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Microscopy is the technical field of using
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible ...
s to view objects and areas of objects that cannot be seen with the naked 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 Scan may refer to: Acronyms * Schedules for Clinical Assessment in Neuropsychiatry (SCAN), a psychiatric diagnostic tool developed by WHO * Shared Check Authorization Network (SCAN), a database of bad check writers and collection agency for b ...
, along with the emerging field of
X-ray microscopy An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations. Unlike visible li ...
. Optical microscopy and electron microscopy involve the
diffraction Diffraction is defined as the interference or bending of waves around the corners of an obstacle or through an aperture into the region of geometrical shadow of the obstacle/aperture. The diffracting object or aperture effectively becomes a s ...
, reflection, or refraction of
electromagnetic radiation In physics, electromagnetic radiation (EMR) consists of waves of the electromagnetic (EM) field, which propagate through space and carry momentum and electromagnetic radiant energy. It includes radio waves, microwaves, infrared, (visible) lig ...
/electron beams interacting with the specimen, and the collection of the 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 Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a ...
) or by scanning a fine beam over the sample (for example confocal laser scanning microscopy and scanning electron microscopy).
Scanning probe microscopy Scan may refer to: Acronyms * Schedules for Clinical Assessment in Neuropsychiatry (SCAN), a psychiatric diagnostic tool developed by WHO * Shared Check Authorization Network (SCAN), a database of bad check writers and collection agency for b ...
involves the interaction of a scanning probe with the surface of the object of interest. The development of microscopy revolutionized
biology Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary in ...
, gave rise to the field of histology and so remains an essential technique in the
life Life is a quality that distinguishes matter that has biological processes, such as signaling and self-sustaining processes, from that which does not, and is defined by the capacity for growth, reaction to stimuli, metabolism, energy tra ...
and
physical science Physical science is a branch of natural science that studies non-living systems, in contrast to life science. It in turn has many branches, each referred to as a "physical science", together called the "physical sciences". Definition Phy ...
s. X-ray microscopy is three-dimensional and non-destructive, allowing for repeated imaging of the same sample for
in situ ''In situ'' (; often not italicized in English) is a Latin phrase that translates literally to "on site" or "in position." It can mean "locally", "on site", "on the premises", or "in place" to describe where an event takes place and is used in ...
or 4D studies, and providing the ability to "see inside" the sample being studied before sacrificing it to higher resolution techniques. A 3D X-ray microscope uses the technique of computed tomography (
microCT X-ray microtomography, like tomography and X-ray computed tomography, uses X-rays to create cross-sections of a physical object that can be used to recreate a virtual model (3D model) without destroying the original object. The prefix ''micro-'' ...
), rotating the sample 360 degrees and reconstructing the images. CT is typically carried out with a flat panel display. A 3D X-ray microscope employs a range of objectives, e.g., from 4X to 40X, and can also include a flat panel.

# History

The field of microscopy ( optical microscopy) dates back to at least the 17th-century. Earlier microscopes, single
lens A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements ...
magnifying glass A magnifying glass is a convex lens that is used to produce a magnified image of an object. The lens is usually mounted in a frame with a handle. A magnifying glass can be used to focus light, such as to concentrate the sun's radiation to crea ...
es with limited magnification, date at least as far back as the wide spread use of lenses in
eyeglasses Glasses, also known as eyeglasses or spectacles, are vision eyewear, with lenses (clear or tinted) mounted in a frame that holds them in front of a person's eyes, typically utilizing a bridge over the nose and hinged arms (known as temples ...
in the 13th century but more advanced
compound microscope The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microsc ...
s first appeared in Europe around 1620 The earliest practitioners of microscopy include
Galileo Galilei Galileo di Vincenzo Bonaiuti de' Galilei (15 February 1564 – 8 January 1642) was an Italian astronomer, physicist and engineer, sometimes described as a polymath. Commonly referred to as Galileo, his name was pronounced (, ). He was ...
, who found in 1610 that he could close focus his telescope to view small objects close up and
Cornelis Drebbel Cornelis Jacobszoon Drebbel ( ) (1572 – 7 November 1633) was a Dutch engineer and inventor. He was the builder of the first operational submarine in 1620 and an innovator who contributed to the development of measurement and control systems, ...
, who may have invented the compound microscope around 1620Raymond J. Seeger, Men of Physics: Galileo Galilei, His Life and His Works, Elsevier - 2016, page 24J. William Rosenthal, Spectacles and Other Vision Aids: A History and Guide to Collecting, Norman Publishing, 1996, page 391 Antonie van Leeuwenhoek developed a very high magnification simple microscope in the 1670s and is often considered to be the first acknowledged
microscopist Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
and microbiologist.

