[转帖]显微镜相关背景知识--英文

郑振寰 · 发表于 2010-3-4 12:57

Optical microscope

The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest and simplest of the microscopes. However, new designs of digital microscopes are now available which use a CCD camera to examine a sample and the image is shown directly on a computer screen without the need for expensive optics such as eye-pieces. Other microscopic methods which do not use visible light include scanning electron microscopy and transmission electron microscopy.

Optical configurations

There are two basic configurations of the conventional optical microscope in use, the simple (one lens) and compound (many lenses). Digital microscopes are based on an entirely different system of collecting the reflected light from a sample.

Light microscope

A simple microscope is a microscope that uses only one lens for magnification, and is the original 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, and the loupe. Light microscopes are able to view specimens in colour, an important advantage when compared with electron microscopes, especially for forensic analysis, where blood traces may be important, for example.

History

See also: Timeline of microscope technology

The oldest published image known to have been made with a microscope: bees by Francesco Stelluti, 1630^[1]

The earliest evidence of magnifying glass forming a magnified image dates back to the Book of Optics published by Ibn al-Haytham (Alhazen) in 1021. After the book was translated into Latin, Roger Bacon described the properties of magnifying glass in 13th-century England, followed by the development of eyeglasses in 13th-century Italy.^[2]

It is difficult to say who invented the compound microscope. Dutch spectacle-makers Hans Janssen and his son Zacharias Janssen 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 1600s. 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. 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 in 1624 and was the first such device to be given the name "microscope" a year latter by fellow Lincean Giovanni Faber. Faber coined the name from the Greek words μικρόν (micron) meaning "small", and σκοπεῖν (skopein) meaning "to look at", a name meant to be analogus with "telescope", another word coined by the Linceans^[3].

Christiaan Huygens, another Dutchman, developed a simple 2-lens ocular system in the late 1600s that was achromatically 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.

Anton van Leeuwenhoek (1632-1723) is credited with bringing the microscope to the attention of biologists, even though simple magnifying lenses were already being produced in the 1500s. 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 timely difficulties of configuring multiple lenses. Still, despite widespread claims, van Leeuwenhoek is not the inventor of the microscope.

Components

Basic optical transmission microscope elements(1990's)

ocular lens, or eyepiece
objective turret
objective lenses
coarse adjustment knob
fine adjustment knob
object holder or stage
mirror or light (illuminator)
diaphragm and condenser

All optical microscopes share the same basic components:

The eyepiece - a cylinder containing two or more lenses to bring the image to 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 5x, 10x and 2x. 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 apochromatic objectives.
The objective lens - a cylinder containing one or more lenses, typically made of glass, to collect light from the sample. At the lower end of the microscope tube one or more objective lenses are screwed into a circular nose piece which may be rotated to select the required objective lens. Typical magnification values of objective lenses are 4x, 5x, 10x, 20x, 40x, 50x and 100x. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance.
The stage - 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 (rectangular glass plates with typical dimensions of 25 mm by 75 mm, on which the specimen is mounted).
The illumination source - below the stage, light is provided and controlled in a variety of ways. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own controllable light source that is focused through an optical device called a condenser, with diaphragms and filters available to manage the quality and intensity of the light.

The whole of the optical assembly is attached to a rigid arm which in turn is attached to a robust U shaped foot to provide the necessary rigidity. The arm is usually able to pivot on its joint with the foot to allow the viewing angle to be adjusted. Mounted on the arm are controls for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine focus.

Updated microscopes may have many more features, including reflected light (incident) illumination, fluorescence microscopy, phase contrast microscopy and differential interference contrast microscopy, spectroscopy, automation, and digital imaging.

On a typical compound optical microscope, there are three objective lenses: a scanning lens (4×), low power lens (10×)and high power lens (ranging from 20 to 100×). Some microscopes have a fourth objective lens, called an oil immersion lens. To use this lens, a drop of immersion oil is placed on top of the cover slip, and the lens is very carefully lowered until the front objective element is immersed in the oil film. Such immersion lenses are designed so that the refractive index of the oil and of the cover slip are closely matched so that the light is transmitted from the specimen to the outer face of the objective lens with minimal refraction. An oil immersion lens usually has a magnification of 50 to 100×.

The actual power or magnification of an optical microscope is the product of the powers of the ocular (eyepiece), usually about 10×, and the objective lens being used.

Compound optical microscopes can produce a magnified image of a specimen up to 1000× and, at high magnifications, are used to study thin specimens as they have a very limited depth of field.

Operation

Optical path in a typical microscope

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 optical 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 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 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 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.

Stereo microscope

Digital display with stereo microscopes

Recently various video dual CCD camera pickups have been fitted to stereo microscopes, allowing the images to be displayed on a high resolution LCD monitor. Software converts the two images to an integrated Anachrome 3D image, for viewing with plastic red/cyan glasses, or to the cross converged process for clear glasses and somewhat better color accuracy. The results are viewable by a group wearing the glasses.

Digital microscopes

A digital microscope.

Low power microscopy is also possible with digital microscopes, with a camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor. Often called "USB" microscopes, they offer high magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. The precise magnification is determined by the working distance between the camera and the object, and good supports are needed to control the image. The images can be recorded and stored in the normal way on the computer. The camera is usually fitted with a light source, although extra sources (such as a fibre-optic light) can be used to highlight features of interest in the object. They also offer a large depth of field, a great advantage at high magnifications.

They are most useful when examining flat objects such as coins, printed circuit boards, or documents such as banknotes. However, they can be used for examining any object which can be studied in a standard stereo-microscope. Such microscopes offer the great advantage of being much less bulky than a conventional microscope, so can be used in the field, attached to a laptop computer.

Special designs

Main article: Microscopy

Other types of optical microscope include:

the inverted microscope for studying samples from below; useful for cell cultures in liquid;
the student microscope designed for low cost, durability, and ease of use;
the research microscope which is an expensive tool with many enhancements;
the petrographic microscope 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.
the polarizing microscope
the fluorescence microscope
the phase contrast microscope

Limitations

At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction 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 of and magnitude of the diffraction patterns are affected by both by the wavelength of light ( $λ$ ), the refractive materials used to manufacture the objective lens and the numerical aperture (NA or $A N$ ) 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, is given by:

Usually, a $λ$ of 550 nm is assumed, corresponding to green light. With air as medium, the highest practical $A N$ is 0.95, and with oil, up to 1.5. In practice the lowest value of d obtainable is around 0.2 micrometres or 200 nanometres.

A modern microscope with a mercury bulb for fluorescence microscopy. The microscope has a digital camera, and is attached to a computer.

Other optical microscope designs can offer an improved resolution. These include ultraviolet microscopes which use shorter wavelengths of light so the diffraction limit is lower, Vertico SMI, near field scanning optical microscopy which uses evanescent waves, and Stimulated Emission Depletion Microscopy which is used for observing self-luminous particles which are not diffraction limited as Abbe's theory (by Ernst Karl Abbe) is based on the fact that a non-self-luminous particle is illuminated by an external source.

