the microscope tube up or down relative to the stand and had a fixed stage.

Frame

The whole of the optical assembly is traditionally attached to a rigid arm which in turn is attached to a robust U shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the viewing angle to be adjusted.

The frame provides a mounting point for various microscope controls. Normally this will include controls for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine focus. Other features may be lamp controls and/or controls for adjusting the condenser.

Light source

Many sources of light can be used. At its simplest, daylight is directed via a mirror

Mirror

A mirror is an object that reflects light or sound in a way that preserves much of its original quality prior to its contact with the mirror. Some mirrors also filter out some wavelengths, while preserving other wavelengths in the reflection...

. Most microscopes, however, have their own adjustable and controllable light source – often a halogen

Halogen

The halogens or halogen elements are a series of nonmetal elements from Group 17 IUPAC Style of the periodic table, comprising fluorine , chlorine , bromine , iodine , and astatine...

lamp, although illumination using LED

LEd

LEd is a TeX/LaTeX editing software working under Microsoft Windows. It is a freeware product....

s and laser

Laser

A laser is a device that emits light through a process of optical amplification based on the stimulated emission of photons. The term "laser" originated as an acronym for Light Amplification by Stimulated Emission of Radiation...

s are becoming a more common provision.

Condenser

The condenser is a lens designed to focus light from the illumination source onto the sample. The condenser may also include other features, such as a diaphragm

Diaphragm (optics)

In optics, a diaphragm is a thin opaque structure with an opening at its center. The role of the diaphragm is to stop the passage of light, except for the light passing through the aperture...

and/or filters, to manage the quality and intensity of the illumination. For illumination techniques like dark field, phase contrast and differential interference contrast microscopy additional optical components must be precisely aligned in the light path.

Stage

The stage is a platform below the objective which supports the specimen being viewed. In the center of the stage is a hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides

Microscope slide

A microscope slide is a thin flat piece of glass, typically 75 by 25 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured on the slide, and then both are inserted together in the microscope for viewing...

(rectangular glass plates with typical dimensions of 25×75 mm, on which the specimen is mounted).

At magnifications higher than 100x moving a slide by hand is not practical. A mechanical stage, typical of medium and higher priced microscopes, allows tiny movements of the slide via control knobs that reposition the sample/slide as desired. If a microscope did not originally have a mechanical stage it may be possible to add one.

All stages move up and down for focus. With a mechanical stage slides move on two horizontal axes for positioning the specimen to examine specimen details.

Focusing starts at lower magnification in order to center the specimen by the user on the stage. Moving to a higher magnification requires the stage to be moved higher vertically for re-focus at the higher magnification and may also require slight horizontal specimen position adjustment. Horizontal specimen position adjustments are the reason for having a mechanical stage.

Due to the difficulty in preparing specimens and mounting them on slides, for children it's best to begin with prepared slides that are centered and focus easily regardless of the focus level used.

Magnification

The actual power or magnification

Magnification

Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called "magnification"...

of a compound optical microscope is the product of the powers of the ocular (eyepiece

Eyepiece

) and the objective lens. The maximum normal magnifications of the ocular and objective are 10× and 100× respectively giving a final magnification of 1000×.

Magnification and micrographs

When using a camera to capture a micrograph

Micrograph

the effective magnification of the image must take into account the size of the image. This is independent of whether it is on a print from a film negative or displayed digitally on a computer screen.

In the case of photographic film cameras the calculation is simple; the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor of the film print relative to the negative. A typical value of the enlargement factor is around 5× (for the case of 35mm film and a 15x10 cm (6×4 inch) print).

In the case of digital cameras the size of the pixels in the CMOS

CMOS

or CCD

Charge-coupled device

detector and the size of the pixels on the screen have to be known. The enlargement factor from the detector to the pixels on screen can then be calculated. As with a film camera the final magnification is the product of: the objective lens magnification, the camera optics magnification and the enlargement factor.

Operation

The optical components of a modern microscope are very complex and for a microscope to work well, the whole optical path has to be very accurately set up and controlled. Despite this, the basic operating principles of a microscope are quite simple.

