Microscopy
Overview
 
Microscopy is the technical field of using 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...

s to view samples and objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy, optical, electron, and scanning probe microscopy
Scanning probe microscopy
Scanning Probe Microscopy is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. An image of the surface is obtained by mechanically moving the probe in a raster scan of the specimen, line by line, and recording the probe-surface interaction as a...

.

Optical and electron microscopy involve the 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...

, reflection
Reflection (physics)
Reflection is the change in direction of a wavefront at an interface between two differentmedia so that the wavefront returns into the medium from which it originated. Common examples include the reflection of light, sound and water waves...

, or refraction
Refraction
Refraction is the change in direction of a wave due to a change in its speed. It is essentially a surface phenomenon . The phenomenon is mainly in governance to the law of conservation of energy. The proper explanation would be that due to change of medium, the phase velocity of the wave is changed...

 of electromagnetic radiation
Electromagnetic radiation
Electromagnetic radiation is a form of energy that exhibits wave-like behavior as it travels through space...

/electron beams interacting with the specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...

, and the subsequent collection of this scattered radiation or another signal in order to create an image.
Discussions
Encyclopedia
Microscopy is the technical field of using 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...

s to view samples and objects that cannot be seen with the unaided eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy, optical, electron, and scanning probe microscopy
Scanning probe microscopy
Scanning Probe Microscopy is a branch of microscopy that forms images of surfaces using a physical probe that scans the specimen. An image of the surface is obtained by mechanically moving the probe in a raster scan of the specimen, line by line, and recording the probe-surface interaction as a...

.

Optical and electron microscopy involve the 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...

, reflection
Reflection (physics)
Reflection is the change in direction of a wavefront at an interface between two differentmedia so that the wavefront returns into the medium from which it originated. Common examples include the reflection of light, sound and water waves...

, or refraction
Refraction
Refraction is the change in direction of a wave due to a change in its speed. It is essentially a surface phenomenon . The phenomenon is mainly in governance to the law of conservation of energy. The proper explanation would be that due to change of medium, the phase velocity of the wave is changed...

 of electromagnetic radiation
Electromagnetic radiation
Electromagnetic radiation is a form of energy that exhibits wave-like behavior as it travels through space...

/electron beams interacting with the specimen
Specimen
A specimen is a portion/quantity of material for use in testing, examination, or study.BiologyA laboratory specimen is an individual animal, part of an animal, a plant, part of a plant, or a microorganism, used as a representative to study the properties of the whole population of that species or...

, and the subsequent collection of this scattered radiation or another signal in order to create 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
Confocal laser scanning microscopy
Confocal laser scanning microscopy is a technique for obtaining high-resolution optical images with depth selectivity. The key feature of confocal microscopy is its ability to acquire in-focus images from selected depths, a process known as optical sectioning...

 and scanning electron microscopy). Scanning probe microscopy involves the interaction of a scanning probe with the surface of the object of interest. The development of microscopy revolutionized biology
Biology
Biology is a natural science concerned with the study of life and living organisms, including their structure, function, growth, origin, evolution, distribution, and taxonomy. Biology is a vast subject containing many subdivisions, topics, and disciplines...

 and remains an essential technique in the life
Life sciences
The life sciences comprise the fields of science that involve the scientific study of living organisms, like plants, animals, and human beings. While biology remains the centerpiece of the life sciences, technological advances in molecular biology and biotechnology have led to a burgeoning of...

 and physical science
Physical science
Physical science is an encompassing term for the branches of natural science and science that study non-living systems, in contrast to the life sciences...

s.

Optical microscopy

Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single or multiple 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 allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate
Photographic plate
Photographic plates preceded photographic film as a means of photography. A light-sensitive emulsion of silver salts was applied to a glass plate. This form of photographic material largely faded from the consumer market in the early years of the 20th century, as more convenient and less fragile...

 or captured digitally
Digital imaging
Digital imaging or digital image acquisition is the creation of digital images, typically from a physical scene. The term is often assumed to imply or include the processing, compression, storage, printing, and display of such images...

. 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
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...

, which uses a CCD camera to focus on the exhibit of interest. The image is shown on a computer screen, so eye-pieces are unnecessary.

Limitations

Limitations of standard optical microscopy (bright field microscopy
Bright field microscopy
Bright field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light and contrast in the sample is caused by absorbance of some of the transmitted light in dense areas of the sample...

) lie in three areas;
  • The technique can only image dark or strongly refracting objects effectively.
  • 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...

     limits resolution to approximately 0.2 micrometre
    Micrometre
    A micrometer , is by definition 1×10-6 of a meter .In plain English, it means one-millionth of a meter . Its unit symbol in the International System of Units is μm...

    s (see: 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...

