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Saturday, May 16, 2026

Interferometry

From Wikipedia, the free encyclopedia
Figure 1. The light path through a Michelson interferometer. The two light rays with a common source combine at the half-silvered mirror to reach the detector. They may either interfere constructively (strengthening in intensity) if their light waves arrive in phase, or interfere destructively (weakening in intensity) if they arrive out of phase, depending on the exact distances between the three mirrors.

Interferometry is a technique which uses the interference of superimposed waves to extract information. Interferometry typically uses electromagnetic waves and is an important investigative technique in the fields of astronomy, fiber optics, engineering metrology, optical metrology, oceanography, seismology, spectroscopy (and its applications to chemistry), quantum mechanics, nuclear and particle physics, plasma physics, biomolecular interactions, surface profiling, microfluidics, mechanical stress/strain measurement, velocimetry, optometry, and making holograms.

Interferometers are devices that extract information from interference. They are widely used in science and industry for the measurement of microscopic displacements, refractive index changes and surface irregularities. In the case with most interferometers, light from a single source is split into two beams that travel in different optical paths, which are then combined again to produce interference; two incoherent sources can also be made to interfere under some circumstances. The resulting interference fringes give information about the difference in optical path lengths. In analytical science, interferometers are used to measure lengths and the shape of optical components with nanometer precision; they are the highest-precision length measuring instruments in existence. In Fourier transform spectroscopy they are used to analyze light containing features of absorption or emission associated with a substance or mixture. An astronomical interferometer consists of two or more separate telescopes that combine their signals, offering a resolution equivalent to that of a telescope of diameter equal to the largest separation between its individual elements.

Basic principles

Figure 2. Formation of fringes in a Michelson interferometer
Figure 3. Colored and monochromatic fringes in a Michelson interferometer: (a) White light fringes where the two beams differ in the number of phase inversions; (b) White light fringes where the two beams have experienced the same number of phase inversions; (c) Fringe pattern using monochromatic light (sodium D lines)

Interferometry makes use of the principle of superposition to combine waves in a way that will cause the result of their combination to have some meaningful property that is diagnostic of the original state of the waves. This works because when two waves with the same frequency combine, the resulting intensity pattern is determined by the phase difference between the two waves—waves that are in phase will undergo constructive interference while waves that are out of phase will undergo destructive interference. Waves which are not completely in phase nor completely out of phase will have an intermediate intensity pattern, which can be used to determine their relative phase difference. Most interferometers use light or some other form of electromagnetic wave.

Typically (see Fig. 1, the well-known Michelson configuration) a single incoming beam of coherent light will be split into two identical beams by a beam splitter (a partially reflecting mirror). Each of these beams travels a different route, called a path, and they are recombined before arriving at a detector. The path difference, the difference in the distance traveled by each beam, creates a phase difference between them. It is this introduced phase difference that creates the interference pattern between the initially identical waves. If a single beam has been split along two paths, then the phase difference is diagnostic of anything that changes the phase along the paths. This could be a physical change in the path length itself or a change in the refractive index along the path.

As seen in Fig. 2a and 2b, the observer has a direct view of mirror M1 seen through the beam splitter, and sees a reflected image M2 of mirror M2. The fringes can be interpreted as the result of interference between light coming from the two virtual images S1 and S2 of the original source S. The characteristics of the interference pattern depend on the nature of the light source and the precise orientation of the mirrors and beam splitter. In Fig. 2a, the optical elements are oriented so that S1 and S2 are in line with the observer, and the resulting interference pattern consists of circles centered on the normal to M1 and M'2. If, as in Fig. 2b, M1 and M2 are tilted with respect to each other, the interference fringes will generally take the shape of conic sections (hyperbolas), but if M1 and M2 overlap, the fringes near the axis will be straight, parallel, and equally spaced. If S is an extended source rather than a point source as illustrated, the fringes of Fig. 2a must be observed with a telescope set at infinity, while the fringes of Fig. 2b will be localized on the mirrors.

Use of white light will result in a pattern of colored fringes (see Fig. 3). The central fringe representing equal path length may be light or dark depending on the number of phase inversions experienced by the two beams as they traverse the optical system. (See Michelson interferometer for a discussion of this.)

History

The law of interference of light was described by Thomas Young in his 1803 Bakerian Lecture to the Royal Society of London. In preparation for the lecture, Young performed a double-aperture experiment in a water ripple tank. His interpretation in terms of the interference of waves was rejected by most scientists at the time because of the dominance of Isaac Newton's corpuscular theory of light proposed a century before.

The French engineer Augustin-Jean Fresnel, unaware of Young's results, began working on a wave theory of light and interference and was introduced to François Arago. Between 1816 and 1818, Fresnel and Arago performed interference experiments at the Paris Observatory. During this time, Arago designed and built the first interferometer, using it to measure the refractive index of moist air relative to dry air, which posed a potential problem for astronomical observations of star positions. The success of Fresnel's wave theory of light was established in his prize-winning memoire of 1819 that predicted and measured diffraction patterns. The Arago interferometer was later employed in 1850 by Leon Foucault to measure the speed of light in air relative to water, and it was used again in 1851 by Hippolyte Fizeau to measure the effect of Fresnel drag on the speed of light in moving water.

Jules Jamin developed the first single-beam interferometer (not requiring a splitting aperture as the Arago interferometer did) in 1856. In 1881, the American physicist Albert A. Michelson, while visiting Hermann von Helmholtz in Berlin, invented the interferometer that is named after him, the Michelson Interferometer, to search for effects of the motion of the Earth on the speed of light. Michelson's null results performed in the basement of the Potsdam Observatory outside of Berlin (the horse traffic in the center of Berlin created too many vibrations), and his later more-accurate null results observed with Edward W. Morley at Case College in Cleveland, Ohio, contributed to the growing crisis of the luminiferous ether. Einstein stated that it was Fizeau's measurement of the speed of light in moving water using the Arago interferometer that inspired his theory of the relativistic addition of velocities.

