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Dark-field microscopy (also called dark-ground microscopy) describes
microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of micr ...
methods, in both
light Light or visible light is electromagnetic radiation that can be perceived by the human eye. Visible light is usually defined as having wavelengths in the range of 400–700 nanometres (nm), corresponding to frequencies of 750–420 t ...
and
electron microscopy An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a hi ...
, which exclude the unscattered beam from the image. As a result, the field around the specimen (i.e., where there is no specimen to
scatter Scatter may refer to: * Scattering, in physics, the study of collisions * Statistical dispersion or scatter * Scatter (modeling), a substance used in the building of dioramas and model railways * Scatter, in computer programming, a parameter in ...
the beam) is generally dark. In optical microscopes a darkfield condenser lens must be used, which directs a cone of light away from the objective lens. To maximize the scattered light-gathering power of the objective lens, oil immersion is used and the
numerical aperture In optics, the numerical aperture (NA) of an optical system is a dimensionless number that characterizes the range of angles over which the system can accept or emit light. By incorporating index of refraction in its definition, NA has the proper ...
(NA) of the objective lens must be less than 1.0. Objective lenses with a higher NA can be used but only if they have an adjustable diaphragm, which reduces the NA. Often these objective lenses have a NA that is variable from 0.7 to 1.25.


Light microscopy applications

In
optical microscopy Optics is the branch of physics that studies the behaviour and properties of light, including its interactions with matter and the construction of instruments that use or detect it. Optics usually describes the behaviour of visible, ultravio ...
, 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, almost black, background with bright objects on it.


The light's path

The steps are illustrated in the figure where an inverted microscope is used. # Light enters the
microscope A microscope () is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisi ...
for illumination of the sample. # A specially sized disc, the ''patch stop'' (see figure), blocks some light from the light source, leaving an outer ring of illumination. A wide phase annulus can also be reasonably substituted at low magnification. # The
condenser lens A condenser is an optical lens which renders a divergent beam from a point source into a parallel or converging beam to illuminate an object. Condensers are an essential part of any imaging device, such as microscopes, enlargers, slide projecto ...
focuses the light towards the sample. # The light enters the sample. Most is directly transmitted, while some is scattered from the sample. # The scattered light enters the objective lens, while the directly transmitted light simply misses the lens and is not collected due to a ''direct-illumination block'' (see figure). # Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.


Advantages and disadvantages

Dark-field microscopy is a very 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. One limitation of dark-field microscopy is the low light levels seen in the final image. This means that the sample must be very strongly illuminated, which can cause damage to the sample. Dark-field microscopy techniques are almost entirely free of halo or relief-style artifacts typical of DIC and phase-contrast imaging. This comes at the expense of sensitivity to phase information. The interpretation of dark-field images must be done with great care, as common dark features of
bright-field microscopy Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by at ...
images may be invisible, and vice versa. In general the dark-field image lacks the low
spatial frequencies In mathematics, physics, and engineering, spatial frequency is a characteristic of any structure that is periodic across position in space. The spatial frequency is a measure of how often sinusoidal components (as determined by the Fourier tra ...
associated with the bright-field image, making the image a high-passed version of the underlying structure. 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, possibly 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. tissue paper Tissue paper or simply tissue is a lightweight paper or, light crêpe paper. Tissue can be made from recycled paper pulp on a paper machine. Tissue paper is very versatile, and different kinds of tissue are made to best serve these purposes, w ...
(1.559 μm/pixel when viewed at full resolution)" align="center"> Image:Paper_Micrograph_Dark.png, Dark-field illumination, sample contrast comes from light
scattered Scattered may refer to: Music * ''Scattered'' (album), a 2010 album by The Handsome Family * "Scattered" (The Kinks song), 1993 * "Scattered", a song by Ace Young * "Scattered", a song by Lauren Jauregui * "Scattered", a song by Green Day from ' ...
by the sample Image:Paper_Micrograph_Bright.png, Bright-field illumination, sample contrast comes from
attenuation In physics, attenuation (in some contexts, extinction) is the gradual loss of flux intensity through a medium. For instance, dark glasses attenuate sunlight, lead attenuates X-rays, and water and air attenuate both light and sound at var ...
of light in the sample Image:Paper_Micrograph_Cross-Polarised.png,
Cross-polarized light Polarized light microscopy can mean any of a number of optical microscopy techniques involving polarized light. Simple techniques include illumination of the sample with polarized light. Directly transmitted light can, optionally, be blocked with ...
illumination, sample contrast comes from rotation of polarized light through the sample Image:Paper_Micrograph_Phase.png,
Phase-contrast __NOTOC__ Phase-contrast microscopy (PCM) is an optical microscopy technique that converts phase shifts in light passing through a transparent specimen to brightness changes in the image. Phase shifts themselves are invisible, but become visibl ...
illumination, sample contrast comes from interference of different path lengths of light through the sample