The objective lens is, at its simplest, a very high powered magnifying glass i.e. a lens with a very short focal length. This is brought very close to the specimen being examined so that the light from the specimen comes to a focus about 160 mm inside the microscope tube. This creates an enlarged image of the subject. This image is inverted and can be seen by removing the eyepiece and placing a piece of tracing paper over the end of the tube. By carefully focusing a brightly lit specimen, a highly enlarged image can be seen. It is this real image

Real image

In optics, a real image is a representation of an object in which the perceived location is actually a point of convergence of the rays of light that make up the image. If a screen is placed in the plane of a real image the image will generally become visible on the screen...

that is viewed by the eyepiece lens that provides further enlargement.

In most microscopes, the eyepiece is a compound lens, with one component lens near the front and one near the back of the eyepiece tube. This forms an air-separated couplet.
In many designs, the virtual image

Virtual image

In optics, a virtual image is an image in which the outgoing rays from a point on the object always diverge. It will appear to converge in or behind the optical device . A simple example is a flat mirror where the image of oneself is perceived at twice the distance from oneself to the mirror...

comes to a focus between the two lenses of the eyepiece, the first lens bringing the real image to a focus and the second lens enabling the eye to focus on the virtual image.

In all microscopes the image is intended to be viewed with the eyes focused at infinity (mind that the position of the eye in the above figure is determined by the eye's focus). Headaches and tired eyes after using a microscope are usually signs that the eye is being forced to focus at a close distance rather than at infinity.

The essential principle of the microscope is that an objective lens with very short focal length (often a few mm) is used to form a highly magnified real image of the object. Here, the quantity of interest is linear magnification, and this number is generally inscribed on the objective lens casing. In practice, today, this magnification is carried out by means
of two lenses: the objective lens which creates an image at infinity, and a second weak tube lens which then forms a real image in its focal plane.

Illumination techniques

Many techniques are available which modify the light path to generate an improved contrast

Contrast (vision)

image from a sample. Major techniques for generating increased contrast from the sample include cross-polarized light

Polarized light microscopy

Polarized light microscopy can mean any of a number of optical microscopy techniques involving polarized light. Simple techniques include illumination of the sample with polarized light. Directly transmitted light can, optionally, be blocked with a polariser orientated at 90 degrees to the...

, dark field, phase contrast and differential interference contrast illumination. A recent technique (Sarfus

Sarfus

Sarfus is an optical quantitative imaging technique based on the association of:*an upright or inverted optical microscope in crossed polarization configuration and*specific supporting plates - called surfs - on which the sample to observe is deposited....

) combines cross-polarized light

Polarized light microscopy

and specific contrast-enhanced slides for the visualization of nanometric samples.

Other techniques

Modern microscopes allow more than just observation of transmitted light image of a sample; there are many techniques which can be used to extract other kinds of data. Most of these require additional equipment in addition to a basic compound microscope.

Reflected light, or incident, illumination (for analysis of surface structures)
Fluorescence microscopy, both:
Epifluorescence microscopy
Confocal microscopy
Confocal microscopy
Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane. It enables the reconstruction of...
Microspectroscopy (where a UV-visible spectrophotometer is integrated with an optical microscope)
Ultraviolet microscopy
Near-Infrared microscopy
Multiple Transmission Microscopy for contrast enhancement and aberration reduction.
Automation (for automatic scanning of a large sample or image capture)

Applications

Optical microscopy is used extensively in microelectronics, nanophysics, biotechnology, pharmaceutic research, mineralogy and microbiology.

Optical microscopy is used for medical diagnosis

Medical diagnosis

Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...

, the field being termed histopathology

Histopathology

Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease...

when dealing with tissues, or in smear tests on free cells or tissue fragments.

In industrial use, binocular microscopes are common. Aside from applications needing true depth perception

Stereo microscope

The stereo or dissecting microscope is an optical microscope variant designed for low magnification observation or a sample using incident light illumination rather than transillumination. It uses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing...

, the use of dual eyepieces reduces eye strain

Asthenopia

Asthenopia or eye strain is an ophthalmological condition that manifests itself through nonspecific symptoms such as fatigue, pain in or around the eyes, blurred vision, headache and occasional double vision...

associated with long workdays at a microscopy station
. In certain applications, long-working-distance or long-focus microscopes are beneficial. An item may need to be examined behind a window

Optical window

The meaning of this term depends on the context:* In astronomy, the optical window is the optical portion of the electromagnetic spectrum that passes through the atmosphere all the way to the ground...

, or industrial subjects may be a hazard to the objective. Such optics resemble telescopes with close-focus capabilities.