    ).
  • 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
Staining (biology)
Staining is an auxiliary technique used in microscopy to enhance contrast in the microscopic image. Stains and dyes are frequently used in biology and medicine to highlight structures in biological tissues for viewing, often with the aid of different microscopes...

 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 that 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

In order to improve specimen 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...

 or highlight certain structures in a sample special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample.

Bright field

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
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...

 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

Dark field microscopy is a technique for improving the contrast of unstained, transparent specimens. 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.

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

In electron microscopy: Phase-contrast imaging
Phase-contrast imaging
Phase-contrast imaging is a method of imaging that has a range of different applications. It exploits differences in the refractive index of different materials to differentiate between structures under analysis...


More sophisticated techniques will show proportional differences in optical density . Phase contrast is a widely used technique that shows differences in refractive index
Refractive index
In optics the refractive index or index of refraction of a substance or medium is a measure of the speed of light in that medium. It is expressed as a ratio of the speed of light in vacuum relative to that in the considered medium....

 as difference in contrast. It was developed by the Dutch physicist Frits Zernike
Frits Zernike
Frits Zernike was a Dutch physicist and winner of the Nobel prize for physics in 1953 for his invention of the phase contrast microscope, an instrument that permits the study of internal cell structure without the need to stain and thus kill the cells....

 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.

one disadvantge of phasecontrast microscopy is halo formation ( halo-light ring)

Differential interference contrast

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
Georges Nomarski
Georges Nomarski was a Polish physicist and optics theoretician. Creator of Nomarski Interference Contrast or differential interference contrast microscopy , the method is widely used to study live biological specimens and unstained tissues.-Biography:Born in Nowy Targ, Nomarski was educated in...

. 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
Nomarski prism
A Nomarski prism is a modification of the Wollaston prism that is used in differential interference contrast microscopy. It is named after its inventor, Polish physicist Georges Nomarski. Like the Wollaston prism, the Nomarski prism consists of two birefringent crystal wedges cemented together at...

, Wollaston prism
Wollaston prism
A Wollaston prism is an optical device, invented by William Hyde Wollaston, that manipulates polarized light. It separates randomly polarized or unpolarized light into two orthogonal linearly polarized outgoing beams....

) 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
Classical interference microscopy
Classical interference microscopy uses two separate light beams with much greater lateral separation than that used in phase contrast microscopy or in differential interference microscopy ....

, which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

Interference reflection microscopy

An additional technique using interference is interference reflection microscopy (also known as reflected interference contrast, or RIC). It is used to examine the adhesion of cells to a glass surface, using polarized light of a narrow range of wavelengths to be reflected whenever there is an interface between two substances with different refractive indices. Whenever a cell is attached to the glass surface, reflected light from the glass and from the attached cell will interfere, while if there is no cell attached to the glass, there will be no interference.

Interference reflection microscopy can be obtained by using the same elements used by DIC, but without the prisms. Also, the light that is being detected is reflected and not transmitted as it is when DIC is employed.

Fluorescence

When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency. This effect is known as fluorescence
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

. Often specimens show their own characteristic autofluorescence
Autofluorescence
Autofluorescence is the natural emission of light by biological entities such as mitochondria and lysosomes, and is used to distinguish the light originating from artificially added fluorescent markers...

 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 dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....

s can be used to stain different structures or chemical compounds. One particularly powerful method is the combination of antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...

 coupled to a fluorophore as in immunostaining
Immunostaining
Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...

. Examples of commonly used fluorophores are fluorescein
Fluorescein
Fluorescein is a synthetic organic compound available as a dark orange/red powder soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications....

 or rhodamine
Rhodamine
Rhodamine is a family of related chemical compounds, fluorone dyes. Examples are Rhodamine 6G and Rhodamine B. They are used as a dye and as a dye laser gain medium. They are often used as a tracer dye within water to determine the rate and direction of flow and transport...

.
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 fluorophore and then used to trace the proteins in the cells under study.

Highly-efficient fluorescent protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...

s such as the green fluorescent protein
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...

 (GFP) have been developed using the molecular biology
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...

 technique of gene fusion
Fusion gene
A fusion gene is a hybrid gene formed from two previously separate genes. It can occur as the result of a translocation, interstitial deletion, or chromosomal inversion...

, a process that links the expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...

 of the fluorescent compound to that of the target protein. This combined fluorescent protein is, in general, 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
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...

.

Growth of protein crystals
Protein crystallization
Most Proteins and many biological macromolecules differ from "small" molecules because the environment in which they function is aqueous. Therefore most biological macromolecules can be prompted to form crystals when the solution in which they are dissolved becomes supersaturated. The manner in...

 results in both protein and salt crystals. Both are colorless and microscopic. Recovery of the protein crystals requires imaging which can be done by the intrinsic fluorescence of the protein or by using transmission microscopy. Both methods require a ultraviolet microscope as protein absorbs light at 280 nm. Protein will also fluorescence at approximately 353 nm when excited with 280 nm light.