Use in computing

Dark-field microscopy has recently been applied in computer mouse pointing devices to allow the mouse to work on transparent glass by imaging microscopic flaws and dust on the glass's surface.


Dark-field microscopy combined with hyperspectral imaging

When coupled to
hyperspectral imaging Hyperspectral imaging collects and processes information from across the electromagnetic spectrum. The goal of hyperspectral imaging is to obtain the spectrum for each pixel in the image of a scene, with the purpose of finding objects, identifyi ...
, dark-field microscopy becomes a powerful tool for the characterization of
nanomaterials * Nanomaterials describe, in principle, materials of which a single unit is sized (in at least one dimension) between 1 and 100 nm (the usual definition of nanoscale). Nanomaterials research takes a materials science-based approach to n ...
embedded in cells. In a recent publication, Patskovsky et al. used this technique to study the attachment of gold
nanoparticles A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 10 ...
(AuNPs) targeting CD44+ cancer cells.


Transmission electron microscope applications

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.


Conventional dark-field imaging

Briefly, imagingP. Hirsch, A. Howie, R. Nicholson, D. W. Pashley and M. J. Whelan (1965/1977) ''Electron microscopy of thin crystals'' (Butterworths/Krieger, London/Malabar FL) . 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 In physics and chemistry , Bragg's law, Wulff–Bragg's condition or Laue–Bragg interference, a special case of Laue diffraction, gives the angles for coherent scattering of waves from a crystal lattice. It encompasses the superposition of wave ...
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 then 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

Weak-beam imaging involves optics similar to conventional dark-field, but uses a diffracted beam ''harmonic'' rather than the diffracted beam itself. In this way, much higher resolution of strained regions around defects can be obtained.


Low- and high-angle annular dark-field imaging

Annular dark-field imaging Annular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope (STEM). These images are formed by collecting scattered electrons with an annular dark-field detector. Conventional TEM 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.


Digital dark-field analysis

This a mathematical technique intermediate between direct and reciprocal (Fourier-transform) 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 also allow one to map the ''Fourier-phase'' of periodicities, and hence phase gradients, which provide quantitative information on vector lattice strain.


See also

*
Annular dark-field imaging Annular dark-field imaging is a method of mapping samples in a scanning transmission electron microscope (STEM). These images are formed by collecting scattered electrons with an annular dark-field detector. Conventional TEM dark-field imaging ...
*
Light field microscopy Light field microscopy (LFM) is a scanning-free 3-dimensional (3D) microscopic imaging method based on the theory of light field. This technique allows sub-second (~10 Hz) large volumetric imaging ( 0.1 to 1 mmsup>3) with ~1 μm spatial ...
*
Wavelets A wavelet is a wave-like oscillation with an amplitude that begins at zero, increases or decreases, and then returns to zero one or more times. Wavelets are termed a "brief oscillation". A taxonomy of wavelets has been established, based on the nu ...


Footnotes


External links


Nikon - Stereomicroscopy > Darkfield Illumination

Molecular Expressions


* Gage SH. 1920
Modern dark-field microscopy and the history of its development
''Transactions of the American Microscopical Society'' 39(2):95–141.
Dark field and phase contrast microscopes
(Université Paris Sud) {{Optical microscopy Electron microscopy Optical microscopy techniques