Categories

Interferometers and interferometric techniques may be categorized by a variety of criteria:

Homodyne versus heterodyne detection

In homodyne detection, the interference occurs between two beams at the same wavelength (or carrier frequency). The phase difference between the two beams results in a change in the intensity of the light on the detector. The resulting intensity of the light after mixing of these two beams is measured, or the pattern of interference fringes is viewed or recorded. Most of the interferometers discussed in this article fall into this category.

The heterodyne technique is used for (1) shifting an input signal into a new frequency range as well as (2) amplifying a weak input signal (assuming use of an active mixer). A weak input signal of frequency f1 is mixed with a strong reference frequency f2 from a local oscillator (LO). The nonlinear combination of the input signals creates two new signals, one at the sum f1 + f2 of the two frequencies, and the other at the difference f1 − f2. These new frequencies are called heterodynes. Typically only one of the new frequencies is desired, and the other signal is filtered out of the output of the mixer. The output signal will have an intensity proportional to the product of the amplitudes of the input signals.

The most important and widely used application of the heterodyne technique is in the superheterodyne receiver (superhet), invented in 1917-18 by U.S. engineer Edwin Howard Armstrong and French engineer Lucien Lévy. In this circuit, the incoming radio frequency signal from the antenna is mixed with a signal from a local oscillator (LO) and converted by the heterodyne technique to a lower fixed frequency signal called the intermediate frequency (IF). This IF is amplified and filtered, before being applied to a detector which extracts the audio signal, which is sent to the loudspeaker.

Optical heterodyne detection is an extension of the heterodyne technique to higher (visible) frequencies. While optical heterodyne interferometry is usually done at a single point it is also possible to perform this widefield.

Double path versus common path

Figure 4. Four examples of common-path interferometers

A double-path interferometer is one in which the reference beam and sample beam travel along divergent paths. Examples include the Michelson interferometer, the Twyman–Green interferometer, and the Mach–Zehnder interferometer. After being perturbed by interaction with the sample under test, the sample beam is recombined with the reference beam to create an interference pattern which can then be interpreted.

A common-path interferometer is a class of interferometer in which the reference beam and sample beam travel along the same path. Fig. 4 illustrates the Sagnac interferometer, the fibre optic gyroscope, the point diffraction interferometer, and the lateral shearing interferometer. Other examples of common path interferometer include the Zernike phase-contrast microscope, Fresnel's biprism, the zero-area Sagnac, and the scatterplate interferometer.

Wavefront splitting versus amplitude splitting

Wavefront splitting inferometers

A wavefront splitting interferometer divides a light wavefront emerging from a point or a narrow slit (i.e. spatially coherent light) and, after allowing the two parts of the wavefront to travel through different paths, allows them to recombine. Fig. 5 illustrates Young's interference experiment and Lloyd's mirror. Other examples of wavefront splitting interferometer include the Fresnel biprism, the Billet Bi-Lens, diffraction-grating Michelson interferometer, and the Rayleigh interferometer.

Figure 5. Two wavefront splitting interferometers

In 1803, Young's interference experiment played a major role in the general acceptance of the wave theory of light. If white light is used in Young's experiment, the result is a white central band of constructive interference corresponding to equal path length from the two slits, surrounded by a symmetrical pattern of colored fringes of diminishing intensity. In addition to continuous electromagnetic radiation, Young's experiment has been performed with individual photons, with electrons, and with buckyball molecules large enough to be seen under an electron microscope.

Lloyd's mirror generates interference fringes by combining direct light from a source (blue lines) and light from the source's reflected image (red lines) from a mirror held at grazing incidence. The result is an asymmetrical pattern of fringes. The band of equal path length, nearest the mirror, is dark rather than bright. In 1834, Humphrey Lloyd interpreted this effect as proof that the phase of a front-surface reflected beam is inverted.

Amplitude-splitting inferometers

Figure 6. Three amplitude-splitting interferometers: Fizeau, Mach–Zehnder, and Fabry Pérot.

An amplitude splitting interferometer uses a partial reflector to divide the amplitude of the incident wave into separate beams which are separated and recombined.

The Fizeau interferometer is shown as it might be set up to test an optical flat. A precisely figured reference flat is placed on top of the flat being tested, separated by narrow spacers. The reference flat is slightly beveled (only a fraction of a degree of beveling is necessary) to prevent the rear surface of the flat from producing interference fringes. Separating the test and reference flats allows the two flats to be tilted with respect to each other. By adjusting the tilt, which adds a controlled phase gradient to the fringe pattern, one can control the spacing and direction of the fringes, so that one may obtain an easily interpreted series of nearly parallel fringes rather than a complex swirl of contour lines. Separating the plates, however, necessitates that the illuminating light be collimated. Fig 6 shows a collimated beam of monochromatic light illuminating the two flats and a beam splitter allowing the fringes to be viewed on-axis.

The Mach–Zehnder interferometer is a more versatile instrument than the Michelson interferometer. Each of the well separated light paths is traversed only once, and the fringes can be adjusted so that they are localized in any desired plane. Typically, the fringes would be adjusted to lie in the same plane as the test object, so that fringes and test object can be photographed together. If it is decided to produce fringes in white light, then, since white light has a limited coherence length, on the order of micrometers, great care must be taken to equalize the optical paths or no fringes will be visible. As illustrated in Fig. 6, a compensating cell would be placed in the path of the reference beam to match the test cell. Note also the precise orientation of the beam splitters. The reflecting surfaces of the beam splitters would be oriented so that the test and reference beams pass through an equal amount of glass. In this orientation, the test and reference beams each experience two front-surface reflections, resulting in the same number of phase inversions. The result is that light traveling an equal optical path length in the test and reference beams produces a white light fringe of constructive interference.