Professor Stefan Hell of the Max Planck Institute for Biophysical Chemistry was awarded the 10th German Future Prize in 2006 for his development of the Stimulated Emission Depletion (STED) microscope.^[7]

Several other optical microscopes have been able to see beyond the theoretical Abbe limit of 200nm. In 2005, Assistant Professor Masaru Kuno and post-graduate students Vladimir Protasenko and Katherine L. Hull of the University of Notre Dame described a single-molecule capable unit that could be constructed cheaply as a teaching tool.^[8] A holographic microscope described by Courjon and Bulabois in 1979 is also capable of breaking this magnification limit, although resolution was restricted in their experimental analysis.^[9]

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 (AFM)
Scanning Electron Microscope (SEM)
Scanning Tunneling Microscope (STM)
Transmission Electron Microscope (TEM)
X-ray microscope

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) 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 5-10nm.

However, all such methods use a vacuum or partial vacuum, which limits their use for live and biological samples (with the exception of ESEM). The specimen chambers needed for all such instruments also limits sample size, and sample manipulation is more difficult. Colour 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 alloys, or the microstructure of polymers.

References

^ "The Lying stones of Marrakech", by Stephen Jay Gould, 2000
^ Kriss, Timothy C.; Kriss, Vesna Martich (April 1998), "History of the Operating Microscope: From Magnifying Glass to Microneurosurgery", Neurosurgery 42 (4): 899–907, doi:10.1097/00006123-199804000-00116
^ brunelleschi.imss.fi.it "Il microscopio di Galileo"
^ ^a ^b "Introduction to Stereomicroscopy" by Paul E. Nothnagle, William Chambers, and Michael W. Davidson, Nikon MicroscopyU.
^ "Illumination for Stereomicroscopy: Reflected (Episcopic) Light" by Paul E. Nothnagle, William Chambers, Thomas J. Fellers, and Michael W. Davidson , Nikon MicroscopyU.
^ "Illumination for Stereomicroscopy: Darkfield Illumination" by William Chambers, Thomas J. Fellers, and Michael W. Davidson , Nikon MicroscopyU.
^ "German Future Prize for crossing Abbe's Limit". https://www.heise.de/english/newsticker/news/81528. Retrieved on Feb 24, 2009.
^ "Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope". https://chemeducator.org/bibs/0010004/1040269mk.htm. Retrieved on Feb 25, 2009.
^ Real Time Holographic Microscopy Using a Peculiar Holographic Illuminating System and a Rotary Shearing Interferometer, By D. Courjon and J. Bulabois, Journal of Optics, Paris, 1979, Vol. 10, No. 3

"Metallographic and Materialographic Specimen Preparation, Light Microscopy, Image Analysis and Hardness Testing", Kay Geels in collaboration with Struers A/S, ASTM International 2006.

External links

A collection of early microscopes
Historical microscopes, an illustrated collection with more than 3000 photos of scientific microscopes by European makers (German)
Metallurgical microscope (SubsTech - free and open knowledge source in Materials Engineering)
Molecular Expressions, concepts in optical microscopy
Online tutorial of practical optical microscopy
Optical microscope videos
Structure Magazine
Microscopy Information Easily understandable articles relating to optics, techniques and specimen preparation.

郑振寰 · 发表于 2010-3-4 12:59

Microscope Uses Small sample observation Notable experiments Discovery of cells Inventor Hans Lippershey
Zacharias Janssen Related items Electron microscope A microscope (from the Greek: μικρός, mikrós, "small" and σκοπεῖν, skopeîn, "to look" or "see") is an instrument for viewing objects that are too small to be seen by the naked or unaided eye. The science of investigating small objects using such an instrument is called microscopy. The term microscopic means minute or very small, not visible with the eye unless aided by a microscope. Anton Van Leeuwenhoek's new, improved microscope allowed people to see things no human had ever seen before.
Contents 1 History 2 Types 2.1 Optical microscopes 2.2 Electron microscopes 2.3 Established types of scanning probe microscopy 2.4 Other microscopes 3 See also 4 References 5 External links //
History See also: History of optics The first true microscope was made around 1595 in Middelburg, The Netherlands.[1] Three different eyeglass makers have been given credit for the invention: Hans Lippershey (who also developed the first real telescope); Sacharias Jansen; and his son, Zacharias. The coining of the name "microscope" has been credited to Giovanni Faber, who gave that name to Galileo Galilei's compound microscope in 1625.[2] (Galileo had called it the "occhiolino" or "little eye".)
The most common type of microscope—and the first to be invented—is the optical microscope. This is an optical instrument containing one or more lenses that produce an enlarged image of an object placed in the focal plane of the lens(es). There are, however, many other microscope designs.

Types Several types of microscopes "Microscopes" can largely be separated into three classes: optical theory microscopes (Light microscope), electron microscopes (e.g.,TEM), and scanning probe microscopes (SPM). Optical microscopes are microscopes which function through the optical theory of lenses in order to magnify the image generated by the passage of a wave through the sample, or reflected by the sample. The waves used are either electromagnetic (in optical microscopes) or electron beams (in electron microscopes). The types are the Compound Light, Stereo, and the electron microscope.

Optical microscopes Main article: Optical microscope Optical microscopes, through their use of visible wavelengths of light, are the simplest and hence most widely used type of microscope. Optical microscopes typically use refractive glass and occasionally of plastic or quartz, to focus light into the eye or 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 1500x with a theoretical resolution limit of around 0.2 micrometres or 200 nanometers. Specialized techniques (e.g., scanning confocal microscopy, Vertico SMI) may exceed this magnification but the resolution is diffraction limited. Using shorter wavelengths of light, such as the ultraviolet, is one way to improve the spatial resolution of the microscope as are techniques such as Near-field scanning optical microscope.
A stereo microscope is often used for lower-power magnification on large subjects. Various wavelengths of light, including those beyond the visible range, are sometimes used for special purposes. Ultraviolet light is used to enable the resolution of smaller features as well as to image samples that are transparent to the eye. Near infrared light is used to image circuitry embedded in bonded silicon devices as silicon is transparent in this region. Many wavelengths of light, ranging from the ultraviolet to the visible are used to excite fluorescence emission from objects for viewing by eye or with sensitive cameras.
Phase contrast microscopy is an optical microscopy 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. A phase contrast microscope does not require staining to view the slide. This microscope made it possible to study the cell cycle.
The Digital microscope appeared a few years ago, using optics and a charge-coupled device (CCD) camera to output a digital image to a monitor.

