A petrographic microscope is a type of optical microscope used in petrology and optical mineralogy to identify rocks and minerals in thin sections. The microscope is used in optical mineralogy and petrography, a branch of petrology which focuses on detailed descriptions of rocks; the method is called "polarized light microscopy". Depending on the grade of observation required, petrological microscopes are derived from conventional brightfield microscopes of similar basic capabilities by: Adding a Nicol prism polarizer filter to the light path beneath the sample slide Replacing the normal stage with a circular rotating stage Adding a second rotatable and removable Nicol prism filter, called the analyzer, to the light path between objective and eyepiece Adding a Phase telescope known as a Bertrand Lens, which allows the viewer to see conoscopic interference patterns Adding a slot for insertion of wave platesPetrographic microscopes are constructed with optical parts that do not add unwanted polarizing effects due to strained glass, or polarization by reflection in prisms and mirrors.
These special parts add to the complexity of the microscope. However, a "simple polarizing" microscope is made by adding inexpensive polarizing filters to a standard biological microscope with one in a filter holder beneath the condenser, a second inserted beneath the head or eyepiece; these can be sufficient for many non-quantitative purposes. The two Nicol prisms of the petrographic microscope have their polarizing planes oriented perpendicular to one another; when only an isotropic material such as air, water, or glass exists between the filters, all light is blocked, but most crystalline materials and minerals change the polarizing light directions, allowing some of the altered light to pass through the analyzer to the viewer. Using one polarizer makes it possible to view the slide in plane polarized light. A particular light pattern on the upper lens surface of the objectives is created as a conoscopic interference pattern characteristic of uniaxial and biaxial minerals, produced with convergent polarized light.
To observe the interference figure, true petrographic microscopes include an accessory called a Bertrand lens, which focuses and enlarges the figure. It is possible to remove an eyepiece lens to make a direct observation of the objective lens surface. In addition to modifications of the microscope's optical system, petrographic microscopes allow for the insertion of specially-cut oriented filters of biaxial minerals, into the optical train between the polarizers to identify positive and negative birefringence, in extreme cases, the mineral order when needed
Bright-field microscopy is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light, contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample. Bright-field microscopy is the simplest of a range of techniques used for illumination of samples in light microscopes, its simplicity makes it a popular technique; the typical appearance of a bright-field microscopy image is a dark sample on a bright background, hence the name. The light path of a bright-field microscope is simple, no additional components are required beyond the normal light-microscope setup; the light path therefore consists of: a transillumination light source a halogen lamp in the microscope stand. Bright-field microscopy may use critical or Köhler illumination to illuminate the sample. Bright-field microscopy has low contrast with most biological samples, as few absorb light to a great extent. Staining is required to increase contrast, which prevents use on live cells in many situations.
Bright-field illumination is useful for samples that have an intrinsic color, for example chloroplasts in plant cells. Comparison of transillumination techniques used to generate contrast in a sample of tissue paper Bright-field microscopy is a standard light-microscopy technique, therefore magnification is limited by the resolving power possible with the wavelength of visible light. Simplicity of setup with only basic equipment required. Living cells can be seen with bright-field microscopes. Low contrast of most biological samples; the practical limit to magnification with a light microscope is around 1300X. Although higher magnifications are possible, it becomes difficult to maintain image clarity as the magnification increases. Low apparent optical resolution due to the blur of out-of-focus material. Samples that are colorless and transparent cannot be seen well, e.g. many types of mammalian cells. These samples have to be stained before viewing. Samples that do have their own color can be seen without preparation, e.g. the observation of cytoplasmic streaming in Chara cells.
Reducing or increasing the amount of the light source by the iris diaphragm. Use of an oil-immersion objective lens and a special immersion oil placed on a glass cover over the specimen. Immersion oil improves the resolution of the observed specimen. Use of sample-staining methods for use in microbiology, such as simple stains and differential stains. Use of a colored or polarizing filter on the light source to highlight features not visible under white light; the use of filters is useful with mineral samples. Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers Microbiology: Principles and Explorations by Jacquelyn G. Black, John Wiley & Sons, Inc. Microscopy and Imaging LiteratureNotes
In physics, interference is a phenomenon in which two waves superpose to form a resultant wave of greater, lower, or the same amplitude. Constructive and destructive interference result from the interaction of waves that are correlated or coherent with each other, either because they come from the same source or because they have the same or nearly the same frequency. Interference effects can be observed with all types of waves, for example, radio, surface water waves, gravity waves, or matter waves; the resulting images or graphs are called interferograms. The principle of superposition of waves states that when two or more propagating waves of same type are incident on the same point, the resultant amplitude at that point is equal to the vector sum of the amplitudes of the individual waves. If a crest of a wave meets a crest of another wave of the same frequency at the same point the amplitude is the sum of the individual amplitudes—this is constructive interference. If a crest of one wave meets a trough of another wave the amplitude is equal to the difference in the individual amplitudes—this is known as destructive interference.