The heart of the Fabry–Pérot interferometer is a pair of partially silvered glass optical flats spaced several millimeters to centimeters apart with the silvered surfaces facing each other. (Alternatively, a Fabry–Pérot etalon uses a transparent plate with two parallel reflecting surfaces.) As with the Fizeau interferometer, the flats are slightly beveled. In a typical system, illumination is provided by a diffuse source set at the focal plane of a collimating lens. A focusing lens produces what would be an inverted image of the source if the paired flats were not present, i.e., in the absence of the paired flats, all light emitted from point A passing through the optical system would be focused at point A'. In Fig. 6, only one ray emitted from point A on the source is traced. As the ray passes through the paired flats, it is multiply reflected to produce multiple transmitted rays which are collected by the focusing lens and brought to point A' on the screen. The complete interference pattern takes the appearance of a set of concentric rings. The sharpness of the rings depends on the reflectivity of the flats. If the reflectivity is high, resulting in a high Q factor (i.e., high finesse), monochromatic light produces a set of narrow bright rings against a dark background.[26] In Fig. 6, the low-finesse image corresponds to a reflectivity of 0.04 (i.e., unsilvered surfaces) versus a reflectivity of 0.95 for the high-finesse image.

Fig. 6 illustrates the Fizeau, Mach–Zehnder, and Fabry–Pérot interferometers. Other examples of amplitude splitting interferometer include the Michelson, Twyman–Green, Laser Unequal Path, and Linnik interferometer.

Michelson-Morley

Michelson and Morley (1887) and other early experimentalists using interferometric techniques in an attempt to measure the properties of the luminiferous aether, used monochromatic light only for initially setting up their equipment, always switching to white light for the actual measurements. The reason is that measurements were recorded visually. Monochromatic light would result in a uniform fringe pattern. Lacking modern means of environmental temperature control, experimentalists struggled with continual fringe drift even though the interferometer might be set up in a basement. Since the fringes would occasionally disappear due to vibrations by passing horse traffic, distant thunderstorms and the like, it would be easy for an observer to "get lost" when the fringes returned to visibility. The advantages of white light, which produced a distinctive colored fringe pattern, far outweighed the difficulties of aligning the apparatus due to its low coherence length. This was an early example of the use of white light to resolve the "2 pi ambiguity".

Applications

Physics and astronomy

Interferometry is used in radio astronomy, with timing offsets of: D sin θ

In physics, one of the most important experiments of the late 19th century was the famous "failed experiment" of Michelson and Morley which provided evidence for special relativity. Recent repetitions of the Michelson–Morley experiment perform heterodyne measurements of beat frequencies of crossed cryogenic optical resonators. Fig 7 illustrates a resonator experiment performed by Müller et al. in 2003. Two optical resonators constructed from crystalline sapphire, controlling the frequencies of two lasers, were set at right angles within a helium cryostat. A frequency comparator measured the beat frequency of the combined outputs of the two resonators. As of 2009, the precision by which anisotropy of the speed of light can be excluded in resonator experiments is at the 10−17 level.


Figure 7. Michelson–Morley experiment with
cryogenic optical resonators

Figure 8. Fourier transform spectroscopy

Figure 9. A picture of the solar corona taken
with the LASCO C1 coronagraph

Michelson interferometers are used in tunable narrow band optical filters and as the core hardware component of Fourier transform spectrometers.

When used as a tunable narrow band filter, Michelson interferometers exhibit a number of advantages and disadvantages when compared with competing technologies such as Fabry–Pérot interferometers or Lyot filters. Michelson interferometers have the largest field of view for a specified wavelength, and are relatively simple in operation, since tuning is via mechanical rotation of waveplates rather than via high voltage control of piezoelectric crystals or lithium niobate optical modulators as used in a Fabry–Pérot system. Compared with Lyot filters, which use birefringent elements, Michelson interferometers have a relatively low temperature sensitivity. On the negative side, Michelson interferometers have a relatively restricted wavelength range and require use of prefilters which restrict transmittance.

Fig. 8 illustrates the operation of a Fourier transform spectrometer, which is essentially a Michelson interferometer with one mirror movable. (A practical Fourier transform spectrometer would substitute corner cube reflectors for the flat mirrors of the conventional Michelson interferometer, but for simplicity, the illustration does not show this.) An interferogram is generated by making measurements of the signal at many discrete positions of the moving mirror. A Fourier transform converts the interferogram into an actual spectrum.

Fig. 9 shows a doppler image of the solar corona made using a tunable Fabry-Pérot interferometer to recover scans of the solar corona at a number of wavelengths near the FeXIV green line. The picture is a color-coded image of the doppler shift of the line, which may be associated with the coronal plasma velocity towards or away from the satellite camera.

Fabry–Pérot thin-film etalons are used in narrow bandpass filters capable of selecting a single spectral line for imaging; for example, the H-alpha line or the Ca-K line of the Sun or stars. Fig. 10 shows an Extreme ultraviolet Imaging Telescope (EIT) image of the Sun at 195 Ångströms (19.5 nm), corresponding to a spectral line of multiply-ionized iron atoms. EIT used multilayer coated reflective mirrors that were coated with alternate layers of a light "spacer" element (such as silicon), and a heavy "scatterer" element (such as molybdenum). Approximately 100 layers of each type were placed on each mirror, with a thickness of around 10 nm each. The layer thicknesses were tightly controlled so that at the desired wavelength, reflected photons from each layer interfered constructively.