Electron microscopes Main article: Electron microscope Three major variants of electron microscopes exist:
Scanning electron microscope (SEM): looks at the surface of bulk objects by scanning the surface with a fine electron beam and measuring reflection. May also be used for spectroscopy. See also environmental scanning electron microscope Transmission electron microscope (TEM): passes electrons completely through the sample, analogous to basic optical microscopy. This requires careful sample preparation, since electrons are scattered so strongly by most materials.This is a scientific device that allows people to see objects that could normally not be seen by the naked or unaided eye. Scanning Tunneling Microscope (STM): is a powerful technique for viewing surfaces at the atomic level. The SEM and STM can also be considered examples of scanning probe microscopy.

Established types of scanning probe microscopy AFM, atomic force microscopy Contact AFM Non-contact AFM Dynamic contact AFM Tapping AFM BEEM, ballistic electron emission microscopy EFM, electrostatic force microscope ESTM electrochemical scanning tunneling microscope FMM, force modulation microscopy KPFM, kelvin probe force microscopy MFM, magnetic force microscopy MRFM, magnetic resonance force microscopy NSOM, near-field scanning optical microscopy (or SNOM, scanning near-field optical microscopy) PFM, Piezo Force Microscopy PSTM, photon scanning tunneling microscopy PTMS, photothermal microspectroscopy/microscopy SAP, scanning atom probe [3] SECM, scanning electrochemical microscopy SCM, scanning capacitance microscopy SGM, scanning gate microscopy SICM, scanning ion-conductance microscopy SPSM spin polarized scanning tunneling microscopy SThM, scanning thermal microscopy[1] STM, scanning tunneling microscopy SVM, scanning voltage microscopy SHPM, scanning Hall probe microscopy SSM, Scanning SQUID microscope Of these techniques AFM and STM are the most commonly used followed by MFM and SNOM/NSOM.

Other microscopes Replica of microscope by Van Leeuwenhoek 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.

See also Different microscopes Wikimedia Commons has media related to: Microscopes Bright field microscopy Condensed Matter Physics Confocal microscopy Dark field microscopy Digital microscope Electron Microscope Fluorescence interference contrast microscopy Fluorescence microscope Microscope image processing Microscopy Optical Microscope Intel Play Phase contrast microscopy Microscope slide Telescope Timeline of microscope technology X-ray microscope Microscopy laboratory in: A Study Guide to the Science of Botany at Wikibooks Laser capture microdissection
References ^ Microscopes: Time Line ^ Stephen Jay Gould(2000). The Lying Stones of Marrakech, ch.2 "The Sharp-Eyed Lynx, Outfoxed by Nature". London: Jonathon Cape. ISBN 0224050443 ^ Morita, Seizo. Roadmap of Scanning Probe Microscopy. 3 January 2007
External links FAQ on Optical Microscopes Nikon MicroscopyU, tutorials from Nikon Molecular Expressions : Exploring the World of Optics and Microscopy, Florida State University. Microscopes made from bamboo at Nature.com Microscope videos

郑振寰 · 发表于 2010-3-4 13:01

Microscopy Microscopy is the technical field of using microscopes to view samples or objects. There are three well-known branches of microscopy, optical, electron and scanning probe microscopy.
Optical and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/electron beam interacting with the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. This process may be carried out by wide-field irradiation of the sample (for example standard light microscopy and transmission electron microscopy) or by scanning of a fine beam over the sample (for example confocal laser scanning microscopy and scanning electron microscopy). Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest. The development of microscopy revolutionized biology and remains an essential tool in that science, along with many others including materials science and numerous engineering disciplines.
Scanning electron microscope image of pollen. Contents 1 Optical microscopy 1.1 Limitations 1.2 Techniques 1.2.1 Bright field 1.2.2 Oblique illumination 1.2.3 Dark field 1.2.4 Dispersion staining 1.2.5 Phase contrast 1.2.6 Differential interference contrast 1.2.7 Fluorescence 1.2.8 Confocal 1.2.9 Deconvolution 1.3 Sub-diffraction techniques 1.3.1 Near-field scanning 1.3.2 Local enhancement / ANSOM / bowties 1.3.3 Stimulated emission depletion 1.3.4 Fitting the point-spread function 1.3.5 PALM, STORM 1.3.6 Structured illumination 1.3.7 Localization Microscopy/Spatially Structured Illumination 1.4 Extensions 1.5 Other enhancements 1.6 X-ray 2 Electron microscopy 2.1 Atomic de Broglie 3 Scanning probe microscopy 3.1 Ultrasonic force 4 Infrared microscopy 5 Digital holographic microscopy 6 Digital Pathology (virtual microscopy) 7 Amateur microscopy 8 See also 9 References 10 Further reading 11 External links 11.1 General 11.2 Techniques 11.3 Organizations //
Optical microscopy See also: Optical microscope Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple lenses to allow a magnified view of the sample.[1] The resulting image can be detected directly by the eye, imaged on a photographic plate or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the basic light microscope. The most recent development is the digital microscope which uses a CCD camera to focus on the exhibit of interest. The image is shown on a computer screen since the camera is attached to it via a USB port, so eye-pieces are unnecessary.

Limitations Limitations of standard optical microscopy (bright field microscopy) lie in three areas;
The technique can only image dark or strongly refracting objects effectively. Diffraction limits resolution to approximately 0.2 micrometre (see: microscope). Out of focus light from points outside the focal plane reduces image clarity. Live cells in particular generally lack sufficient contrast to be studied successfully, internal structures of the cell are colourless and transparent. The most common way to increase contrast is to stain the different structures with selective dyes, but this involves killing and fixing the sample. Staining may also introduce artifacts, apparent structural details that are caused by the processing of the specimen and are thus not a legitimate feature of the specimen.
These limitations have all been overcome to some extent by specific microscopy techniques which can non-invasively increase the contrast of the image. In general, these techniques make use of differences in the refractive index of cell structures. It is comparable to looking through a glass window: you (bright field microscopy) don't see the glass but merely the dirt on the glass. There is however a difference as glass is a denser material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase but clever optical solutions have been thought out to change this difference in phase into a difference in amplitude (light intensity).

Techniques Main article: Optical microscope
Bright field Main article: Bright field microscopy Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, i.e. illuminated from below and observed from above. Limitations include low contrast of most biological samples and low apparent resolution due to the blur of out of focus material. The simplicity of the technique and the minimal sample preparation required are significant advantages.

Oblique illumination The use of oblique (from the side) illumination gives the image a 3-dimensional appearance and can highlight otherwise invisible features. A more recent technique based on this method is Hoffmann's modulation contrast, a system found on inverted microscopes for use in cell culture. Oblique illumination suffers from the same limitations as bright field microscopy (low contrast of many biological samples; low apparent resolution due to out of focus objects), but may highlight otherwise invisible structures.