# Optical microscopy

Optical or light microscopy involves passing
visible light Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 tera ...
transmitted through or reflected from the sample through a single lens or multiple
lenses A lens is a transmissive optical device which focuses or disperses a light beam by means of refraction. A simple lens consists of a single piece of transparent material, while a compound lens consists of several simple lenses (''elements'' ...
to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a
photographic plate Photographic plates preceded photographic film as a capture medium in photography, and were still used in some communities up until the late 20th century. The light-sensitive emulsion of silver salts was coated on a glass plate, typically thin ...
, 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, 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. * There is a diffraction-limited resolution depending on incident wavelength; in visible range, the resolution of optical microscopy is limited to approximately 0.2
micrometre The micrometre ( international spelling as used by the International Bureau of Weights and Measures; SI symbol: μm) or micrometer ( American spelling), also commonly known as a micron, is a unit of length in the International System of Un ...
s (''see:
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible ...
'') and the practical magnification limit to ~1500x. * 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, since the internal structures of the cell are colorless and transparent. The most common way to increase contrast is to
stain A stain is a discoloration that can be clearly distinguished from the surface, material, or medium it is found upon. They are caused by the chemical or physical interaction of two dissimilar materials. Accidental staining may make materials ap ...
the structures with selective dyes, but this often involves killing and
fixing Fixing may refer to: * The present participle of the verb "to fix", an action meaning maintenance, repair, and operations * "fixing someone up" in the context of arranging or finding a social date for someone * "Fixing", craving an addictive drug ...
the sample. Staining may also introduce artifacts, which are apparent structural details that are caused by the processing of the specimen and are thus not features of the specimen. In general, these techniques make use of differences in the refractive index of cell structures. Bright-field microscopy is comparable to looking through a glass window: one sees not the glass but merely the dirt on the glass. There is 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 devised to change this difference in phase into a difference in amplitude (light intensity).

## Techniques

To improve specimen contrast or highlight structures in a sample, special techniques must be used. A huge selection of microscopy techniques are available to increase contrast or label a sample. tissue paper Tissue paper or simply tissue is a lightweight paper or, light crêpe paper. Tissue can be made from recycled paper pulp on a paper machine. Tissue paper is very versatile, and different kinds of tissue are made to best serve these purposes, ...
. 1.559 μm/pixel." align="center"> Image:Paper_Micrograph_Bright.png, Bright field illumination, sample contrast comes from
absorbance Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative lo ...
of light in the sample Image:Paper_Micrograph_Cross-Polarised.png, Cross-polarized light illumination, sample contrast comes from rotation of polarized light through the sample Image:Paper_Micrograph_Dark.png,
Dark field Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is ...
illumination, sample contrast comes from light scattered by the sample Image:Paper_Micrograph_Phase.png, Phase contrast illumination, sample contrast comes from
interference Interference is the act of interfering, invading, or poaching. Interference may also refer to: Communications * Interference (communication), anything which alters, modifies, or disrupts a message * Adjacent-channel interference, caused by extr ...
of different path lengths of light through the 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 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 three-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 enhances contrast even in clear specimens, however, because light enters off-axis, the position of an object will appear to shift as the focus is changed. This limitation makes techniques like optical sectioning or accurate measurement on the z-axis impossible.

### 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. Dark field can dramatically improve image contrast – especially of transparent objects – while requiring little equipment setup or sample preparation. However, the technique suffers from low light intensity in the final image of many biological samples and continues to be affected by low apparent resolution. ''Rheinberg illumination'' is a 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 red). 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'' More sophisticated techniques will show proportional differences in optical density. Phase contrast is a widely used technique that shows differences in
refractive index In optics, the refractive index (or refraction index) of an optical medium is a dimensionless number that gives the indication of the light bending ability of that medium. The refractive index determines how much the path of light is bent, o ...
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. One disadvantage of phase-contrast 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. 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 Polarization ( also polarisation) is a property applying to transverse waves that specifies the geometrical orientation of the oscillations. In a transverse wave, the direction of the oscillation is perpendicular to the direction of motion of t ...
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, also called quantitative interference microscopy, uses two separate light beams with much greater lateral separation than that used in phase contrast microscopy or in differential interference microscopy (DIC). ...
, which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