The Laser Interferometer Gravitational-Wave Observatory (LIGO) uses two 4-km Michelson–Fabry–Pérot interferometers for the detection of gravitational waves. In this application, the Fabry–Pérot cavity is used to store photons for almost a millisecond while they bounce up and down between the mirrors. This increases the time a gravitational wave can interact with the light, which results in a better sensitivity at low frequencies. Smaller cavities, usually called mode cleaners, are used for spatial filtering and frequency stabilization of the main laser. The first observation of gravitational waves occurred on September 14, 2015.

The Mach–Zehnder interferometer's relatively large and freely accessible working space, and its flexibility in locating the fringes has made it the interferometer of choice for visualizing flow in wind tunnels, and for flow visualization studies in general. It is frequently used in the fields of aerodynamics, plasma physics and heat transfer to measure pressure, density, and temperature changes in gases.

Mach–Zehnder interferometers are also used to study one of the most counterintuitive predictions of quantum mechanics, the phenomenon known as quantum entanglement.

Figure 11. The VLA interferometer

An astronomical interferometer achieves high-resolution observations using the technique of aperture synthesis, mixing signals from a cluster of comparatively small telescopes rather than a single very expensive monolithic telescope.

Early radio telescope interferometers used a single baseline for measurement. Later astronomical interferometers, such as the Very Large Array illustrated in Fig 11, used arrays of telescopes arranged in a pattern on the ground. A limited number of baselines will result in insufficient coverage. This was alleviated by using the rotation of the Earth to rotate the array relative to the sky. Thus, a single baseline could measure information in multiple orientations by taking repeated measurements, a technique called Earth-rotation synthesis. Baselines thousands of kilometers long were achieved using very long baseline interferometry.

ALMA is an astronomical interferometer located in Chajnantor Plateau

Astronomical optical interferometry has had to overcome a number of technical issues not shared by radio telescope interferometry. The short wavelengths of light necessitate extreme precision and stability of construction. For example, spatial resolution of 1 milliarcsecond requires 0.5 μm stability in a 100 m baseline. Optical interferometric measurements require high sensitivity, low noise detectors that did not become available until the late 1990s. Astronomical "seeing", the turbulence that causes stars to twinkle, introduces rapid, random phase changes in the incoming light, requiring data collection rates to be faster than the rate of turbulence. Despite these technical difficulties, three major facilities are now in operation offering resolutions down to the fractional milliarcsecond range.

The wave character of matter can be exploited to build interferometers. The first examples of matter interferometers were electron interferometers, later followed by neutron interferometers. Around 1990 the first atom interferometers were demonstrated, later followed by interferometers employing molecules.

Electron holography is an imaging technique that photographically records the electron interference pattern of an object, which is then reconstructed to yield a greatly magnified image of the original object. This technique was developed to enable greater resolution in electron microscopy than is possible using conventional imaging techniques. The resolution of conventional electron microscopy is not limited by electron wavelength, but by the large aberrations of electron lenses.

Neutron interferometry has been used to investigate the Aharonov–Bohm effect, to examine the effects of gravity acting on an elementary particle, and to demonstrate a strange behavior of fermions that is at the basis of the Pauli exclusion principle: Unlike macroscopic objects, when fermions are rotated by 360° about any axis, they do not return to their original state, but develop a minus sign in their wave function. In other words, a fermion needs to be rotated 720° before returning to its original state.

Atom interferometry techniques are reaching sufficient precision to allow laboratory-scale tests of general relativity.

Interferometers are used in atmospheric physics for high-precision measurements of trace gases via remote sounding of the atmosphere. There are several examples of interferometers that utilize either absorption or emission features of trace gases. A typical use would be in continual monitoring of the column concentration of trace gases such as ozone and carbon monoxide above the instrument.

Engineering and applied science

Figure 13. Optical flat interference fringes. (left) flat surface, (right) curved surface.
How interference fringes are formed by an optical flat resting on a reflective surface. The gap between the surfaces and the wavelength of the light waves are greatly exaggerated.

Newton (test plate) interferometry is frequently used in the optical industry for testing the quality of surfaces as they are being shaped and figured. Fig. 13 shows photos of reference flats being used to check two test flats at different stages of completion, showing the different patterns of interference fringes. The reference flats are resting with their bottom surfaces in contact with the test flats, and they are illuminated by a monochromatic light source. The light waves reflected from both surfaces interfere, resulting in a pattern of bright and dark bands. The surface in the left photo is nearly flat, indicated by a pattern of straight parallel interference fringes at equal intervals. The surface in the right photo is uneven, resulting in a pattern of curved fringes. Each pair of adjacent fringes represents a difference in surface elevation of half a wavelength of the light used, so differences in elevation can be measured by counting the fringes. The flatness of the surfaces can be measured to millionths of an inch by this method. To determine whether the surface being tested is concave or convex with respect to the reference optical flat, any of several procedures may be adopted. One can observe how the fringes are displaced when one presses gently on the top flat. If one observes the fringes in white light, the sequence of colors becomes familiar with experience and aids in interpretation. Finally one may compare the appearance of the fringes as one moves ones head from a normal to an oblique viewing position. These sorts of maneuvers, while common in the optical shop, are not suitable in a formal testing environment. When the flats are ready for sale, they will typically be mounted in a Fizeau interferometer for formal testing and certification.

Fabry-Pérot etalons are widely used in telecommunications, lasers and spectroscopy to control and measure the wavelengths of light. Dichroic filters are multiple layer thin-film etalons. In telecommunications, wavelength-division multiplexing, the technology that enables the use of multiple wavelengths of light through a single optical fiber, depends on filtering devices that are thin-film etalons. Single-mode lasers employ etalons to suppress all optical cavity modes except the single one of interest.