Dark field Main article: Dark field microscopy Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens.[2] Dark field illumination uses a carefully aligned light source to minimize the quantity of directly-transmitted (unscattered) light entering the image plane, collecting only the light scattered by the sample. Darkfield can dramatically improve image contrast—especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique does suffer from low light intensity in final image of many biological samples, and continues to be affected by low apparent resolution.
Rheinberg illumination is a special variant of dark field illumination in which transparent, colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture (i.e. the background to the specimen may be blue while the object appears self-luminous yellow). Other color combinations are possible but their effectiveness is quite variable.[3]

Dispersion staining Main article: Dispersion staining Dispersion staining is an optical technique that results in a colored image of a colorless object. This is an optical staining technique and requires no stains or dyes to produce a color effect. There are five different microscope configurations used in the broader technique of dispersion staining. They include brightfield Becke` line, oblique, darkfield, phase contrast, and objective stop dispersion staining.

Phase contrast Main articles: Phase contrast microscope and Phase contrast microscopy In electron microscopy: Phase-contrast imaging More sophisticated techniques will show proportional differences in optical density . Phase contrast is a widely used technique that shows differences in refractive index as difference in contrast. It was developed by the Dutch physicist Frits Zernike in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of a circular annulus in the condenser which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical properties: it first of all reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about a quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image.

Differential interference contrast Main article: Differential interference contrast microscopy Superior and much more expensive is the use of interference contrast. Differences in optical density will show up as differences in relief. A nucleus within a cell will actually show up as a globule in the most often used differential interference contrast system according to Georges Nomarski. However, it has to be kept in mind that this is an optical effect, and the relief does not necessarily resemble the true shape! Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximizing resolution.
The system consists of a special prism (Nomarski prism, Wollaston prism) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective.
In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast requires a polarized light source to function; two polarizing filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyzer).
Note: In cases where the optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of classical interference microscopy, which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

Fluorescence Main article: Fluorescence microscopy When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency. This effect is known as fluorescence. Often specimens show their own characteristic autofluorescence image, based on their chemical makeup.
This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many different fluorescent dyes can be used to stain different structures or chemical compounds. One particularly powerful method is the combination of antibodies coupled to a fluorochrome as in immunostaining. Examples of commonly used fluorochromes are fluorescein or rhodamine. The antibodies can be made tailored specifically for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, which then form antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorochrome and then used to trace the proteins in the cells under study.
Highly-efficient fluorescent proteins such as the green fluorescent protein (GFP) have been developed using the molecular biology technique of gene fusion, a process which links the expression of the fluorescent compound to that of the target protein. Piston DW, Patterson GH, Lippincott-Schwartz J, Claxton NS, Davidson MW (2007). "Nikon MicroscopyU: Introduction to Fluorescent Proteins". Nikon MicroscopyU. https://www.microscopyu.com/articles/livecellimaging/fpintro.html. Retrieved on 2007-08-22. This combined fluorescent protein is generally non-toxic to the organism and rarely interferes with the function of the protein under study. Genetically modified cells or organisms directly express the fluorescently-tagged proteins, which enables the study of the function of the original protein in vivo.
Since fluorescence emission differs in wavelength (color) from the excitation light, a fluorescent image ideally only shows the structure of interest that was labeled with the fluorescent dye. This high specificity led to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye.
To block the excitation light from reaching the observer or the detector, filter sets of high quality are needed. These typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.
See also total internal reflection fluorescence microscope.

Confocal Main article: Confocal microscopy Confocal microscopy generates the image in a completely different way to normal "wide-field" microscopes. Using a scanning point of light instead of full sample illumination confocal microscopy gives slightly higher resolution, and significant improvements in optical sectioning by blocking the influence of out-of-focus light which would otherwise degrading the image. Confocal microscopy is therefore commonly used where 3D structure is important.

Deconvolution Fluorescence microscopy is extremely powerful due to its ability to show specifically labeled structures within a complex environment and also because of its inherent ability to provide three dimensional information of biological structures. Unfortunately this information is blurred by the fact that upon illumination all fluorescently labeled structures emit light no matter if they are in focus or not. This means that an image of a certain structure is always blurred by the contribution of light from structures which are out of focus. This phenomenon becomes apparent as a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture.
Fortunately though, this phenomenon is not caused by random processes such as light scattering but can be relatively well defined by the optical properties of the image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out the further out of focus one is. Under ideal conditions this produces a sort of "hourglass" shape of this point source in the third (axial) dimension. This shape is called the point spread function (PSF) of the microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources the image is said to be "convolved by the point spread function".
Knowing this point spread function means that it is possible to reverse this process to a certain extent by computer based methods commonly known as deconvolution microscopy.[4] There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in non restorative and restorative methods. While the non restorative methods can improve contrast by removing out of focus light from focal planes, only the restorative methods can actually reassign light to it proper place of origin. This can be an advantage over other types of 3D microscopy such as confocal microscopy, because light is not thrown away but reused. For 3D deconvolution one typically provides a series of images derived from different focal planes (called a Z-stack) plus the knowledge of the PSF which can be either derived experimentally or theoretically from knowing all contributing parameters of the microscope.

Sub-diffraction techniques It is well known that there is a spatial limit to which light can focus: approximately half of the wavelength of the light you are using. But this is not a true barrier, because this diffraction limit is only true in the far-field and localization precision can be increased with many photons and careful analysis (although two objects still cannot be resolved); and like the sound barrier, the diffraction barrier is breakable. This section explores some approaches to imaging objects smaller than ~250 nm. Most of the following information was gathered (with permission) from a chemistry blog's review of sub-diffraction microscopy techniques Part I and Part II. For a review, see also reference [5].

Near-field scanning Near-field scanning is also called NSOM. Probably the most conceptual way to break the diffraction barrier is to use a light source and/or a detector that is itself nanometer in scale. Diffraction as we know it is truly a far-field effect: the light from an aperture is the Fourier transform of the aperture in the far-field.[6] But in the near-field, all of this is not necessarily the case. Near-field scanning optical microscopy (NSOM) forces light through the tiny tip of a pulled fiber—and the aperture can be on the order of tens of nanometers.[7] When the tip is brought to nanometers away from a molecule, the resolution is not limited by diffraction but by the size of the tip aperture (because only that one molecule will see the light coming out of the tip). An image can be built by a raster scan of the tip over the surface to create an image.
The main down-side to NSOM is the limited number of photons you can force out a tiny tip, and the minuscule collection efficiency (if you are trying to collect fluorescence in the near-field). Other techniques such as ANSOM (see below) try to avoid this drawback.