### Interference reflection

An additional technique using interference is interference reflection microscopy (also known as reflected interference contrast, or RIC). It relies on cell adhesion to the slide to produce an interference signal. 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 emit light of a lower frequency. This effect is known as
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
. Often specimens show their characteristic
autofluorescence Autofluorescence is the natural emission of light by biological structures such as mitochondria and lysosomes when they have absorbed light, and is used to distinguish the light originating from artificially added fluorescent markers (fluorophores) ...
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 fluorescent
dye A dye is a colored substance that chemically bonds to the substrate to which it is being applied. This distinguishes dyes from pigments which do not chemically bind to the material they color. Dye is generally applied in an aqueous solution and ...
s can be used to stain structures or chemical compounds. One powerful method is the combination of
antibodies An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
coupled to a fluorophore as in immunostaining. Examples of commonly used fluorophores are
fluorescein Fluorescein is an organic compound and dye based on the xanthene tricyclic structural motif, formally belonging to triarylmethine dyes family. It is available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used ...
or
rhodamine Rhodamine is a family of related dyes, a subset of the triarylmethane dyes. They are derivatives of xanthene. Important members of the rhodamine family are Rhodamine 6G, Rhodamine 123, and Rhodamine B. They are mainly used to dye paper and ...
. The antibodies can be tailor-made 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, forming antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorophore and used to trace the proteins in the cells under study. Highly efficient fluorescent proteins such as the
green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish '' Aequ ...
(GFP) have been developed using the
molecular biology Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physica ...
technique of gene fusion, a process that links the
expression Expression may refer to: Linguistics * Expression (linguistics), a word, phrase, or sentence * Fixed expression, a form of words with a specific meaning * Idiom, a type of fixed expression * Metaphorical expression, a particular word, phrase, o ...
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 Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and p ...
. Growth of protein crystals 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 an 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 differs in wavelength (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 of high quality are needed. These typically consist of an
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. Most excitation filters select light of re ...
selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible ...
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 A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cel ...
Neuroscience Neuroscience is the scientific study of the nervous system (the brain, spinal cord, and peripheral nervous system), its functions and disorders. It is a multidisciplinary science that combines physiology, anatomy, molecular biology, developmen ...

### Confocal

Confocal laser scanning microscopy uses a focused laser beam (e.g. 488 nm) that is scanned across the sample to excite
fluorescence Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, ...
in a point-by-point fashion. The emitted light is directed through a pinhole to prevent out-of-focus light from reaching the detector, typically a
photomultiplier tube Photomultiplier tubes (photomultipliers or PMTs for short) are extremely sensitive detectors of light in the ultraviolet, visible, and near-infrared ranges of the electromagnetic spectrum. They are members of the class of vacuum tubes, more speci ...
. The image is constructed in a computer, plotting the measured fluorescence intensities according to the position of the excitation laser. Compared to full sample illumination, confocal microscopy gives slightly higher lateral resolution and significantly improves optical sectioning (axial resolution). Confocal microscopy is, therefore, commonly used where 3D structure is important. A subclass of confocal microscopes are spinning disc microscopes which are able to scan multiple points simultaneously across the sample. A corresponding disc with pinholes rejects out-of-focus light. The light detector in a spinning disc microscope is a digital camera, typically EM-CCD or
sCMOS sCMOS (''scientific Complementary Metal–Oxide–Semiconductor'') are a type of CMOS image sensor (CIS). These sensors are commonly used as components in specific observational scientific instruments, such as microscopes and telescopes. sCMOS ...
.