Figure 14. Twyman–Green Interferometer

The Twyman–Green interferometer, invented by Twyman and Green in 1916, is a variant of the Michelson interferometer widely used to test optical components. The basic characteristics distinguishing it from the Michelson configuration are the use of a monochromatic point light source and a collimator. Michelson (1918) criticized the Twyman–Green configuration as being unsuitable for the testing of large optical components, since the light sources available at the time had limited coherence length. Michelson pointed out that constraints on geometry forced by limited coherence length required the use of a reference mirror of equal size to the test mirror, making the Twyman–Green impractical for many purposes. Decades later, the advent of laser light sources answered Michelson's objections. (A Twyman–Green interferometer using a laser light source and unequal path length is known as a Laser Unequal Path Interferometer, or LUPI.) Fig. 14 illustrates a Twyman–Green interferometer set up to test a lens. Light from a monochromatic point source is expanded by a diverging lens (not shown), then is collimated into a parallel beam. A convex spherical mirror is positioned so that its center of curvature coincides with the focus of the lens being tested. The emergent beam is recorded by an imaging system for analysis.

Mach–Zehnder interferometers are being used in integrated optical circuits, in which light interferes between two branches of a waveguide that are externally modulated to vary their relative phase. A slight tilt of one of the beam splitters will result in a path difference and a change in the interference pattern. Mach–Zehnder interferometers are the basis of a wide variety of devices, from RF modulators to sensors to optical switches.

Some proposed and future extremely large astronomical telescopes, such as the Thirty Meter Telescope and the Extremely Large Telescope, will be of segmented design. Their primary mirrors will comprise hundreds of hexagonal mirror-segments. Polishing and figuring these highly aspheric and non-rotationally symmetric mirror segments presents a major challenge. Traditional means of optical testing compare a surface against a spherical reference with the aid of a null corrector. Computer-generated holograms supplement null correctors in test setups for complex aspheric surfaces. Fig. 15 illustrates how this is done. (Unlike the figure, actual CGHs have line spacing on the order of 1 to 10 μm.) When laser light is passed through the hologram, the zero-order diffracted beam experiences no wavefront modification. The wavefront of the first-order diffracted beam, however, is modified to match the desired shape of the test surface. In the illustrated Fizeau interferometer test setup, the zero-order diffracted beam is directed towards the spherical reference surface, and the first-order diffracted beam is directed towards the test surface in such a way that the two reflected beams combine to form interference fringes.

Figure 15. Optical testing with a Fizeau interferometer and a computer generated hologram

Ring laser gyroscopes (RLGs) and fibre optic gyroscopes (FOGs) are interferometers used in navigation systems. They operate on the principle of the Sagnac effect. The distinction between RLGs and FOGs is that in a RLG, the entire ring is part of the laser while in a FOG, an external laser injects counter-propagating beams into an optical fiber ring, and rotation of the system then causes a relative phase shift between those beams. In a RLG, the observed phase shift is proportional to the accumulated rotation, while in a FOG, the observed phase shift is proportional to the angular velocity.

In telecommunication networks, heterodyning is used to move frequencies of individual signals to different channels which may share a single physical transmission line. This is called frequency division multiplexing (FDM). For example, a coaxial cable used by a cable television system can carry 500 television channels at the same time because each one is given a different frequency, so they don't interfere with one another. Continuous wave (CW) doppler radar detectors are basically heterodyne detection devices that compare transmitted and reflected beams.

Optical heterodyne detection is used for coherent Doppler lidar measurements capable of detecting very weak light scattered in the atmosphere and monitoring wind speeds with high accuracy. It has application in optical fiber communications, in various high resolution spectroscopic techniques, and the self-heterodyne method can be used to measure the linewidth of a laser.

Figure 16. Frequency comb of a mode-locked laser. The dashed lines represent an extrapolation of the mode frequencies towards the frequency of the carrier–envelope offset (CEO). The vertical grey line represents an unknown optical frequency. The horizontal black lines indicate the two lowest beat frequency measurements.

Optical heterodyne detection is an essential technique used in high-accuracy measurements of the frequencies of optical sources, as well as in the stabilization of their frequencies. Until relatively few years ago, lengthy frequency chains were needed to connect the microwave frequency of a cesium or other atomic time source to optical frequencies. At each step of the chain, a frequency multiplier would be used to produce a harmonic of the frequency of that step, which would be compared by heterodyne detection with the next step (the output of a microwave source, far infrared laser, infrared laser, or visible laser). Each measurement of a single spectral line required several years of effort in the construction of a custom frequency chain. Currently, optical frequency combs have provided a much simpler method of measuring optical frequencies. If a mode-locked laser is modulated to form a train of pulses, its spectrum is seen to consist of the carrier frequency surrounded by a closely spaced comb of optical sideband frequencies with a spacing equal to the pulse repetition frequency (Fig. 16). The pulse repetition frequency is locked to that of the frequency standard, and the frequencies of the comb elements at the red end of the spectrum are doubled and heterodyned with the frequencies of the comb elements at the blue end of the spectrum, thus allowing the comb to serve as its own reference. In this manner, locking of the frequency comb output to an atomic standard can be performed in a single step. To measure an unknown frequency, the frequency comb output is dispersed into a spectrum. The unknown frequency is overlapped with the appropriate spectral segment of the comb and the frequency of the resultant heterodyne beats is measured.

