Red blood cells as seen by darkfield microscopy x 1000Operating principle of dark-field and phase-contrast microscopies
Dark-field microscopy (also called dark-ground microscopy) describes microscopy methods, in both light and electron microscopy, which exclude the unscattered beam from the image. Consequently, the field around the specimen (i.e., where there is no specimen to scatter 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 (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.[1]
Light microscopy applications
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, 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.
Diagram illustrating the light path through a dark-field microscope
Light enters the microscope 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 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 produces an image with a dark background
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 images may be invisible, and vice versa. In general the dark-field image lacks the low spatial frequencies 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.
Comparison of transillumination techniques used to generate contrast in a sample of tissue paper (1.559 μm/pixel when viewed at full resolution)
Dark-field illumination, sample contrast comes from light scattered by the sample
Phase-contrast 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, 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 (AuNPs) targeting CD44+ cancer cells.[2]
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, imaging[3] 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 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.
Animation: dark-field imaging of crystals
Digital dark-field simulation of 2nm metal particles on a nano-cylinder This animation illustrates movement of an aperture (centered in the orange figure at left) over the power spectrum (a digital substitute for the back focal-plane's optical diffraction pattern) shown with the DC peak (or unscattered beam) below center. Only nanocrystals with projected periodicities that diffract into the aperture light up in the dark-field image at right. The aperture is moving by 1.25° increments around the ring associated with diffraction from gold 2.3Å (111) lattice spacings.
Weak-beam imaging
Digital dark-field image of internal twins
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 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.
↑ S. Patskovsky; etal. (2014). "Wide-field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy". Journal of Biophotonics. 8 (5): 1–7. doi:10.1002/jbio.201400025. PMID24961507.
↑ P. Hirsch, A. Howie, R. Nicholson, D. W. Pashley and M. J. Whelan (1965/1977) Electron microscopy of thin crystals (Butterworths/Krieger, London/Malabar FL) ISBN0-88275-376-2.
Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye. There are three well-known branches of microscopy: optical, electron, and scanning probe microscopy, along with the emerging field of X-ray microscopy.
A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope.
The optical microscope, also referred to as a light microscope, is a type of microscope that commonly uses visible light and a system of lenses to generate magnified images of small objects. Optical microscopes are the oldest design of microscope and were possibly invented in their present compound form in the 17th century. Basic optical microscopes can be very simple, although many complex designs aim to improve resolution and sample contrast.
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.
In optics, any optical instrument or system – a microscope, telescope, or camera – has a principal limit to its resolution due to the physics of diffraction. An optical instrument is said to be diffraction-limited if it has reached this limit of resolution performance. Other factors may affect an optical system's performance, such as lens imperfections or aberrations, but these are caused by errors in the manufacture or calculation of a lens, whereas the diffraction limit is the maximum resolution possible for a theoretically perfect, or ideal, optical system.
Confocal microscopy, most frequently confocal laser scanning microscopy (CLSM) or laser scanning confocal microscopy (LSCM), is an optical imaging technique for increasing optical resolution and contrast of a micrograph by means of using a spatial pinhole to block out-of-focus light in image formation. Capturing multiple two-dimensional images at different depths in a sample enables the reconstruction of three-dimensional structures within an object. This technique is used extensively in the scientific and industrial communities and typical applications are in life sciences, semiconductor inspection and materials science.
A scanning transmission electron microscope (STEM) is a type of transmission electron microscope (TEM). Pronunciation is [stɛm] or [ɛsti:i:ɛm]. As with a conventional transmission electron microscope (CTEM), images are formed by electrons passing through a sufficiently thin specimen. However, unlike CTEM, in STEM the electron beam is focused to a fine spot which is then scanned over the sample in a raster illumination system constructed so that the sample is illuminated at each point with the beam parallel to the optical axis. The rastering of the beam across the sample makes STEM suitable for analytical techniques such as Z-contrast annular dark-field imaging, and spectroscopic mapping by energy dispersive X-ray (EDX) spectroscopy, or electron energy loss spectroscopy (EELS). These signals can be obtained simultaneously, allowing direct correlation of images and spectroscopic data.
Metallography is the study of the physical structure and components of metals, by using microscopy.
Selected area (electron) diffraction, is a crystallographic experimental technique typically performed using a transmission electron microscope (TEM). It is a specific case of electron diffraction used primarily in material science and solid state physics as one of the most common experimental techniques. Especially with appropriate analytical software, SAD patterns (SADP) can be used to determine crystal orientation, measure lattice constants or examine its defects.
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.