### Two-photon microscopy

A two-photon microscope is also a laser-scanning microscope, but instead of UV, blue or green laser light, a pulsed infrared laser is used for excitation. Only in the tiny focus of the laser is the intensity high enough to generate fluorescence by two-photon excitation, which means that no out-of-focus fluorescence is generated, and no pinhole is necessary to clean up the image. This allows imaging deep in scattering tissue, where a confocal microscope would not be able to collect photons efficiently. Two-photon microscopes with wide-field detection are frequently used for functional imaging, e.g.
calcium imaging Calcium imaging is a microscopy technique to optically measure the calcium (Ca2+) status of an isolated cell, tissue or medium. Calcium imaging takes advantage of calcium indicators, fluorescent molecules that respond to the binding of Ca2+ ions b ...
, in brain tissue. They are marketed as Multiphoton microscopes by several companies, although the gains of using 3-photon instead of 2-photon excitation are marginal.

### Single plane illumination microscopy and light sheet fluorescence 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, or light sheet 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.

### Wide-field multiphoton microscopy

Wide-field multiphoton microscopy refers to an optical non-linear imaging technique in which a large area of the object is illuminated and imaged without the need for scanning. High intensities are required to induce non-linear optical processes such as two-photon fluorescence or second harmonic generation. In scanning multiphoton microscopes the high intensities are achieved by tightly focusing the light, and the image is obtained by beam scanning. In wide-field multiphoton microscopy the high intensities are best achieved using an optically amplified pulsed laser source to attain a large field of view (~100 µm). The image in this case is obtained as a single frame with a CCD camera without the need of scanning, making the technique particularly useful to visualize dynamic processes simultaneously across the object of interest. With wide-field multiphoton microscopy the frame rate can be increased up to a 1000-fold compared to multiphoton scanning microscopy. In scattering tissue, however, image quality rapidly degrades with increasing depth.

### Deconvolution

Fluorescence microscopy is a powerful technique to show specifically labeled structures within a complex environment and 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, irrespective of whether they are in focus or not. So an image of a certain structure is always blurred by the contribution of light from structures that are out of focus. This phenomenon results in a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture. However, blurring is not caused by random processes, such as light scattering, but can be 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 further from our perspective as the spot becomes more out of focus. Under ideal conditions, this produces an "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". The mathematically modeled PSF of a terahertz laser pulsed imaging system is shown on the right. The output of an imaging system can be described using the equation: $s\left(x,y\right) = PSF\left(x,y\right) * o\left(x,y\right) + n$ Where is the additive noise. 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 In mathematics, deconvolution is the operation inverse to convolution. Both operations are used in signal processing and image processing. For example, it may be possible to recover the original signal after a filter (convolution) by using a deco ...
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. Processing fluorescent images in this manner can be an advantage over directly acquiring images without out-of-focus light, such as images from confocal microscopy, because light signals otherwise eliminated become useful information. For 3D deconvolution, one typically provides a series of images taken 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 have been developed in recent times which circumvent the diffraction limit. This is mostly achieved by imaging a sufficiently static sample multiple times and either modifying the excitation light or observing stochastic changes in the image. The deconvolution methods described in the previous section, which removes the PSF induced blur and assigns a mathematically 'correct' origin of light, are used, albeit with slightly different understanding of what the value of a pixel mean. Assuming ''most of the time'', one single fluorophore contributes to one single blob on one single taken image, the blobs in the images can be replaced with their calculated position, vastly improving resolution to well below the diffraction limit. To realize such assumption, Knowledge of and chemical control over fluorophore photophysics is at the core of these techniques, by which resolutions of ~20 nanometers are obtained.

## Serial time-encoded amplified microscopy

Serial time encoded amplified microscopy (STEAM) 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 Photodetectors, also called photosensors, are sensors of light or other electromagnetic radiation. There is a wide variety of photodetectors which may be classified by mechanism of detection, such as photoelectric or photochemical effects, or ...
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 CCD may refer to: Science and technology * Charge-coupled device, an electronic light sensor used in various devices including digital cameras * .ccd, the filename extension for CloneCD's CD image file * Carbonate compensation depth, a property o ...
and
CMOS Complementary metal–oxide–semiconductor (CMOS, pronounced "sea-moss", ) is a type of metal–oxide–semiconductor field-effect transistor (MOSFET) fabrication process that uses complementary and symmetrical pairs of p-type and n-type MOSF ...
cameras. Consequently, it is potentially useful for scientific, industrial, and biomedical applications that require high image acquisition rates, including real-time diagnosis and evaluation of shockwaves, microfluidics,
MEMS Microelectromechanical systems (MEMS), also written as micro-electro-mechanical systems (or microelectronic and microelectromechanical systems) and the related micromechatronics and microsystems constitute the technology of microscopic devices, ...
, and
laser surgery Laser surgery is a type of surgery that uses a laser (in contrast to using a scalpel) to cut tissue. Examples include the use of a laser scalpel in otherwise conventional surgery, and soft-tissue laser surgery, in which the laser beam vapori ...
.