One of the most common industrial applications of optical interferometry is as a versatile measurement tool for the high precision examination of surface topography. Popular interferometric measurement techniques include phase shifting interferometry (PSI), and vertical scanning interferometry (VSI), also known as scanning white light interferometry (SWLI) or by the ISO term coherence scanning interferometry (CSI), CSI exploits coherence to extend the range of capabilities for interference microscopy. These techniques are widely used in micro-electronic and micro-optic fabrication. PSI uses monochromatic light and provides very precise measurements; however it is only usable for surfaces that are very smooth. CSI often uses white light and high numerical apertures, and rather than looking at the phase of the fringes, as does PSI, looks for best position of maximum fringe contrast or some other feature of the overall fringe pattern. In its simplest form, CSI provides less precise measurements than PSI but can be used on rough surfaces. Some configurations of CSI, variously known as Enhanced VSI (EVSI), high-resolution SWLI or Frequency Domain Analysis (FDA), use coherence effects in combination with interference phase to enhance precision.

Figure 17. Phase shifting and Coherence scanning interferometers

Phase Shifting Interferometry addresses several issues associated with the classical analysis of static interferograms. Classically, one measures the positions of the fringe centers. As seen in Fig. 13, fringe deviations from straightness and equal spacing provide a measure of the aberration. Errors in determining the location of the fringe centers provide the inherent limit to precision of the classical analysis, and any intensity variations across the interferogram will also introduce error. There is a trade-off between precision and number of data points: closely spaced fringes provide many data points of low precision, while widely spaced fringes provide a low number of high precision data points. Since fringe center data is all that one uses in the classical analysis, all of the other information that might theoretically be obtained by detailed analysis of the intensity variations in an interferogram is thrown away. Finally, with static interferograms, additional information is needed to determine the polarity of the wavefront: In Fig. 13, one can see that the tested surface on the right deviates from flatness, but one cannot tell from this single image whether this deviation from flatness is concave or convex. Traditionally, this information would be obtained using non-automated means, such as by observing the direction that the fringes move when the reference surface is pushed.

Phase shifting interferometry overcomes these limitations by not relying on finding fringe centers, but rather by collecting intensity data from every point of the CCD image sensor. As seen in Fig. 17, multiple interferograms (at least three) are analyzed with the reference optical surface shifted by a precise fraction of a wavelength between each exposure using a piezoelectric transducer (PZT). Alternatively, precise phase shifts can be introduced by modulating the laser frequency. The captured images are processed by a computer to calculate the optical wavefront errors. The precision and reproducibility of PSI is far greater than possible in static interferogram analysis, with measurement repeatabilities of a hundredth of a wavelength being routine. Phase shifting technology has been adapted to a variety of interferometer types such as Twyman–Green, Mach–Zehnder, laser Fizeau, and even common path configurations such as point diffraction and lateral shearing interferometers. More generally, phase shifting techniques can be adapted to almost any system that uses fringes for measurement, such as holographic and speckle interferometry.

Figure 18. Lunate cells of Nepenthes khasiana visualized by Scanning White Light Interferometry (SWLI)
Figure 19. Twyman–Green interferometer set up as a white light scanner

In coherence scanning interferometry, interference is only achieved when the path length delays of the interferometer are matched within the coherence time of the light source. CSI monitors the fringe contrast rather than the phase of the fringes. Fig. 17 illustrates a CSI microscope using a Mirau interferometer in the objective; other forms of interferometer used with white light include the Michelson interferometer (for low magnification objectives, where the reference mirror in a Mirau objective would interrupt too much of the aperture) and the Linnik interferometer (for high magnification objectives with limited working distance). The sample (or alternatively, the objective) is moved vertically over the full height range of the sample, and the position of maximum fringe contrast is found for each pixel. The chief benefit of coherence scanning interferometry is that systems can be designed that do not suffer from the 2 pi ambiguity of coherent interferometry, and as seen in Fig. 18, which scans a 180μm x 140μm x 10μm volume, it is well suited to profiling steps and rough surfaces. The axial resolution of the system is determined in part by the coherence length of the light source. Industrial applications include in-process surface metrology, roughness measurement, 3D surface metrology in hard-to-reach spaces and in hostile environments, profilometry of surfaces with high aspect ratio features (grooves, channels, holes), and film thickness measurement (semi-conductor and optical industries, etc.).

Fig. 19 illustrates a Twyman–Green interferometer set up for white light scanning of a macroscopic object.

Holographic interferometry is a technique which uses holography to monitor small deformations in single wavelength implementations. In multi-wavelength implementations, it is used to perform dimensional metrology of large parts and assemblies and to detect larger surface defects.

Holographic interferometry was discovered by accident as a result of mistakes committed during the making of holograms. Early lasers were relatively weak and photographic plates were insensitive, necessitating long exposures during which vibrations or minute shifts might occur in the optical system. The resultant holograms, which showed the holographic subject covered with fringes, were considered ruined.

Eventually, several independent groups of experimenters in the mid-60s realized that the fringes encoded important information about dimensional changes occurring in the subject, and began intentionally producing holographic double exposures. The main Holographic interferometry article covers the disputes over priority of discovery that occurred during the issuance of the patent for this method.

Double- and multi- exposure holography is one of three methods used to create holographic interferograms. A first exposure records the object in an unstressed state. Subsequent exposures on the same photographic plate are made while the object is subjected to some stress. The composite image depicts the difference between the stressed and unstressed states.

Real-time holography is a second method of creating holographic interferograms. A holograph of the unstressed object is created. This holograph is illuminated with a reference beam to generate a hologram image of the object directly superimposed over the original object itself while the object is being subjected to some stress. The object waves from this hologram image will interfere with new waves coming from the object. This technique allows real time monitoring of shape changes.

The third method, time-average holography, involves creating a holograph while the object is subjected to a periodic stress or vibration. This yields a visual image of the vibration pattern.