Optical microscope variants

There are many variants of the basic compound optical microscope design for specialized purposes. Some of these are physical design differences allowing specialization for certain purposes:

Stereo microscope
Stereo microscope
The stereo or dissecting microscope is an optical microscope variant designed for low magnification observation or a sample using incident light illumination rather than transillumination. It uses two separate optical paths with two objectives and two eyepieces to provide slightly different viewing...

, a low powered microscope which provides a stereoscopic view of the sample, commonly used for dissection.
Comparison microscope
Comparison microscope
A comparison microscope is a device used to analyze side-by-side specimens. It consists of two microscopes connected by an optical bridge, which results in a split view window enabling two separate objects to be viewed simultaneously...

, which has two separate light paths allowing direct comparison of two samples via one image in each eye.
Inverted microscope
Inverted microscope
An inverted microscope is a microscope with its light source and condenser on the top, above the stage pointing down, while the objectives and turret are below the stage pointing up. It was invented in 1850 by J. Lawrence Smith, a faculty member of Tulane University...

, for studying samples from below; useful for cell cultures in liquid.
Student microscope
Student microscope
A Student microscope is a low power, durable optical microscope typically sold in bulk for use in school science classes. Although university science students use microscopes, the term typically refers to the type of instrument used in primary and secondary schools...

, designed for low cost, durability, and ease of use.
Fiber optic connector inspection microscope, designed for connector end-face inspection

Other microscope variants are designed for different illumination techniques:

Petrographic microscope
Petrographic microscope
A petrographic microscope is a type of optical microscope used in petrology and optical mineralogy to identify rocks and minerals in thin sections. The microscope is used in optical mineralogy and petrography, a branch of petrology which focuses on detailed descriptions of rocks...

, whose design usually includes a polarizing filter, rotating stage and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation.
Polarizing microscope, similar to the petrographic microscope.
Phase contrast microscope, which applies the phase contrast illumination method.
Epifluorescence microscope, designed for analysis of samples which include fluorophores.
Confocal microscope, a widely used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence.

Digital microscope

A digital microscope

Digital microscope

is a microscope equipped with a digital camera

Digital camera

A digital camera is a camera that takes video or still photographs, or both, digitally by recording images via an electronic image sensor. It is the main device used in the field of digital photography...

allowing observation of a sample via a computer

Computer

A computer is a programmable machine designed to sequentially and automatically carry out a sequence of arithmetic or logical operations. The particular sequence of operations can be changed readily, allowing the computer to solve more than one kind of problem...

. Microscopes can also be partly or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and quantitaton of a fluorescent or histological

Histology

Histology is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope...

stain.

Low-powered digital microscopes, USB microscope

USB microscope

A USB microscope is a low-powered digital microscope which connects to a computer, normally via a USB port. They are widely available at low cost commercially. In essence USB microscopes are a webcam with a high-powered macro lens and generally do not use transillumination but rely on incident...

s, are also commercially available. These are essentially webcam

Webcam

A webcam is a video camera that feeds its images in real time to a computer or computer network, often via USB, ethernet, or Wi-Fi.Their most popular use is the establishment of video links, permitting computers to act as videophones or videoconference stations. This common use as a video camera...

s with a high-powered macro lens and generally do not use transillumination

Transillumination

Transillumination is the technique of sample illumination by transmission of light through the sample. Transillumination is used in a variety of methods of imaging.-Microscopy:...

. The camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor. They offer modest magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. The lack of illumination optics limits their use in a similar manner to stereo microscope

Stereo microscope

Limitations

At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction

Diffraction

Diffraction refers to various phenomena which occur when a wave encounters an obstacle. Italian scientist Francesco Maria Grimaldi coined the word "diffraction" and was the first to record accurate observations of the phenomenon in 1665...

rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks (or, in other words the ability of the microscope to reveal adjacent structural detail as distinct and separate). It is these impacts of diffraction that limit the ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by both the wavelength

Wavelength

of light

Light

Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...