Local enhancement / ANSOM / bowties Instead of forcing photons down a tiny tip, some techniques create a local bright spot in an otherwise diffraction-limited spot. ANSOM is apertureless NSOM: it uses a tip very close to a fluorophore to enhance the local electric field the fluorophore sees.[8] Basically, the ANSOM tip is like a lightning rod which creates a hot spot of light.
Bowtie nanoantennas have been used to greatly and reproducibly enhance the electric field in the nanometer gap between the tips two gold triangles. Again, the point is to enhance a very small region of a diffraction-limited spot, thus improving the mismatch between light and nanoscale objects—and breaking the diffraction barrier.[9]

Stimulated emission depletion Stefan Hell at the Max Planck Institute for Biophysical Chemistry - Goettingen (Germany) developed STED microscopy (stimulated emission depletion), which uses two laser pulses. The first pulse is a diffraction-limited spot that is tuned to the absorption wavelength, so excites any fluorophores in that region; an immediate second pulse is red-shifted to the emission wavelength and stimulates emission back to the ground state before, thus depleting the excited state of any fluorophores in this depletion pulse. The trick is that the depletion pulse goes through a phase modulator that makes the pulse illuminate the sample in the shape of a donut, so the outer part of the diffraction limited spot is depleted and the small center can still fluoresce. By saturating the depletion pulse, the center of the donut gets smaller and smaller until they can get resolution of tens of nanometers.[10]
This technique also requires a raster scan like NSOM and standard confocal laser scanning microscopy.

Fitting the point-spread function Fitting the point-spread function is also called PSF. The methods above (and below) use experimental techniques to circumvent the diffraction barrier, but one can also use crafty analysis to increase the ability to know where a nanoscale object is located. The image of a point source on a charge-coupled device camera is called a point-spread function (PSF), which is limited by diffraction to be no less than approximately half the wavelength of the light. But it is possible to simply fit that PSF with a Gaussian to locate the center of the PSF—and thus the location of the fluorophore. The precision by which this technique can locate the center depends on the number of photons collected (as well as the CCD pixel size and other factors).[11] Regardless, groups like the Selvin lab and many others have employed this analysis to localize single fluorophores to a few nanometers. This, of course, requires careful measurements and collecting many photons.

PALM, STORM What fitting a PSF is to localization, photo-activated localization microscopy (PALM) is to "resolution"—this term is here used loosely to mean measuring the distance between objects, not true optical resolution. Eric Betzig and colleagues developed PALM;[12] Xiaowei Zhuang at Harvard used a similar techniques and calls it STORM: stochastic optical reconstruction microscopy.[13] Sam Hess at University of Maine developed the technique simultaneously. The basic premise of both techniques is to fill the imaging area with many dark fluorophores that can be photoactivated into a fluorescing state by a flash of light. Because photoactivation is stochastic, only a few, well separated molecules "turn on." Then Gaussians are fit to their PSFs to high precision (see section above). After the few bright dots photobleach, another flash of the photoactivating light activates random fluorophores again and the PSFs are fit of these different well spaced objects. This process is repeated many times, building up an image molecule-by-molecule; and because the molecules were localized at different times, the "resolution" of the final image can be much higher than that limited by diffraction.
The major problem with these techniques is that to get these beautiful pictures, it takes on the order of hours to collect the data. This is certainly not the technique to study dynamics (fitting the PSF is better for that).

Structured illumination Comparison of the resolution obtained by confocal laser scanning microscopy (top) and 3D structured illumination microscopy (3D-SIM-Microscopy, bottom). Shown are details of a nuclear envelope. Nuclear pores (anti-NPC) red, nuclear envelope (anti-Lamin) green, chromatin (DAPI-staining) blue. Scale bars: 1µm. There is also the wide-field structured-illumination (SI) approach to breaking the diffraction limit of light.[14][15] SI—or patterned illumination—relies on both specific microscopy protocols and extensive software analysis post-exposure. But, because SI is a wide-field technique, it is usually able to capture images at a higher rate than confocal-based schemes like STED. (This is only a generalization, because SI isn't actually super fast. I'm sure someone could make STED fast and SI slow!) The main concept of SI is to illuminate a sample with patterned light and increase the resolution by measuring the fringes in the Moiré pattern (from the interference of the illumination pattern and the sample). "Otherwise-unobservable sample information can be deduced from the fringes and computationally restored."[16]
SI enhances spatial resolution by collecting information from frequency space outside the observable region. This process is done in reciprocal space: the Fourier transform (FT) of an SI image contains superimposed additional information from different areas of reciprocal space; with several frames with the illumination shifted by some phase, it is possible to computationally separate and reconstruct the FT image, which has much more resolution information. The reverse FT returns the reconstructed image to a super-resolution image.
But this only enhances the resolution by a factor of 2 (because the SI pattern cannot be focused to anything smaller than half the wavelength of the excitation light). To further increase the resolution, you can introduce nonlinearities, which show up as higher-order harmonics in the FT. In reference [16], Gustafsson uses saturation of the fluorescent sample as the nonlinear effect. A sinusoidal saturating excitation beam produces the distorted fluorescence intensity pattern in the emission. This nonpolynomial nonlinearity yields a series of higher-order harmonics in the FT.
Each higher-order harmonic in the FT allows another set of images that can be used to reconstruct a larger area in reciprocal space, and thus a higher resolution. In this case, Gustafsson achieves less than 50-nm resolving power, more than five times that of the microscope in its normal configuration.
The main problems with SI are that, in this incarnation, saturating excitation powers cause more photodamage and lower fluorophore photostability, and sample drift must be kept to below the resolving distance. The former limitation might be solved by using a different nonlinearity (such as stimulated emission depletion or reversible photoactivation, both of which are used in other sub-diffraction imaging schemes); the latter limits live-cell imaging and may require faster frame rates or the use of some fiduciary markers for drift subtraction. Nevertheless, SI is certainly a strong contender for further application in the field of super-resolution microscopy.

Localization Microscopy/Spatially Structured Illumination Around 1995, Christoph Cremer commenced with the development of a light microscopic process, which achieved a substantially improved size resolution of cellular nanostructures stained with a fluorescent marker. This time he employed the principle of wide field microscopy combined with structured laser illumination (spatially modulated illumination, SMI[17]. Currently, a size resolution of 30 – 40 nm (approximately 1/16 – 1/13 of the wave length used) is being achieved. In addition, this technology is no longer subjected to the speed limitations of the focusing microscopy so that it becomes possible to undertake 3D analyses of whole cells within short observation times (at the moment around a few seconds). Also since around 1995, Christoph Cremer developed and realized new fluorescence based wide field microscopy approaches which had as their goal the improvement of the effective optical resolution (in terms of the smallest detectable distance between two localized objects) down to a fraction of the conventional resolution (spectral precision distance/position determination microscopy, SPDM). Combining SPDM and SMI, known as Vertico-SMI microscopy[18] Christoph Cremer can currently achieve a resolution of approx. 10 nm in 2D and 40 nm in 3D in wide field images of whole living cells[19]. Widefield 3D “nanoimages” of whole living cells currently still take about two minutes, but work to reduce this further is currently under way. Vertico-SMI is currently the fastest optical 3D nanoscope for the three dimensional structural analysis of whole cells world-wide.
Images of cell nuclei and mitotic stages recorded with 3D-SIM Microscopy. Comparison confocal microscopy - 3D-SIM Cell nucleus in prophase from various angles Two mouse cell nuclei in prophase. mouse cell in telophase
Extensions Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and the more experienced microscopist will, in many cases, still prefer a hand drawn image rather than a photograph. This is because a microscopist with knowledge of the subject can accurately convert a three dimensional image into a precise two dimensional drawing . In a photograph or other image capture system however, only one thin plane is ever in good focus.
The creation of careful and accurate micrographs requires a microscopical technique using a monocular eyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is possible to accurately record the observed details by tracing round the observed shapes by simultaneously "seeing" the pencil point in the microscopical image.
Practicing this technique also establishes good general microscopical technique. It is always less tiring to observe with the microscope focused so that the image is seen at infinity and with both eyes open at all times.