Interferometric synthetic aperture radar (InSAR) is a radar technique used in geodesy and remote sensing. Satellite synthetic aperture radar images of a geographic feature are taken on separate days, and changes that have taken place between radar images taken on the separate days are recorded as fringes similar to those obtained in holographic interferometry. The technique can monitor centimeter- to millimeter-scale deformation resulting from earthquakes, volcanoes and landslides, and also has uses in structural engineering, in particular for the monitoring of subsidence and structural stability. Fig 20 shows Kilauea, an active volcano in Hawaii. Data acquired using the space shuttle Endeavour's X-band Synthetic Aperture Radar on April 13, 1994 and October 4, 1994 were used to generate interferometric fringes, which were overlaid on the X-SAR image of Kilauea.

Electronic speckle pattern interferometry (ESPI), also known as TV holography, uses video detection and recording to produce an image of the object upon which is superimposed a fringe pattern which represents the displacement of the object between recordings. (see Fig. 21) The fringes are similar to those obtained in holographic interferometry.

When lasers were first invented, laser speckle was considered to be a severe drawback in using lasers to illuminate objects, particularly in holographic imaging because of the grainy image produced. It was later realized that speckle patterns could carry information about the object's surface deformations. Butters and Leendertz developed the technique of speckle pattern interferometry in 1970, and since then, speckle has been exploited in a variety of other applications. A photograph is made of the speckle pattern before deformation, and a second photograph is made of the speckle pattern after deformation. Digital subtraction of the two images results in a correlation fringe pattern, where the fringes represent lines of equal deformation. Short laser pulses in the nanosecond range can be used to capture very fast transient events. A phase problem exists: In the absence of other information, one cannot tell the difference between contour lines indicating a peak versus contour lines indicating a trough. To resolve the issue of phase ambiguity, ESPI may be combined with phase shifting methods.

A method of establishing precise geodetic baselines, invented by Yrjö Väisälä, exploited the low coherence length of white light. Initially, white light was split in two, with the reference beam "folded", bouncing back-and-forth six times between a mirror pair spaced precisely 1 m apart. Only if the test path was precisely 6 times the reference path would fringes be seen. Repeated applications of this procedure allowed precise measurement of distances up to 864 meters. Baselines thus established were used to calibrate geodetic distance measurement equipment, leading to a metrologically traceable scale for geodetic networks measured by these instruments. (This method has been superseded by GPS.)

Other uses of interferometers have been to study dispersion of materials, measurement of complex indices of refraction, and thermal properties. They are also used for three-dimensional motion mapping including mapping vibrational patterns of structures.

Potential military applications of laser interferometry, documented as of 1964, included "precision positioning in research, manufacture, and in field use of equipment, such as missile alignment or turret aiming equipment; in geodesy and mapping, where one does not need a map accurate to microinches, but where the accurate establishment of base lines is essential; and in the remote measuring of variations of temperature in the atmosphere [...] which affect optical seeing ability."

Biology and medicine

Optical interferometry, applied to biology and medicine, provides sensitive metrology capabilities for the measurement of biomolecules, subcellular components, cells and tissues. Many forms of label-free biosensors rely on interferometry because the direct interaction of electromagnetic fields with local molecular polarizability eliminates the need for fluorescent tags or nanoparticle markers. At a larger scale, cellular interferometry shares aspects with phase-contrast microscopy, but comprises a much larger class of phase-sensitive optical configurations that rely on optical interference among cellular constituents through refraction and diffraction. At the tissue scale, partially-coherent forward-scattered light propagation through the micro aberrations and heterogeneity of tissue structure provides opportunities to use phase-sensitive gating (optical coherence tomography) as well as phase-sensitive fluctuation spectroscopy to image subtle structural and dynamical properties.


Figure 22. Typical optical setup of single point OCT
     
Figure 23. Central serous retinopathy, imaged using
optical coherence tomography

Optical coherence tomography (OCT) is a medical imaging technique using low-coherence interferometry to provide tomographic visualization of internal tissue microstructures. As seen in Fig. 22, the core of a typical OCT system is a Michelson interferometer. One interferometer arm is focused onto the tissue sample and scans the sample in an X-Y longitudinal raster pattern. The other interferometer arm is bounced off a reference mirror. Reflected light from the tissue sample is combined with reflected light from the reference. Because of the low coherence of the light source, interferometric signal is observed only over a limited depth of sample. X-Y scanning therefore records one thin optical slice of the sample at a time. By performing multiple scans, moving the reference mirror between each scan, an entire three-dimensional image of the tissue can be reconstructed. Recent advances have striven to combine the nanometer phase retrieval of coherent interferometry with the ranging capability of low-coherence interferometry.

Phase contrast and differential interference contrast (DIC) microscopy are important tools in biology and medicine. Most animal cells and single-celled organisms have very little color, and their intracellular organelles are almost totally invisible under simple bright field illumination. These structures can be made visible by staining the specimens, but staining procedures are time-consuming and kill the cells. As seen in Figs. 24 and 25, phase contrast and DIC microscopes allow unstained, living cells to be studied. DIC also has non-biological applications, for example in the analysis of planar silicon semiconductor processing.

Angle-resolved low-coherence interferometry (a/LCI) uses scattered light to measure the sizes of subcellular objects, including cell nuclei. This allows interferometry depth measurements to be combined with density measurements. Various correlations have been found between the state of tissue health and the measurements of subcellular objects. For example, it has been found that as tissue changes from normal to cancerous, the average cell nuclei size increases.