(λ), the refractive materials used to manufacture the objective lens and the numerical aperture

Numerical aperture

(NA) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field, known as the diffraction limit. Assuming that optical aberrations in the whole optical set-up are negligible, the resolution d, can be stated as:

Usually a wavelength of 550 nm is assumed, which corresponds to green

Green

Green is a color, the perception of which is evoked by light having a spectrum dominated by energy with a wavelength of roughly 520–570 nanometres. In the subtractive color system, it is not a primary color, but is created out of a mixture of yellow and blue, or yellow and cyan; it is considered...

light. With air

Earth's atmosphere

The atmosphere of Earth is a layer of gases surrounding the planet Earth that is retained by Earth's gravity. The atmosphere protects life on Earth by absorbing ultraviolet solar radiation, warming the surface through heat retention , and reducing temperature extremes between day and night...

as the external medium, the highest practical NA is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable with conventional lenses is about 200 nm. A new type of lens using multiple scattering of light allowed to improve the resolution to below 100 nm.

Surpassing the resolution limit

Multiple techniques are available for reaching resolutions higher than the transmitted light limit described above. Techniques for surpassing the resolution limit for bright field microscopy include ultraviolet microscopes, which use shorter wavelengths of light so the diffraction limit is lower. Holographic techniques, as described by Courjon and Bulabois in 1979, are also capable of breaking this resolution limit, although resolution was restricted in their experimental analysis.

Using fluorescent samples more techniques are available. Examples include Vertico SMI

Vertico SMI

Vertico-SMI is currently the fastest light microscope for the 3D analysis of complete cells in the nanometer range. It is based on two technologies developed in 1996, SMI and SPDM...

, near field scanning optical microscopy which uses evanescent waves, and stimulated emission depletion. In 2005, a microscope capable of detecting a single molecule was described as a teaching tool.

While most techniques focus on increases in lateral resolution there are also some techniques which aim to allow analysis of extremely thin samples. For example sarfus

Sarfus

methods place the thin sample on a contrast-enhancing surface and thereby allows to directly visualize films as thin as 0.3 nanometers.

Structured Illumination SMI

SMI (spatially modulated illumination microscopy) is a light optical process of the so-called point spread function

Point spread function

The point spread function describes the response of an imaging system to a point source or point object. A more general term for the PSF is a system's impulse response, the PSF being the impulse response of a focused optical system. The PSF in many contexts can be thought of as the extended blob...

(PSF) engineering. These are processes which modify the PSF of a microscope

Microscope

A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...

in a suitable manner to either increase the optical resolution, to maximize the precision of distance

Distance

Distance is a numerical description of how far apart objects are. In physics or everyday discussion, distance may refer to a physical length, or an estimation based on other criteria . In mathematics, a distance function or metric is a generalization of the concept of physical distance...

measurements of fluorescent objects that are small relative to the wavelength

Wavelength

of the illuminating light, or to extract other structural parameters in the nanometer range.

Localization Microscopy SPDMphymod

SPDM (spectral precision distance microscopy), the basic localization microscopy technology is a light optical process of fluorescence microscopy which allows position, distance and angle measurements on "optically isolated" particles (e.g. molecules) well below the theoretical limit of resolution for light microscopy. "Optically isolated" means that at a given point in time, only a single particle/molecule within a region of a size determined by conventional optical resolution (typically approx. 200–250 nm diameter

Diameter

In geometry, a diameter of a circle is any straight line segment that passes through the center of the circle and whose endpoints are on the circle. The diameters are the longest chords of the circle...

) is being registered. This is possible when molecules within such a region all carry different spectral markers (e.g. different colors or other usable differences in the light emission of different particles).

Many standard fluorescent dyes like GFP

Green fluorescent protein

, Alexa dyes, Atto dyes, Cy2/Cy3 and fluorescein molecules can be used for localization microscopy, provided certain photo-physical conditions are present. Using this so-called SPDMphymod (physically modifiable fluorophores) technology a single laser wavelength of suitable intensity is sufficient for nanoimaging.

3D super resolution microscopy

3D super resolution microscopy with standard fluorescent dyes can be achieved by combination of localization microscopy for standard fluorescent dyes SPDMphymod and structured illumination SMI.

STED

Stimulated emission depletion is a simple example of how higher resolution surpassing the diffraction limit is possible, but it has major limitations. STED is a fluorescence microscopy technique which uses a combination of light pulses to induce fluorescence in a small sub-population of fluorescent molecules in a sample. Each molecule produces a diffraction-limited spot of light in the image, and the centre of each of these spots corresponds to the location of the molecule. As the number of fluorescing molecules is low the spots of light are unlikely to overlap and therefore can be placed accurately. This process is then repeated many times to generate the image. Stefan Hell

Stefan Hell

Stefan W. Hell is a physicist and one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany as well as the head of the department "Optical Nanoscopy" at the German Cancer Research Center in Heidelberg.- Life :In 1981 Hell began his studies at the...

of the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 for his development of the STED microscope.