Since fluorescence emission
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...

 differs in 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...

 (color) from the excitation light, an ideal fluorescent image shows only 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
Filter (optics)
Optical filters are devices which selectively transmit light of different wavelengths, usually implemented as plane glass or plastic devices in the optical path which are either dyed in the mass or have interference coatings....

 of high quality are needed. These typically consist of an excitation filter
Excitation filter
An excitation filter is a high quality optical-glass filter commonly used in fluorescence microscopy and spectroscopic applications for selection of the excitation wavelength of light from a light source...

 selecting the range of excitation 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...

s, a dichroic
Dichroism
Dichroism has two related but distinct meanings in optics. A dichroic material is either one which causes visible light to be split up into distinct beams of different wavelengths , or one in which light rays having different polarizations are absorbed by different amounts.The original meaning of...

 mirror, and an emission filter blocking the excitation light. Most fluorescence 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...

s 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
Total internal reflection fluorescence microscope
A total internal reflection fluorescence microscope is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.-Background:...

.

Confocal

Using a scanning point of light instead of full sample illumination confocal microscopy gives slightly higher resolution, and significant improvements in optical sectioning
Optical sectioning
Optical sectioning is the process by which a suitably designed microscope can produce clear images of a focal planes deep within a thick sample. This is used to reduce the need for thin sectioning using instruments such as the microtome...

 . Confocal microscopy is, therefore, commonly used where 3D structure is important.

Single plane illumination microscopy and Light sheet florescence microscopy

Using a plane of light formed by focusing light through a cylindrical lens at a narrow angle or by scanning a line of light in a plane perpendicular to the axis of objective, high resolution optical sections can be taken. Single plane illumination is also accomplished using beam shaping techniques incorporating multiple-prism beam expanders. The images are captured by CCDs. These variants allow very fast and high signal to noise ratio image capture.

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.
However, this information is blurred by the fact that, upon illumination, all fluorescently labeled structures emit light no matter whether 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 that 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.

However, 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
Point source
A point source is a localised, relatively small source of something.Point source may also refer to:*Point source , a localised source of pollution**Point source water pollution, water pollution with a localized source...

 in the third (axial) dimension. This shape is called the 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) 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
Deconvolution
In mathematics, deconvolution is an algorithm-based process used to reverse the effects of convolution on recorded data. The concept of deconvolution is widely used in the techniques of signal processing and image processing...

 microscopy. There are various algorithms available for 2D or 3D deconvolution. They can be roughly classified in nonrestorative and restorative methods. While the nonrestorative methods can improve contrast by removing out-of-focus light from focal planes, only the restorative methods can actually reassign light to its 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 derived either experimentally or theoretically from knowing all contributing parameters of the microscope.

Sub-diffraction techniques

A multitude of super-resolution microscopy techniques has been developed in recent times which circumvent the diffraction barrier.
This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastical changes in the image.
Knowledge of and chemical control over fluorophore photophysics is at the core of these techniques, by which resolutions of ~20 nanometers are regularly obtained.

Serial time-encoded amplified microscopy (STEAM)

Serial time encoded amplified microscopy (STEAM)
Serial time encoded amplified microscopy (STEAM)
Serial time-encoded amplified imaging/microscopy is an ultrafast optical imaging method that provides ~10 MHz frame rate, ~100 ps shutter speed, and ~30 dB optical image gain. As of today, STEAM holds world records for shutter speed and frame rate in continuous real-time imaging...

 is an imaging method that provides ultrafast shutter speed and frame rate, by using optical image amplification to circumvent the fundamental trade-off between sensitivity and speed, and a single-pixel photodetector
Photodetector
Photosensors or photodetectors are sensors of light or other electromagnetic energy. There are several varieties:*Active pixel sensors are image sensors consisting of an integrated circuit that contains an array of pixel sensors, each pixel containing a both a light sensor and an active amplifier...

 to eliminate the need for a detector array and readout time limitations The method is at least 1000 times faster than the state-of-the-art CCD
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...

 and 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...

 cameras. Consequently, it is potentially useful for a broad range of scientific, industrial, and biomedical applications that require high image acquisition rates, including real-time diagnosis and evaluation of shockwaves, microfluidics
Microfluidics
Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-millimeter, scale.Typically, micro means one of the following features:* small volumes...

, MEMS, and laser surgery
Laser surgery
Laser surgery is surgery using a laser to cut tissue instead of a scalpel. Examples include the use of a laser scalpel in otherwise conventional surgery, and soft tissue laser surgery, in which the laser beam vaporizes soft tissue with high water content...

.

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.
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