## 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 may prefer a hand drawn image to 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 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 trace the observed shapes by simultaneously "seeing" the pencil point in the microscopical image. 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

Microspectroscopy:spectroscopy with a microscope

## X-ray

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 An X-ray microscope uses electromagnetic radiation in the soft X-ray band to produce magnified images of objects. Since X-rays penetrate most objects, there is no need to specially prepare them for X-ray microscopy observations. Unlike visible li ...
has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and electron microscopy.

# Electron microscopy

Until the invention of sub-diffraction microscopy, the wavelength of the light limited the resolution of traditional microscopy 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 Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a ...
(TEM) is 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 specimens and gives a very nice 3D view. It gives results much like those of the stereo light microscope. The best resolution for SEM in 2011 was 0.4 nanometer. Electron microscopes equipped for X-ray spectroscopy can provide qualitative and quantitative elemental analysis. This type of electron microscope, also known as analytical electron microscope, can be a very powerful tool for investigation of nanomaterials.

# Scanning probe microscopy

This is a sub-diffraction technique. Examples of scanning probe microscopes are the atomic force microscope (AFM), the
scanning tunneling microscope A scanning tunneling microscope (STM) is a type of microscope used for imaging surfaces at the atomic level. Its development in 1981 earned its inventors, Gerd Binnig and Heinrich Rohrer, then at IBM Zürich, the Nobel Prize in Physics in 1986. ...
, the photonic force microscope and the recurrence tracking microscope. All such methods use the physical contact of a solid probe tip to scan the surface 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 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. To analyze 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.

# Ultraviolet microscopy

Ultraviolet microscopes have two main purposes. The first is to use the shorter wavelength of ultraviolet electromagnetic energy to improve the image resolution beyond that of the diffraction limit of standard optical microscopes. This technique is used for non-destructive inspection of devices with very small features such as those found in modern semiconductors. The second application for UV microscopes is contrast enhancement where the response of individual samples is enhanced, relative to their surrounding, due to the interaction of light with the molecules within the sample itself. One example is in the growth of protein crystals. Protein crystals are formed in salt solutions. As salt and protein crystals are both formed in the growth process, and both are commonly transparent to the human eye, they cannot be differentiated with a standard optical microscope. As the
tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromati ...
of protein absorbs light at 280 nm, imaging with a UV microscope with 280 nm bandpass filters makes it simple to differentiate between the two types of crystals. The protein crystals appear dark while the salt crystals are transparent.

# Infrared microscopy

The term ''infrared microscopy'' refers to microscopy performed at
infrared Infrared (IR), sometimes called infrared light, is electromagnetic radiation (EMR) with wavelengths longer than those of visible light. It is therefore invisible to the human eye. IR is generally understood to encompass wavelengths from around ...
wavelengths. In the typical instrument configuration, a Fourier Transform Infrared Spectrometer (FTIR) is combined with an optical microscope and an infrared detector. The infrared detector can be a single point detector, a linear array or a 2D focal plane array. FTIR provides the ability to perform chemical analysis via
infrared spectroscopy Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the interaction of infrared radiation with matter by absorption, emission, or reflection. It is used to study and identify chemical substances or functio ...
and the microscope and point or array detector enable this chemical analysis to be spatially resolved, i.e. performed at different regions of the sample. As such, the technique is also called infrared microspectroscopyH M Pollock and S G Kazarian, Microspectroscopy in the Mid-Infrared, in Encyclopedia of Analytical Chemistry (Robert A. Meyers, Ed, 1-26 (2014), John Wiley & Sons Ltd, An alternative architecture called Laser Direct Infrared (LDIR) Imaging involves the combination of a tuneable infrared light source and single point detector on a flying objective. This technique is frequently used 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, usually specific IR absorption bands and associated molecular resonances. A key limitation of conventional infrared microspectroscopy is that the spatial resolution is diffraction-limited. Specifically the spatial resolution is limited to a figure related to the wavelength of the light. For practical IR microscopes, the spatial resolution is limited to 1-3x the wavelength, depending on the specific technique and instrument used. For mid-IR wavelengths, this sets a practical spatial resolution limit of ~3-30 μm. IR versions of sub-diffraction microscopy also exist. These include IR Near-field scanning optical microscope (NSOM), photothermal microspectroscopy and atomic force microscope based infrared spectroscopy (AFM-IR), as well as scattering-type Scanning Near-field Optical Microscopy (s-SNOM) &
nano-FTIR Nano-FTIR (nanoscale Fourier transform infrared spectroscopy) is a scanning probe technique that utilizes as a combination of two techniques: Fourier transform infrared spectroscopy (FTIR) and scattering-type scanning near-field optical microsco ...
that provide nanoscale spatial resolution at IR wavelengths.

