When very thin and transparent samples are imaged, such as cells or tissue cultures fixed on glass slides, transmitted illumination is the preferred technique. Transmitted illumination, represented by the blue optical path in Figure 2, directs light through the specimen and into the objective, allowing for better observation of a transparent sample’s morphology. In transmission mode, since the incoming light is not directed through a focusing objective like in reflection mode, a condenser located on the opposite side of the stage from the objective is used to gather the light from the transmission lamp and concentrate it into a cone that illuminates the sample. In microscopes that have both illumination modes available, the direction of illumination light is altered by pressing a switch on the frame. Figure 3 shows an example of a sample where transmitted illumination gives better contrast to visualise the sample than reflected.

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.

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.

Briefly, imaging[5] 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.

There are two main contrast modes in optical microscopy, brightfield and darkfield. In the reflected brightfield mode shown in Figure 4, light is reflected into the centre of the objective via a half-silvered mirror. In reflected darkfield, a light stop is engaged, and the mirror is changed to a full silvered ring mirror that causes the illuminating beam to be directed down the edges of the objective.

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.

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.

In this Spectral School tutorial, we introduce some of the most common optical microscopy terms and techniques, including upright and inverted microscopes, reflected and transmitted illumination modes, and brightfield and darkfield contrast mechanisms, and discuss their applicability with respect to different research areas.

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 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. Optical dark fields usually done with an condenser that features a central light-stop in front of the light source to prevent direct illumination of the focal plane, and at higher numerical apertures may require oil or water between the condenser and the specimen slide to provide an optimal refractive index.[2][3]

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Upright microscopes are used to visualise samples, with the surface of interest facing upward towards an objective lens that is located above the stage. Upright microscopes can be used to image most sample types and can support slides, well plates, and specialised temperature, pressure, or electrochemical stages. There are, however, two circumstances in which this configuration is not suitable, these are when the sample is too large to fit underneath the objective and when sample access is required from above during imaging. In both cases, an inverted microscope would be required.

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Figure 1. Upright and inverted microscope configurations showing the orientation of the objective relative to the stage and sample.

In this Spectral School tutorial, we discussed some of the most common optical microscopy terms and techniques, such as brightfield microscopy, and how they can all be used to optimally visualise various sample types prior to performing spectroscopic analysis. This is an important consideration in experiments that utilise techniques that merge optical microscopy with spectroscopy because it allows the user to be sure of sample alignment, and it enables them to find interesting features on the sample prior to chemical imaging.

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At the sample, the two techniques differ primarily in the angle at which the illuminating beam is incident; generally, the brightfield geometry involves illumination from an acute angle between 45 and 90 degrees from the horizontal plane of the sample, whereas the darkfield geometry involves illumination from an oblique angle between 0 and 45 degrees, Figure 5. In brightfield microscopy, the acute angle light will primarily be reflected back into the objective lens if it is not absorbed. This means that absorbing features will appear dark against a bright background. Due to the oblique incident angle in darkfield illumination, the light is typically not reflected into the objective lens, resulting in a dark background. Instead, darkfield is employed to detect defects and/or edges in the sample, which cause the oblique incident ray to be scattered into the objective.

Reflected illumination, represented by the red optical path in Figure 2, is considered the best mode for visualising any opaque sample that light is unable to fully penetrate such as semiconductor wafers, polymers, paint, paper, metal, and pharmaceuticals. When a sample is viewed using reflected illumination, light is directed onto the surface of the sample through the objective, and it then re-enters the objective after being reflected off the sample. Upon re-entering the objective, the light is directed into a camera for visualisation of the sample.

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dark-field microscopy

Darkfield contrast requires specialised optics, for the reflected darkfield (as shown in Figure 4), a specialised darkfield objective and nosepiece are required. Darkfield transmission is also possible and for that, a darkfield condenser is required. Figure 6 shows an example of a sample, consisting of various diatoms fixed on a glass microscope slide, in which darkfield illumination provides better contrast than brightfield. It can be seen in the darkfield image that the edges and fine structure of the diatoms that scatter light into the objective contrast very well against the background, and that the improvement in contrast over brightfield is significant.

In an inverted configuration, the objective is located underneath the stage facing upwards towards the sample. Here, the surface of interest on the sample must face downwards towards the objective, which means that at least this side of the sample must be flat. One example where an inverted microscope is better suited is in live cell imaging because this application makes use of stage-top incubators where cells sink to the bottom of the sample chamber and access is required from above for the exchange of liquid media.

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.

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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.

Dark fieldimage

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.[4]

An important consideration when choosing a microscope is the location of the objective lens relative to the sample. In an upright microscope, the objective is located above the sample, whereas in an inverted it is below. Despite differences in construction and appearance, they have the same ability to image the sample and are designed to work best with different sample types.

Dark-field microscopy techniques are almost entirely free of halo or relief-style artifacts typical of differential interference contrast microscopy. This comes at the expense of sensitivity to phase information.

Figure 5. A) Differences in the incident angle of brightfield and darkfield and B) the detection of defect scattered light in darkfield.

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]

Brightfield microscopy uses light from the lamp source under the microscope stage to illuminate the specimen. This light is gathered in the condenser, then ...

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.

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Bright field dark field

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.

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.

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.

Since a range of samples with widely varying optical properties, from opaque semiconductor chips to semi-transparent tissue cultures, can be observed with a microscope, most modern microscopes enable the user to illuminate the sample in both reflection and transmission modes depending on the specific application, Figure 2. Note that this schematic illustrates an upright microscope and that both illumination modes are also available on inverse microscope frames.