Phase-contrast X-ray imaging (Fig. 26) refers to a variety of techniques that use phase information of a coherent x-ray beam to image soft tissues. (For an elementary discussion, see Phase-contrast x-ray imaging (introduction). For a more in-depth review, see Phase-contrast X-ray imaging.) It has become an important method for visualizing cellular and histological structures in a wide range of biological and medical studies. There are several technologies being used for x-ray phase-contrast imaging, all utilizing different principles to convert phase variations in the x-rays emerging from an object into intensity variations. These include propagation-based phase contrast, Talbot interferometry, Moiré-based far-field interferometry, refraction-enhanced imaging, and x-ray interferometry. These methods provide higher contrast compared to normal absorption-contrast x-ray imaging, making it possible to see smaller details. A disadvantage is that these methods require more sophisticated equipment, such as synchrotron or microfocus x-ray sources, x-ray optics, or high resolution x-ray detectors.

Friday, May 15, 2026

Microscopy

From Wikipedia, the free encyclopedia
Scanning electron microscope image of pollen (false colors)
Microscopic examination in a biochemical laboratory

Microscopy is the technical field of using microscopes to view subjects too small to 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, along with the emerging field of X-ray microscopy.

Optical microscopy and electron microscopy involve the diffraction, reflection, or refraction of electromagnetic radiation/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) or by scanning a fine beam over the sample (for example confocal laser scanning microscopy and scanning electron microscopy). Scanning probe microscopy involves the interaction of a scanning probe with the surface of the object of interest. The development of microscopy revolutionized biology, gave rise to the field of histology and so remains an essential technique in the life and physical sciences. X-ray microscopy is three-dimensional and non-destructive, allowing for repeated imaging of the same sample for in situ 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), 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

Antonie van Leeuwenhoek (1632–1723)

The field of microscopy (optical microscopy) dates back to at least the 17th-century. Earlier microscopes, single lens magnifying glasses with limited magnification, date at least as far back as the wide spread use of lenses in eyeglasses in the 13th century but more advanced compound microscopes first appeared in Europe around 1620 The earliest practitioners of microscopy include Galileo Galilei, who found in 1610 that he could close focus his telescope to view small objects close up and Cornelis Drebbel, who may have invented the compound microscope around 1620. Antonie van Leeuwenhoek developed a very high magnification simple microscope in the 1670s and is often considered to be the first acknowledged microscopist and microbiologist.

Optical microscopy

Stereo microscope

Optical or light microscopy involves passing visible light transmitted through or reflected from the sample through a single lens or multiple lenses to allow a magnified view of the sample. The resulting image can be detected directly by the eye, imaged on a photographic plate, or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage, and support, makes up the basic light microscope. The most recent development is the digital microscope, which uses a CCD camera to focus on the exhibit of interest. The image is shown on a computer screen, 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  micrometres (see: microscope) 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 the structures with selective dyes, but this often involves killing and fixing 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.

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.

A diatom under Rheinberg illumination

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

Phase-contrast light micrograph of undecalcified hyaline cartilage showing chondrocytes and organelles, lacunae and extracellular matrix

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

Note: In cases where the optical design of a microscope produces an appreciable lateral separation of the two beams we have the case of classical interference microscopy, which does not result in relief images, but can nevertheless be used for the quantitative determination of mass-thicknesses of microscopic objects.

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

Images may also contain artifacts. This is a confocal laser scanning fluorescence micrograph of thale cress anther (part of stamen). The picture shows among other things a nice red flowing collar-like structure just below the anther. However, an intact thale cress stamen does not have such collar, this is a fixation artifact: the stamen has been cut below the picture frame, and epidermis (upper layer of cells) of stamen stalk has peeled off, forming a non-characteristic structure. Photo: Heiti Paves from Tallinn University of Technology.

When certain compounds are illuminated with high energy light, they emit light of a lower frequency. This effect is known as fluorescence. Often specimens show their characteristic autofluorescence image, based on their chemical makeup.

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules. Many fluorescent dyes can be used to stain structures or chemical compounds. One powerful method is the combination of antibodies coupled to a fluorophore as in immunostaining. Examples of commonly used fluorophores are fluorescein or rhodamine.

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 (GFP) have been developed using the molecular biology technique of gene fusion, a process that links the expression 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.

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 proteins 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 selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.

See also: total internal reflection fluorescence microscope Neuroscience

Confocal

Confocal laser scanning microscopy uses a focused laser beam (e.g. 488 nm) that is scanned across the sample to excite fluorescence 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. 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.

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, 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.

Mathematically modeled Point Spread Function of a pulsed THz laser imaging system.

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:

Where n 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 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

Example of super-resolution microscopy. Image of Her3 and Her2, target of the breast cancer drug Trastuzumab, within a cancer cell.

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 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 and CMOS 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, and laser surgery.

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.

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 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 (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, 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 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 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 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 microspectroscopy 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 that provide nanoscale spatial resolution at IR wavelengths.

Digital holographic microscopy

Human cells imaged by DHM phase shift (left) and phase contrast microscopy (right)

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. 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 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 lasers, in a number of techniques labeled as nonlinear microscopy, saturation microscopy, and two-photon excitation microscopy.

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

Photoacoustic micrograph of human red blood cells.

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 . 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 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 which is proportional to

Here is the Grüneisen coefficient, is the laser's photon energy and 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 dx is limited by the Abbe diffraction limit:

where is the wavelength of the excitation laser and NA 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 and non-biological specimens for recreational purposes. Collectors of minerals, insects, seashells, and plants may use microscopes as tools to uncover features that help them classify their collected items. Other amateurs may be interested in observing the life found in pond water and of other samples. Microscopes may also prove useful for the water quality assessment for people that keep a home aquarium. Photographic documentation and drawing of the microscopic images are additional pleasures. There are competitions for photomicrograph 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 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:

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.

Bodymind

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