Constructive interference occurs when the phase difference between the waves is an multiple of π, whereas destructive interference occurs when the difference is an odd multiple of π. If the difference between the phases is intermediate between these two extremes the magnitude of the displacement of the summed waves lies between the minimum and maximum values. Consider, for example, what happens when two identical stones are dropped into a still pool of water at different locations; each stone generates a circular wave propagating outwards from the point where the stone was dropped. When the two waves overlap, the net displacement at a particular point is the sum of the displacements of the individual waves. At some points, these will be in phase, will produce a maximum displacement. In other places, the waves will be in anti-phase, there will be no net displacement at these points. Thus, parts of the surface will be stationary—these are seen in the figure above and to the right as stationary blue-green lines radiating from the centre.
Interference of light is a common phenomenon that can be explained classically by the superposition of waves, however a deeper understanding of light interference requires knowledge of wave-particle duality of light, due to quantum mechanics. Prime examples of light interference are the famous double-slit experiment, laser speckle, anti-reflective coatings and interferometers. Traditionally the classical wave model is taught as a basis for understanding optical interference, based on the Huygens–Fresnel principle; the above can be demonstrated in one dimension by deriving the formula for the sum of two waves. The equation for the amplitude of a sinusoidal wave traveling to the right along the x-axis is W 1 = A cos where A is the peak amplitude, k = 2 π / λ is the wavenumber and ω = 2 π f is the angular frequency of the wave. Suppose a second wave of the same frequency and amplitude but with a different phase is traveling to the right W 2 = A cos where φ is the phase difference between the waves in radians.
The two waves will superpose and add: the sum of the two waves is W 1 + W 2 = A. Using the trigonometric identity for the sum of two cosines: cos a + cos b = 2 cos cos , this can be written W 1 + W 2 = 2 A cos cos ; this represents a wave at the original frequency, traveling to the right like the components, whose amplitude is proportional to the cosine of φ / 2. Constructive interference: If the phase difference is an multiple of π: φ = …, − 4 π, − 2 π, 0, 2 π, 4 π, …
The optical microscope referred to as the light microscope, is a type of microscope that uses visible light and a system of lenses to magnify images of small objects. Optical microscopes are the oldest design of microscope and were invented in their present compound form in the 17th century. Basic optical microscopes can be simple, although many complex designs aim to improve resolution and sample contrast. Used in the classroom and at home unlike the electron microscope, used for closer viewing; the image from an optical microscope can be captured by normal, photosensitive cameras to generate a micrograph. Images were captured by photographic film, but modern developments in CMOS and charge-coupled device cameras allow the capture of digital images. Purely digital microscopes are now available which use a CCD camera to examine a sample, showing the resulting image directly on a computer screen without the need for eyepieces. Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy and scanning probe microscopy.
On 8 October 2014, the Nobel Prize in Chemistry was awarded to Eric Betzig, William Moerner and Stefan Hell for "the development of super-resolved fluorescence microscopy," which brings "optical microscopy into the nanodimension". There are two basic types of optical microscopes: compound microscopes. A simple microscope is one. A compound microscope uses several lenses to enhance the magnification of an object; the vast majority of modern research microscopes are compound microscopes while some cheaper commercial digital microscopes are simple single lens microscopes. Compound microscopes can be further divided into a variety of other types of microscopes which differ in their optical configurations and intended purposes. A regular microscope uses a lens or set of lenses to enlarge an object through angular magnification alone, giving the viewer an erect enlarged virtual image; the use of a single convex lens or groups of lenses are found in simple magnification devices such as the magnifying glass and eyepieces for telescopes and microscopes.
A compound microscope uses a lens close to the object being viewed to collect light which focuses a real image of the object inside the microscope. That image is magnified by a second lens or group of lenses that gives the viewer an enlarged inverted virtual image of the object; the use of a compound objective/eyepiece combination allows for much higher magnification. Common compound microscopes feature exchangeable objective lenses, allowing the user to adjust the magnification. A compound microscope enables more advanced illumination setups, such as phase contrast. There are many variants of the compound optical microscope design for specialized purposes; some of these are physical design differences allowing specialization for certain purposes: Stereo microscope, a low-powered microscope which provides a stereoscopic view of the sample used for dissection. Comparison microscope, which has two separate light paths allowing direct comparison of two samples via one image in each eye. Inverted microscope, for studying samples from below.