Alternatives

In order to overcome the limitations set by the diffraction limit of visible light other microscopes have been designed which use other waves.

Atomic force microscope
Atomic force microscope
Atomic force microscopy or scanning force microscopy is a very high-resolution type of scanning probe microscopy, with demonstrated resolution on the order of fractions of a nanometer, more than 1000 times better than the optical diffraction limit...

(AFM)
Scanning electron microscope
Scanning electron microscope
A scanning electron microscope is a type of electron microscope that images a sample by scanning it with a high-energy beam of electrons in a raster scan pattern...

(SEM)
Scanning ion-conductance microscopy
Scanning ion-conductance microscopy
The scanning ion-conductance microscope consists of an electrically charged glass micro- or nanopipette probe filled with electrolyte lowered toward the surface of the sample in an oppositely charged bath of electrolyte...

(SICM)
Scanning tunneling microscope
Scanning tunneling microscope
A scanning tunneling microscope is an instrument for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer , the Nobel Prize in Physics in 1986. For an STM, good resolution is considered to be 0.1 nm lateral resolution and...

(STM)
Transmission electron microscopy
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...

(TEM)
Ultraviolet microscope
X-ray microscope
X-ray microscope
An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce images of very small objects.Unlike visible light, X-rays do not reflect or refract easily, and they are invisible to the human eye. Therefore the basic process of an X-ray microscope is to expose film or use a...

The use of electrons and x-rays in place of light allows much higher resolution – the wavelength of the radiation is shorter so the diffraction limit is lower. To make the
short-wavelength probe non-destructive, the atomic beam imaging system (atomic nanoscope

Atomic nanoscope

The atomic de Broglie microscope is an imaging system which is expected to provide resolution at the nanometer scale....

) has been proposed and widely discussed in the literature, but it is not yet competitive with conventional imaging systems.

STM and AFM are scanning probe techniques using a small probe which is scanned over the sample surface. Resolution in these cases is limited by the size of the probe; micromachining techniques can produce probes with tip radii of 5–10 nm.

Additionally, methods such as electron or X-ray microscopy use a vacuum or partial vacuum, which limits their use for live and biological samples (with the exception of an environmental scanning electron microscope). The specimen chambers needed for all such instruments also limits sample size, and sample manipulation is more difficult. Color cannot be seen in images made by these methods, so some information is lost. They are however, essential when investigating molecular or atomic effects, such as age hardening in aluminium alloy

Aluminium alloy

Aluminium alloys are alloys in which aluminium is the predominant metal. The typical alloying elements are copper, magnesium, manganese, silicon and zinc. There are two principal classifications, namely casting alloys and wrought alloys, both of which are further subdivided into the categories...

s, or the microstructure

Microstructure

Microstructure is defined as the structure of a prepared surface or thin foil of material as revealed by a microscope above 25× magnification...

of polymers.

External links

Antique Microscopes.com A collection of early microscopes
Historical microscopes, an illustrated collection with more than 3000 photos of scientific microscopes by European makers
The Golub Collection, A collection of 17th through 19th Century microscopes, including extensive descriptions
Molecular Expressions, concepts in optical microscopy
Online tutorial of practical optical microscopy
OpenWetWare
Cell Centered Database
Antonie van Leeuwenhoek: Father of Microscopy and Microbiology

The source of this article is wikipedia, the free encyclopedia. The text of this article is licensed under the GFDL.

Optical configurations

Single lens (simple) microscope

Compound microscope

History

Invention

Popularisation

Lighting techniques

Fluorescence microscopy

Components

Eyepiece (ocular)

Objective turret or Revolver or Revolving nose piece

Objective

Oil immersion objective

Focus wheels

Frame

Light source

Condenser

Stage

Magnification

Magnification and micrographs

Operation

Illumination techniques

Other techniques

Applications

Optical microscope variants

Digital microscope

Limitations

Surpassing the resolution limit

Structured Illumination SMI

Localization Microscopy SPDMphymod

3D super resolution microscopy

STED

Alternatives

See also

Further reading

External links