Other enhancements Main article: stereomicroscope
X-ray Main article: X-ray microscopy As resolution depends on the wavelength of the light. Electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much smaller wavelength of the electron beam, resolution is far higher.
Though less common, X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and the electron microscopy.

Electron microscopy Main article: Electron microscope For light microscopy the wavelength of the light limits the resolution to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in electron microscopes.
Transmission electron microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit in 2005 was around 0.05 nanometer and has not increased appreciably since that time. Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 3D view. It gives results much like the stereo light microscope and akin to that its most useful magnification is in the lower range than that of the transmission electron microscope.
Atomic de Broglie Main article: Atomic de Broglie microscope The atomic de Broglie microscope is an imaging system which is expected to provide resolution at the nanometer scale using neutral He atoms as probe particles. [20][21]. Such a device could provide the resolution at nanometer scale and be absolutely non-destructive, but it is not developed so well as optical microscope or an electron microscope.

Scanning probe microscopy Main article: Scanning probe microscopy This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the Scanning tunneling microscope and the photonic force microscope. All such methods imply a solid probe tip in the vicinity (near field) of an object, which is supposed to be almost flat.

Ultrasonic force Ultrasonic Force Microscopy (UFM) has been developed in order to improve the details and image contrast on "flat" areas of interest where the AFM images are limited in contrast. The combination of AFM-UFM allows a near field acoustic microscopic image to be generated. The AFM tip is used to detect the ultrasonic waves and overcomes the limitation of wavelength that occurs in acoustic microscopy. By using the elastic changes under the AFM tip, an image of much greater detail than the AFM topography can be generated.
Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by the application of ultrasonic vibration to the cantilever or sample. In an attempt to analyse the results of ultrasonic force microscopy in a quantitative fashion, a force-distance curve measurement is done with ultrasonic vibration applied to the cantilever base, and the results are compared with a model of the cantilever dynamics and tip-sample interaction based on the finite-difference technique.

Infrared microscopy The term infrared microscope covers two main types of diffraction-limited microscopy. The first provides optical visualization plus IR spectroscopic data collection. The second (more recent and more advanced) technique employs focal plane array detection for infrared chemical imaging, where the image contrast is determined by the response of individual sample regions to particular IR wavelengths selected by the user.
IR versions of sub-diffraction microscopy (see above) exist also. These include IR NSOM [22] and photothermal microspectroscopy.

Digital holographic microscopy In digital holographic microscopy (DHM), interfering wave-fronts from a coherent light-source are recorded on a sensor and the image digitally reconstructed by a computer. The image yielded provides a quantitative measurement of the optical thickness of the specimen. DHM can be used with many different optical set-ups. In reflecting DHM, the sensor is positioned on the same side of the specimen as the light source. In transmitting DHM, the sensor and the light source are positioned on opposite sides of the specimen.
One unique feature of DHM is the ability to adjust focus after the image is recorded, since all focus planes are recorded simultaneously by the hologram.

Digital Pathology (virtual microscopy) Main article: Digital Pathology Digital Pathology is an image-based information environment enabled by computer technology that allows for the management of information generated from a digital slide. Digital pathology is enabled in part by virtual microscopy, which is the practice of converting glass slides into digital slides that can be viewed, managed, and analyzed.