Fiber optic connector inspection microscope, designed for connector end-face inspection Traveling microscope, for studying samples of high optical resolution. Other microscope variants are designed for different illumination techniques: Petrographic microscope, whose design includes a polarizing filter, rotating stage and gypsum plate to facilitate the study of minerals or other crystalline materials whose optical properties can vary with orientation. Polarizing microscope, similar to the petrographic microscope. Phase contrast microscope, which applies the phase contrast illumination method. Epifluorescence microscope, designed for analysis of samples which include fluorophores. Confocal microscope, a used variant of epifluorescent illumination which uses a scanning laser to illuminate a sample for fluorescence. Two-photon microscope, used to image fluorescence deeper in scattering media and reduce photobleaching in living samples. Student microscope – an low-power microscope with simplified controls and sometimes low quality optics designed for school use or as a starter instrument for children.
Ultramicroscope, an adapted light microscope that uses light scattering to allow viewing of tiny particles whose diameter is below or near the wavelength of visible light. Microscopes can be or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and quantitaton of a fluorescent or histological stain. Low-powered digital microscopes, USB microscopes, are commercially available; these are webcams with a high-powered macro lens and do not use transillumination. The camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor, they offer modest magnifications without the need to use eyepieces, at low cost. High power illumination is provided by an LED source or sources adjacent to the camera lens. Digital microscopy with low light levels to avoid damage to vulnerable biological samples is available using sensitive photon-counting digital
Birefringence is the optical property of a material having a refractive index that depends on the polarization and propagation direction of light. These optically anisotropic materials are said to be birefringent; the birefringence is quantified as the maximum difference between refractive indices exhibited by the material. Crystals with non-cubic crystal structures are birefringent, as are plastics under mechanical stress. Birefringence is responsible for the phenomenon of double refraction whereby a ray of light, when incident upon a birefringent material, is split by polarization into two rays taking different paths; this effect was first described by the Danish scientist Rasmus Bartholin in 1669, who observed it in calcite, a crystal having one of the strongest birefringences. However it was not until the 19th century that Augustin-Jean Fresnel described the phenomenon in terms of polarization, understanding light as a wave with field components in transverse polarizations. A mathematical description of wave propagation in a birefringent medium is presented below.
Following is a qualitative explanation of the phenomenon. The simplest type of birefringence is described as uniaxial, meaning that there is a single direction governing the optical anisotropy whereas all directions perpendicular to it are optically equivalent, thus rotating the material around this axis does not change its optical behavior. This special direction is known as the optic axis of the material. Light propagating parallel to the optic axis is governed by a refractive index no. Light whose polarization is in the direction of the optic axis sees an optical index ne. For any ray direction there is a linear polarization direction perpendicular to the optic axis, this is called an ordinary ray. However, for ray directions not parallel to the optic axis, the polarization direction perpendicular to the ordinary ray's polarization will be in the direction of the optic axis, this is called an extraordinary ray. I.e. when unpolarized light enters an uniaxial birefringent material it is split into two beams travelling different directions.
The ordinary ray will always experience a refractive index of no, whereas the refractive index of the extraordinary ray will be in between no and ne, depending on the ray direction as described by the index ellipsoid. The magnitude of the difference is quantified by the birefringence: Δ n = n e − n o; the propagation of the ordinary ray is described by no as if there were no birefringence involved. However the extraordinary ray, as its name suggests, propagates unlike any wave in a homogenous optical material, its refraction at a surface can be understood using the effective refractive index. However it is in fact an inhomogeneous wave whose power flow is not in the direction of the wave vector; this causes an additional shift in that beam when launched at normal incidence, as is popularly observed using a crystal of calcite as photographed above. Rotating the calcite crystal will cause one of the two images, that of the extraordinary ray, to rotate around that of the ordinary ray, which remains fixed.
When the light propagates either along or orthogonal to the optic axis, such a lateral shift does not occur. In the first case, both polarizations see the same effective refractive index, so there is no extraordinary ray. In the second case the extraordinary ray propagates at a different phase velocity but is not an inhomogeneous wave. A crystal with its optic axis in this orientation, parallel to the optical surface, may be used to create a waveplate, in which there is no distortion of the image but an intentional modification of the state of polarization of the incident wave. For instance, a quarter-wave plate is used to create circular polarization from a linearly polarized source; the case of so-called biaxial crystals is more complex. These are characterized by three refractive indices corresponding to three principal axes of the crystal. For most ray directions, both polarizations would be classified as extraordinary rays but with different effective refractive indices. Being extraordinary waves, the direction of power flow is not identical to the direction of the wave vector in either case.