# Digital holographic microscopy

In digital holographic microscopy (DHM), interfering wave fronts from a coherent (monochromatic) light-source are recorded on a sensor. The image is digitally reconstructed by a computer from the recorded
hologram Holography is a technique that enables a wavefront to be recorded and later re-constructed. Holography is best known as a method of generating real three-dimensional images, but it also has a wide range of other applications. In principle, ...
. Besides the ordinary bright field image, a phase shift image is created. DHM can operate both in reflection and transmission mode. In reflection mode, the phase shift image provides a relative distance measurement and thus represents a
topography Topography is the study of the forms and features of land surfaces. The topography of an area may refer to the land forms and features themselves, or a description or depiction in maps. Topography is a field of geoscience and planetary sci ...
map of the reflecting surface. In transmission mode, the phase shift image provides a label-free quantitative measurement of the optical thickness of the specimen. Phase shift images of biological cells are very similar to images of stained cells and have successfully been analyzed by high content analysis software. A 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. This feature makes it possible to image moving particles in a volume or to rapidly scan a surface. Another attractive feature is The ability of DHM to use low cost optics by correcting optical aberrations by software.

# Digital pathology (virtual microscopy)

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.

# Laser microscopy

Laser microscopy is a rapidly growing field that uses laser illumination sources in various forms of microscopy. For instance, laser microscopy focused on biological applications uses
ultrashort pulse In optics, an ultrashort pulse, also known as an ultrafast event, is an electromagnetic pulse whose time duration is of the order of a picosecond (10−12 second) or less. Such pulses have a broadband optical spectrum, and can be created by ...
lasers, in a number of techniques labeled as nonlinear microscopy, saturation microscopy, and
two-photon excitation microscopy Two-photon excitation microscopy (TPEF or 2PEF) is a fluorescence imaging technique that allows imaging of living tissue up to about one millimeter in thickness, with 0.64 μm lateral and 3.35 μm axial spatial resolution. Unlike traditional flu ...
. High-intensity, short-pulse laboratory x-ray lasers have been under development for several years. When this technology comes to fruition, it will be possible to obtain magnified three-dimensional images of elementary biological structures in the living state at a precisely defined instant. For optimum contrast between water and protein and for best sensitivity and resolution, the laser should be tuned near the nitrogen line at about 0.3 nanometers. Resolution will be limited mainly by the hydrodynamic expansion that occurs while the necessary number of photons is being registered. Thus, while the specimen is destroyed by the exposure, its configuration can be captured before it explodes. Scientists have been working on practical designs and prototypes for x-ray holographic microscopes, despite the prolonged development of the appropriate laser.

# Photoacoustic microscopy

A microscopy technique relying on the photoacoustic effect, i.e. the generation of (ultra)sound caused by light absorption. A focused and intensity modulated laser beam is raster scanned over a sample. The generated (ultra)sound is detected via an ultrasound transducer. Commonly piezoelectric ultrasound transducers are employed. The image contrast is related to the sample's absorption coefficient $\alpha$. This is in contrast to bright or dark field microscopy, where the image contrast is due to transmittance or scattering. In principle, the contrast of fluorescence microscopy is proportional to the sample's absorption too. However, in fluorescence microscopy the fluorescence quantum yield $\eta_$ needs to be unequal to zero in order that a signal can be detected. In photoacoustic microscopy, however, every absorbing substance gives a photoacoustic signal $PA$ which is proportional to $PA \propto \alpha * \Gamma *\left( \left(1-\eta_\right)*E_g + \left(E_ - E_g\right) \right)$ Here $\Gamma$ is the Grüneisen coefficient, $E_$ is the laser's photon energy and $E_g$ is the sample's band gap energy. Therefore, photoacoustic microscopy seems well suited as a complementary technique to fluorescence microscopy, as a high fluorescence quantum yield leads to high fluorescence signals and a low fluorescence quantum yield leads to high photoacoustic signals. Neglecting non-linear effects, the lateral resolution is limited by the Abbe diffraction limit: $dx =\lambda / \left(2*NA\right)$ where $\lambda$ is the wavelength of the excitation laser and is the numerical aperture of the objective lens. The Abbe diffraction limit holds if the incoming wave front is parallel. In reality, however, the laser beam profile is Gaussian. Therefore, in order to the calculate the achievable resolution, formulas for truncated Gaussian beams have to be used.