High-resolution transmission electron microscopy is an imaging mode of specialized transmission electron microscopes that allows for direct imaging of the atomic structure of samples. It is a powerful tool to study properties of materials on the atomic scale, such as semiconductors, metals, nanoparticles and sp2-bonded carbon. While this term is often also used to refer to high resolution scanning transmission electron microscopy, mostly in high angle annular dark field mode, this article describes mainly the imaging of an object by recording the two-dimensional spatial wave amplitude distribution in the image plane, similar to a "classic" light microscope. For disambiguation, the technique is also often referred to as phase contrast transmission electron microscopy, although this term is less appropriate. At present, the highest point resolution realised in high resolution transmission electron microscopy is around 0.5 ångströms (0.050 nm). At these small scales, individual atoms of a crystal and defects can be resolved. For 3-dimensional crystals, it is necessary to combine several views, taken from different angles, into a 3D map. This technique is called electron tomography.
Differential interference contrast (DIC) microscopy, also known as Nomarski interference contrast (NIC) or Nomarski microscopy, is an optical microscopy technique used to enhance the contrast in unstained, transparent samples. DIC works on the principle of interferometry to gain information about the optical path length of the sample, to see otherwise invisible features. A relatively complex optical system produces an image with the object appearing black to white on a grey background. This image is similar to that obtained by phase contrast microscopy but without the bright diffraction halo. The technique was invented by Francis Hughes Smith. The "Smith DIK" was produced by Ernst Leitz Wetzlar in Germany and was difficult to manufacture. DIC was then developed further by Polish physicist Georges Nomarski in 1952.
Bright-field microscopy (BF) is the simplest of all the optical microscopy illumination techniques. Sample illumination is transmitted white light, and 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, and 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.
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 visible when shown as brightness variations.
Low-energy electron microscopy, or LEEM, is an analytical surface science technique used to image atomically clean surfaces, atom-surface interactions, and thin (crystalline) films. In LEEM, high-energy electrons are emitted from an electron gun, focused using a set of condenser optics, and sent through a magnetic beam deflector. The “fast” electrons travel through an objective lens and begin decelerating to low energies near the sample surface because the sample is held at a potential near that of the gun. The low-energy electrons are now termed “surface-sensitive” and the near-surface sampling depth can be varied by tuning the energy of the incident electrons. The low-energy elastically backscattered electrons travel back through the objective lens, reaccelerate to the gun voltage, and pass through the beam separator again. However, now the electrons travel away from the condenser optics and into the projector lenses. Imaging of the back focal plane of the objective lens into the object plane of the projector lens produces a diffraction pattern at the imaging plane and recorded in a number of different ways. The intensity distribution of the diffraction pattern will depend on the periodicity at the sample surface and is a direct result of the wave nature of the electrons. One can produce individual images of the diffraction pattern spot intensities by turning off the intermediate lens and inserting a contrast aperture in the back focal plane of the objective lens, thus allowing for real-time observations of dynamic processes at surfaces. Such phenomena include : tomography, phase transitions, adsorption, reaction, segregation, thin film growth, etching, strain relief, sublimation, and magnetic microstructure. These investigations are only possible because of the accessibility of the sample; allowing for a wide variety of in situ studies over a wide temperature range. LEEM was invented by Ernst Bauer in 1962; however, not fully developed until 1985.
Ptychography is a computational method of microscopic imaging. It generates images by processing many coherent interference patterns that have been scattered from an object of interest. Its defining characteristic is translational invariance, which means that the interference patterns are generated by one constant function moving laterally by a known amount with respect to another constant function. The interference patterns occur some distance away from these two components, so that the scattered waves spread out and "fold" into one another as shown in the figure.
A condenser is an optical lens which renders a divergent light beam from a point light source into a parallel or converging beam to illuminate an object to be imaged.
Lattice light-sheet microscopy is a modified version of light sheet fluorescence microscopy that increases image acquisition speed while decreasing damage to cells caused by phototoxicity. This is achieved by using a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images which can provide information about dynamic biological processes.
4D scanning transmission electron microscopy is a subset of scanning transmission electron microscopy (STEM) which utilizes a pixelated electron detector to capture a convergent beam electron diffraction (CBED) pattern at each scan location. This technique captures a 2 dimensional reciprocal space image associated with each scan point as the beam rasters across a 2 dimensional region in real space, hence the name 4D STEM. Its development was enabled by evolution in STEM detectors and improvements computational power. The technique has applications in visual diffraction imaging, phase orientation and strain mapping, phase contrast analysis, among others.
Weak beam dark field (WBDF) microscopy is a type of transmission electron microscopy (TEM) dark field imaging technique that allows for the visualization of crystal defects with high resolution and contrast. Specifically, the technique is mainly used to study crystal defects such as dislocations, stacking faults, and interfaces in crystalline materials. WBDF is a valuable tool for studying the microstructure of materials, as it can provide detailed information about the nature and distribution of defects in crystals. These characteristics can have a significant impact on material properties such as strength, ductility, and corrosion resistance.
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