The two refractive indices can be determined using the index ellipsoids for given directions of the polarization. Note that for biaxial crystals the index ellipsoid will not be an ellipsoid of revolution but is described by three unequal principle refractive indices nα, nβ and nγ, thus there is no axis. Although there is no axis of symmetry, there are two optical axes or binormals which are defined as directions along which light may propagate without birefringence, i.e. directions along which the wavelength is independent of polarization. For this reason, birefringent materials with three distinct refractive indices are called biaxial. Additionally, there are two distinct axes known as optical ray axes or biradials along which the group velocity of the light is independent of polarization; when an arbitrary beam of light strikes the surface of a b
Dark-field 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 is dark. In optical microscopy, dark-field describes an illumination technique used to enhance the contrast in unstained samples, it works by illuminating the sample with light that will not be collected by the objective lens and thus will not form part of the image. This produces the classic appearance of a dark black, background with bright objects on it; the steps are illustrated in the figure. Light enters the microscope for illumination of the sample. A specially sized disc, the patch stop, blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can be reasonably substituted at low magnification; the condenser lens focuses the light towards the sample. The light enters the sample. Most is directly transmitted; the scattered light enters the objective lens, while the directly transmitted light misses the lens and is not collected due to a direct-illumination block.
Only the scattered light goes on to produce the image, while the directly transmitted light is omitted. Dark-field microscopy is a simple yet effective technique and well suited for uses involving live and unstained biological samples, such as a smear from a tissue culture or individual, water-borne, single-celled organisms. Considering the simplicity of the setup, the quality of images obtained from this technique is impressive; the main limitation of dark-field microscopy is the low light levels seen in the final image. This means that the sample must be strongly illuminated, which can cause damage to the sample. Dark-field microscopy techniques are entirely free of artifacts, due to the nature of the process. However, the interpretation of dark-field images must be done with great care, as common dark features of bright-field microscopy images may be invisible, vice versa. While the dark-field image may first appear to be a negative of the bright-field image, different effects are visible in each.
In bright-field microscopy, features are visible where either a shadow is cast on the surface by the incident light or a part of the surface is less reflective by the presence of pits or scratches. Raised features that are too smooth to cast shadows will not appear in bright-field images, but the light that reflects off the sides of the feature will be visible in the dark-field images. Comparison of transillumination techniques used to generate contrast in a sample of tissue paper Dark-field microscopy has been used in computer mouse pointing devices, in order to allow an optical mouse to work on transparent glass by imaging microscopic flaws and dust on its surface; when coupled to hyperspectral imaging, dark-field microscopy becomes a powerful tool for the characterization of nanomaterials embedded in cells. In a recent publication, Patskovsky et al. used this technique to study the attachment of gold nanoparticles targeting CD44+ cancer cells. Dark-field studies in transmission electron microscopy play a powerful role in the study of crystals and crystal defects, as well as in the imaging of individual atoms.
Imaging involves tilting the incident illumination until a diffracted, rather than the incident, beam passes through a small objective aperture in the objective lens back focal plane. Dark-field images, under these conditions, allow one to map the diffracted intensity coming from a single collection of diffracting planes as a function of projected position on the specimen and as a function of specimen tilt. In single-crystal specimens, single-reflection dark-field images of a specimen tilted just off the Bragg condition allow one to "light up" only those lattice defects, like dislocations or precipitates, that bend a single set of lattice planes in their neighborhood. Analysis of intensities in such images may be used to estimate the amount of that bending. In polycrystalline specimens, on the other hand, dark-field images serve to light up only that subset of crystals that are Bragg-reflecting at a given orientation. Weak-beam imaging involves optics similar to conventional dark-field, but use of a diffracted beam harmonic rather than the diffracted beam itself.
Much higher resolution of strained regions around defects can be obtained in this way. Annular dark-field imaging requires one to form images with electrons diffracted into an annular aperture centered on, but not including, the unscattered beam. For large scattering angles in a scanning transmission electron microscope, this is sometimes called Z-contrast imaging because of the enhanced scattering from high-atomic-number atoms; this a mathematical technique intermediate between direct and reciprocal space for exploring images with well-defined periodicities, like electron microscope lattice-fringe images. As with analog dark-field imaging in a transmission electron microscope, it allows one to "light up" those objects in the field of view where periodicities of interest reside. Unlike analog dark-field imaging it may allow one to map the Fourier-phase of periodicities, hence phase gradients, which provide quantitative information on vector lattice strain. Annular dark-field imaging Light field microscopy Wavelets Nikon - Stereomicroscopy > Darkfield Illumination Molecular Expressions Darkfield Illumination Primer Gage SH. 1920.
Modern dark-field the history of its development. Transactions of the American Microscopical Society 39:95–141. Dark field and phas