# Amateur microscopy

''Amateur Microscopy'' is the investigation and observation of
biological Biology is the scientific study of life. It is a natural science with a broad scope but has several unifying themes that tie it together as a single, coherent field. For instance, all organisms are made up of cells that process hereditary ...
and non-biological specimens for recreational purposes. Collectors of minerals, insects,
seashell A seashell or sea shell, also known simply as a shell, is a hard, protective outer layer usually created by an animal or organism that lives in the sea. The shell is part of the body of the animal. Empty seashells are often found washe ...
s, and
plant Plants are predominantly photosynthetic eukaryotes of the kingdom Plantae. Historically, the plant kingdom encompassed all living things that were not animals, and included algae and fungi; however, all current definitions of Plantae exclude ...
s may use
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible ...
s 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 pleasures. There are competitions for
photomicrograph A micrograph or photomicrograph is a photograph or digital image taken through a microscope or similar device to show a magnified image of an object. This is opposed to a macrograph or photomacrograph, an image which is also taken on a mi ...
art. Participants of this pastime may use commercially prepared microscopic slides or prepare their own slides. While microscopy is a central tool in the documentation of biological specimens, it is often 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 A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...
and access to academic literature is a necessity. There is, however, one 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." Examples of amateur microscopy images: Image:Housebeemouth100x.jpg, "house bee" Mouth 100X Image:RiceStemcs400x1.jpg, Rice Stem cs 400X Image:Rabbitttestis100x2.jpg, Rabbit Testis 100X Image:FernProthallium400x.jpg, Fern Prothallium 400X

# Application in forensic science

Microscopy has applications in the forensic sciences. The microscope can detect, resolve and image the smallest items of evidence, often without any alteration or destruction. The microscope is used to identify and compare fibers, hairs, soils, and dust...etc. In ink markings, blood stains or bullets, no specimen treatment is required and the evidence shows directly from microscopical examination. For traces of particular matter, the sample preparation must be done before microscopical examination occurs. Light microscopes are the most use in forensics, using photons to form images, microscopes which are most applicable for examining forensic specimens are as follows: 1. The compound microscope 2. The comparison microscope 3. The stereoscopic microscope 4. The polarizing microscope 5. The micro spectrophotometer This diversity of the types of microscopes in forensic applications comes mainly from their magnification ranges, which are (1- 1200X), (50 -30,000X) and (500- 250,000X) for the optical microscopy, SEM and TEM respectively.

# References

* * * * * Theoretical basis of super resolution 4Pi microscopy & design of a confocal laser scanning fluorescence microscope * , a feature article on sub-diffraction microscopy from the March 1, 2007 issue of ''Analytical Chemistry''

## General

Microscopy glossary
Common terms used in amateur light microscopy.
Nikon MicroscopyU
Extensive information on light microscopy

Microscopy Resource center
Carl Zeiss "Microscopy from the very beginning"
a step by step tutorial into the basics of microscopy.

- A resource with many illustrations elaborating the most common microscopy techniques
Manawatu Microscopy
- first known collaboration environment for Microscopy and Image Analysis.

## Techniques

Examples of Ratiometric Imaging Work on a Microscope
Interactive Fluorescence Dye and Filter Database
Carl Zeiss Interactive Fluorescence Dye and Filter Database.
New approaches to microscopy
Eric Betzig: Beyond the Nobel Prize—New approaches to microscopy. {{Authority control Microbiology techniques Laboratory techniques Cell imaging Laboratory equipment Optical microscopy