Amateur microscopy Amateur Microscopy is the investigation and observation of biological and non-biological specimens for recreational purposes. Collectors of minerals, insects, seashells and plants may use microscopes as tools to uncover features that help them classify their collected items. Other amateurs may be interested in observing the life found in pond water and of other samples. Microscopes may also prove useful for the water quality assessment for people that keep a home aquarium. Photographic documentation and drawing of the microscopic images are additional tasks that augment the spectrum of tasks of the amateur. There are even competitions for photomicrograph art. Participants of this pastime may either use commercially prepared microscopic slides or may engage in the task of specimen preparation.
While microscopy is a central tool in the documentation of biological specimens, it is generally insufficient to justify the description of a new species based on microscopic investigations alone. Often genetic and biochemical tests are necessary to confirm the discovery of a new species. A laboratory and access to academic literature is a necessity, which is specialized and generally not available to amateurs. There is however one huge advantage that amateurs have above professionals: time to explore their surroundings. Often, advanced amateurs team up with professionals to validate their findings and (possibly) describe new species.
In the late 1800s amateur microscopy became a popular hobby in the United States and Europe. Several 'professional amateurs' were being paid for their sampling trips and microscopic explorations by philanthropists, to keep them amused on the Sunday afternoon (e.g. the diatom specialist A. Grunow, being paid by (among others) a Belgian industrialist). Professor John Phin published "Practical Hints on the Selection and Use of the Microscope (Second Edition, 1878)," and was also the editor of the “American Journal of Microscopy.”
In 1995, a loose group of amateur microscopists, drawn from several organizations in the UK and USA, founded a site for microscopy based on the knowledge and input of amateur (perhaps better referred to as 'enthusiast') microscopists. This was historically the first attempt to establish 'amateur' microscopy as a serious subject[citation needed]in the then emerging new media of the Internet. Today, it remains as a powerful established international resource for all ages, to input their findings and share information. It is a non-profit making web presence dedicated to the pursuit of science and understanding of the small-scale world: [1]
Examples of amateur microscopy images:
"house bee" Mouth 100X Rice Stem cs 400X Rabbit Testis 100X Fern Porothallium 400X
See also Acronyms in microscopy Digital Pathology Interferometric microscopy Köhler illumination Timeline of microscope technology Two-photon excitation microscopy
References ^ Abramowitz M, Davidson MW (2007). "Introduction to Microscopy". Molecular Expressions. https://micro.magnet.fsu.edu/primer/anatomy/introduction.html. Retrieved on 2007-08-22. ^ Abramowitz M, Davidson MW (2003-08-01). "Darkfield Illumination". https://micro.magnet.fsu.edu/primer/techniques/darkfield.html. Retrieved on 2008-10-21. ^ Abramowitz M, Davidson MW (2003-08-01). "Rheinberg Illumination". https://micro.magnet.fsu.edu/primer/techniques/rheinberg.html. Retrieved on 2008-10-21. ^ Wallace W, Schaefer LH, Swedlow JR (2001). "A workingperson's guide to deconvolution in light microscopy". BioTechniques 31 (5): 1076–8, 1080, 1082 passim. PMID 11730015. ^ WEM News and Views ^ "Fresnel Diffraction Applet" (Java applet). https://www.falstad.com/diffraction/. Retrieved on 2007-08-22. ^ Cummings JR, Fellers TJ, Davidson MW (2007). "Specialized Microscopy Techniques - Near-Field Scanning Optical Microscopy". Olympus Microscopy Resource Center. https://www.olympusmicro.com/primer/techniques/nearfield/nearfieldintro.html. Retrieved on 2007-08-22. ^ Sánchez EJ, Novotny L, Xie XS (1999). "Near-Field Fluorescence Microscopy Based on Two-Photon Excitation with Metal Tips". Phys Rev Lett 82: 4014–7. doi:10.1103/PhysRevLett.82.4014. https://link.aps.org/abstract/PRL/v82/p4014. ^ Schuck PJ, Fromm DP, Sundaramurthy A, Kino GS, Moerner WE (2005). "Improving the Mismatch between Light and Nanoscale Objects with Gold Bowtie Nanoantennas". Phys Rev Lett 94: 017402. doi:10.1103/PhysRevLett.94.017402. ^ STED ^ Webb paper ^ PALM ^ STORM ^ Bailey, B.; Farkas, D. L.; Taylor, D. L.; Lanni, F. Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 1993, 366, 44–48. ^ Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. of Microsc. 2000, 198(2), 82–87. ^ a b Gustafsson, M. G. L. https://dx.doi.org/10.1073/pnas.0406877102 Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. PNAS 2005, 102(37), 13081–13086. ^ Nano-structure analysis using Spatially Modulated Illumination microscopy: D. Baddeley, C. Batram, Y. Weiland, C. Cremer, U.J. Birk in NATURE PROTOCOLS, Vol 2, pp. 2640 – 2646 (2007) ^ High precision structural analysis of subnuclear complexes in fixed and live cells via Spatially Modulated Illumination (SMI) microscopy: J. Reymann, D. Baddeley, P. Lemmer, W. Stadter, T. Jegou, K. Rippe, C. Cremer, U. Birk in CHROMOSOME RESEARCH, Vol. 16, pp. 367 –382 (2008) ^ SPDM – Light Microscopy with Single Molecule Resolution at the Nanoscale: P. Lemmer, M.Gunkel, D.Baddeley, R. Kaufmann, A. Urich, Y. Weiland, J.Reymann, P. Müller, M. Hausmann, C. Cremer in APPLIED PHYSICS B, Vol 93, pp. 1-12 (2008). ^ D.Kouznetsov; H. Oberst, K. Shimizu, A. Neumann, Y. Kuznetsova, J.-F. Bisson, K. Ueda, S. R. J. Brueck (2006). "Ridged atomic mirrors and atomic nanoscope". JOPB 39: 1605–1623. doi:10.1088/0953-4075/39/7/005. https://stacks.iop.org/0953-4075/39/1605. ^ Atom Optics and Helium Atom Microscopy. Cambridge University, https://www-sp.phy.cam.ac.uk/research/mirror.php3 ^ H M Pollock and D A Smith, The use of near-field probes for vibrational spectroscopy and photothermal imaging, in Handbook of vibrational spectroscopy, J.M. Chalmers and P.R. Griffiths (eds), John Wiley & Sons Ltd, Vol. 2, pp. 1472 - 1492 (2002)
Further reading Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier (1988) Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier (1989) Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers (1998) Video Microscopy by Shinya Inoue, Plenum Press (1986) Portraits of life, one molecule at a time, a feature article on sub-diffraction microscopy from the March 1, 2007 issue of Analytical Chemistry
External links
General Olympus Microscopy Resource Center Nikon MicroscopyU Andor Microscopy Techniques - Various techniques used in microscopy. Carl Zeiss "Microscopy from the very beginning", a step by step tutorial into the basics of microscopy. Microscopy in Detail - A resource with many illustrations elaborating the most common microscopy techniques Microscopy Information - Microscopy information and techniques for teachers, educators and enthusiasts. WITec SNOM System - NSOM/SNOM and Hybrid Microscopy techniques in combination with AFM, RAMAN, Confocal, Dark-field, DIC & Fluorescence Microscopy techniques. Manawatu Microscopy - first known collaboration environment for Microscopy and Image Analysis.
Techniques Ratio-metric Imaging Applications For Microscopes Examples of Ratiometric Imaging Work on a Microscope Interactive Fluorescence Dye and Filter Database Carl Zeiss Interactive Fluorescence Dye and Filter Database. Images formed by simple microscopes - examples of observations with single-lens microscopes.
Organizations Royal Microscopical Society (RMS) Microscopy Society of America (MSA) European Microscopy Society (EMS) Non-membership International online organisation (Mic-UK)

郑振寰 · 发表于 2010-3-4 13:03

Objective (optics)

Several objective lenses on a microscope.

In optics, an objective is the lens or mirror in a microscope, telescope, camera or other optical instrument that gathers the light coming from the object being observed, and focuses the rays to produce a real image. The objective is also called the object lens, object glass, and objective glass.

Microscope objectives are typically designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in focus. Microscope objectives are characterized by two parameters, namely, magnification and numerical aperture. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. For high magnification applications, an oil-immersion objective or water-immersion objective has to be used. The objective is specially designed and refractive index matching oil or water must fill the air gap between the front element and the object to allow the numerical aperture to exceed 1, and hence give greater resolution at high magnification. Numerical apertures as high as 1.6 can be achieved with oil immersion.^[1]

To find the total magnification of a microscope, one multiplies the magnification of the objective lenses by that of the eyepiece.

References

^ Kenneth, Spring; Keller, H. Ernst; Davidson, Michael W.. "Microscope objectives". Olympus Microscopy Resource Center. https://www.olympusmicro.com/primer/anatomy/objectives.html. Retrieved on 29 Oct 2008.

This optics-related article is a stub. You can help Wikipedia by expanding it

郑振寰 · 发表于 2010-3-4 13:05

Oil immersion

Principle of immersion microscopy. Path of rays with immersion medium (yellow) (left half) and without (right half). Rays (black) coming from the object (red) at a certain angle and going through the coverslip (orange, as the slide at the bottom) can enter the objective (dark blue) only when immersion is used. Otherwise, the refraction at the coverslip - air interface causes the ray to miss the objective and its information is lost.

In light microscopy, oil immersion is a technique used to increase the resolution of a microscope. This is achieved by immersing both the objective lens and the specimen in a transparent oil of high refractive index, thereby increasing the numerical aperture of the objective lens.

Immersion oils are transparent oils that have specific optical and viscosity characteristics necessary for use in microscopy. An oil immersion objective is an objective lens specially designed to be used in this way. Many condensers also give optimal resolution when the condenser lens is immersed in oil.

Theoretical background

The resolution of a microscope is defined as the minimum separation needed between two objects under examination in order for the microscope to discern them as separate objects. This minimum distance is labeled δ. If two objects are separated by a distance shorter than δ, they will appear as a single object in the microscope.

A measure of the resolving power of a lens is given by its numerical aperture, NA:

where λ is the wavelength of light. From this it is clear that a good resolution (small δ) is connected with a high numerical aperture.

The numerical aperture of a lens is defined as

NA = nsin α 0

where α₀ is the angle spanned by the objective lens seen from the sample, and n is the refractive index of the medium between the lens and specimen (≈1 for air).

State of the art objectives can have a numerical aperture of up to 0.95. Because sin α₀ is always less than or equal to unity, the numerical aperture can never be greater than unity for an objective lens in air. If the space between the objective lens and the specimen is filled with oil however, the numerical aperture can obtain values greater than unity. This is because oil has a refractive index greater than 1.

Oil immersion objectives

From the above it is understood that oil between the specimen and the objective lens improves the resolving power by a factor 1/n. Objectives specifically designed for this purpose are known as oil immersion objectives.

Oil immersion objectives are used only at very large magnifications that require high resolving power. Objectives with high power magnification have short focal lengths, facilitating the use of oil. The oil is applied to the specimen (conventional microscope), and the stage is raised, immersing the objective in oil. (In inverted microscopes the oil is applied to the objective).

The refractive indices of the oil and of the glass in the first lens element are nearly the same, which means that the refraction of light will be small upon entering the lens (the oil and glass are optically very similar). Oil immersion objectives are designed with this in mind, and can not be used without oil, because in this case there will be much refraction of light at the glass/air interface, drastically changing the path of light entering the lens and making it impossible to achieve focus. Nevertheless, in situations in which not maximal clarity but a closer general view is preferred (for instance, where magnifications must be changed back and forth during the observation of a delicately mounted slide), the oil can be skipped at the cost of image sharpness. Using the oil immersion objective with oil can only be done on fixed specimens.

Another advantage of using oil is that it reduces reflective losses as light enters the lens (again because the oil and glass are optically alike).

Oil immersion and the condenser

Immersion oil

Cedar tree oil, has an index of refraction of approximately 1.516. The numerical aperture of cedar tree oil objectives is generally around 1.3. In modern microscopy, synthetic immersion oils are more commonly used. NA values of 1.6 can be achieved with different oils.

References

Practical Microscopy by L.C. Martin and B.K. Johnson , Glasgow (1966).
Light Microscopy by J.K. Solberg, Tapir Trykk (2000).

External links

"Microscope Objectives: Immersion Media" by Mortimer Abramowitz and Michael W. Davidson, Olympus Microscopy Resource Center (website), 2002.
"Immersion Oil Microscopy" by David B. Fankhauser, Biology at University of Cincinnati, Clermont College (website), December 30, 2004.
"History of Oil Immersion Lenses" by Jim Solliday, Southwest Museum of Engineering, Communications, and Computation (website), 2007.
"Immersion Oil and the Microscope" by John J. Cargille, New York Microscopical Society Yearbook, 1964 (revised, 1985). (Archived at Cargille Labs (website).)

郑振寰 · 发表于 2010-3-4 13:07

Digital microscope

A digital microscope uses optics and a charge-coupled device (CCD) camera to output a digital image to a monitor. A digital microscope differs from an optical microscope in that there is no provision to observe the sample directly through an eyepiece. Since the optical image is projected directly on the CCD camera, the entire system is designed for the monitor image. The optics for the human eye are omitted.

History

A digital microscope.

The first digital microscope was made by a lens company in Tokyo, Japan in 1986. This company is now know as Hirox Co LTd. Hirox's main industry is digital microscopes, but still makes lenses. Hirox's current digital microscope systems are the KH-7700 and the KH-1300. The KH-7700 system has such features as 3D rotation and High Dynamic Range. Shortly after the invention of the digital microscope, a digital sensor company in Osaka, Japan created a digital microscope. This company is now know as the Keyence Corporation. Keyence offers digital automated sensors, barcode readers, industrial laser markers, laser displacement sensors, optical micrometers, digital microscopes, and confocal laser scanning microscopes.

With the invention of the USB port, there came a flood of digital microscopes that connected directly to the computer. This invention has resulted in numerous companies creating some form of USB digital microscope that range in quality and magnification. They continue to fall in price, especially compared with conventional optical microscopes.

The digital microscope continues to evolve as the technology improves.

Optical and Digital Microscopes

A primary difference between an optical microscope and a digital microscope is the magnification. With an optical microscope the magnification is found by multiplying the lens magnification by the eyepiece magnification. Since the digital microscope does not have an eyepiece, the magnification cannot be found using this method. Instead the magnification for a digital microscope is found by how many times larger the sample is reproduced on the monitor. Therefore the magnification will depend on the size of the monitor. The average digital microscope system has a 15" monitor, would result in a average difference in magnification between an optical microscope and a digital microscope of about 60%. Thus the magnification number of an optical microscope is usually 60% larger than the magnification number of an digital microscope.

Since the digital microscope has the image projected directly on to the CCD camera, it is possible to have higher quality recorded images than with an optical microscope. With the optical microscope, the lenses are made for the optics of the eye. Attaching a CCD camera to an optical microscope will result in a image that has compromises made for the eyepiece. Although the monitor image and recorded image may be of higher quality compared with the digital microscope, the application for the microscope may dictate which microscope is preferred.

Resolution

Resolution of the image is dependent on the CCD used in the camera. Using a typical 2 Megapixel CCD, an image with 1600 x 1200 pixels is generated. The resolution of the image is dependent on the field of view of the lens used with the camera. The approximate pixel resolution can be determined by dividing the horizontal field of view (FOV) by 1600. Most common instruments have a relatively low resolution of 1.3 Megapixels, but higher resolution cameras are available.

Increased resolution can be accomplished by creating a sub-pixel image. The Pixel Shift Method uses an actuator to physically move the CCD in order to take multiple overlapping images. By combining the images within the microscope, sub-pixel resolution can be generated. This method provides real sub-pixel information, as compared to simply averaging a standard image. (This is also called Pixel Extrapolation.)

Limitations

Few current models are supplied with stands capable of supporting the camera in a stable way, so it can be racked up and down, for example. But this is only true with USB microscopes. The higher end digital microscopes do not suffer this limitation.

左岸 · 发表于 2010-4-4 14:14

right

		自动登录	找回密码
密码			立即注册

[转帖]显微镜相关背景知识--英文

Optical microscope

Contents

Optical configurations

Light microscope

History

Components

Operation

Stereo microscope

Digital display with stereo microscopes

Digital microscopes

Special designs

Limitations

Alternatives

See also

References

External links

本帖子中包含更多资源

本帖子中包含更多资源

本帖子中包含更多资源

Objective (optics)

References

本帖子中包含更多资源

Oil immersion

Contents

Theoretical background

Oil immersion objectives

Oil immersion and the condenser

Immersion oil

See also

References

External links

本帖子中包含更多资源

Digital microscope

Contents

History

Optical and Digital Microscopes

Resolution

Limitations

See also

External links

本帖子中包含更多资源