Other Types of Microscopy

Dark-Field Microscopy

Dark-field microscopes show a light silhouette of an organism against a dark background.

Learning Objectives

Generalize the process of dark-field microscopy

Key Takeaways

Key Points

  • In dark-field microscopy, the light reaches the specimen from an angle with the help of an opaque disk.
  • The specimen appears lit up agains a dark background.
  • Dark-field microscopy is most useful for extremely small living organisms that are invisible in the light microscope.

Key Terms

  • condenser: A lens (or combination of lenses) designed to gather light and focus it onto a specimen or part of a mechanism.

Radiance Against a Dark Background

Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear brightly lit against a dark background. This type of microscope contains a special condenser that scatters light and causes it to reflect off the specimen at an angle. Rather than illuminating the sample with a filled cone of light, the condenser is designed to form a hollow cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this light moves past the specimen plane it spreads again into a hollow cone. The objective lens sits in the dark hollow of this cone; although the light travels around and past the objective lens, no rays enter it.

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Visualization of live bacteria: Spirochetes bacteria observed under dark field microscopy.

The entire field appears dark when there is no sample on the microscope stage; thus the name dark-field microscopy. When a sample is on the stage, the light at the apex of the cone strikes it. The rays scattered by the sample and captured in the objective lens thus make the image.

Samples observed under dark-field microscopy should be carefully prepared since dust and other particles also scatter the light and are easily detected. Glass slides need to be thoroughly cleaned of extraneous dust and dirt. It may be necessary to filter sample media (agar, water, saline) to exclude confusing contaminants. Sample materials need to be spread thinly; too much material on the slide creates many overlapping layers and edges, making it difficult to interpret structures.

Dark-field microscopy has many applications in microbiology. It allows the visualization of live bacteria, and distinguishes some structure (rods, curved rods, spirals, or cocci) and movement.

Phase-Contrast Microscopy

Phase-contrast microscopy visualizes differences in the refractive indexes of different parts of a specimen relative to unaltered light.

Learning Objectives

Describe the mechanics, advantages, and disadvantages of phase-contrast microscopy

Key Takeaways

Key Points

  • A phase-contrast microscope splits a beam of light into 2 types of light, direct and refracted (reflected) and brings them together to form an image of the specimen.
  • Where the lights are “in-phase” the image is brighter, where the lights are “out of phase” the image is darker, and by amplifying these differences in the light, it enhances contrast.
  • Phase-contrast microscopy allows for the detailed observation of living organisms, especially the internal structures.

Key Terms

  • refractive index: the ratio of the speed of light in air or vacuum to that in another medium.

Phase-contrast microscopy is a method of manipulating light paths through the use of strategically placed rings in order to illuminate transparent objects. Dutch physicist Fritz Zernike developed the technique in the 1930s; for his efforts he was awarded the Nobel Prize in 1953.

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Phase-contrast microscopy: Phase-contrast image of a cheek epithelial cell.

In phase-contrast microscopy, parallel beams of light are passed through objects of different densities. The microscope contains special condensers that throw light “out of phase” causing it to pass through the object at different speeds. Internal details and organelles of live, unstained organisms (e.g. mitochondria, lysosomes, and the Golgi body) can be seen clearly with this microscope.

A phase ring in condenser allows a cylinder of light to pass through it while still in phase. Unaltered light hits the phase ring in the lens and is excluded. Light that is slightly altered by passing through a different refractive index is allowed to pass through. Light passing through cellular structures, such as chromosomes or mitochondria is retarded because they have a higher refractive index than the surrounding medium. Elements of lower refractive index advance the wave. Much of the background light is removed and light that constructively or destructively interfered is let through with enhanced contrast.

Phase-contrast microscopy allows the visualization of living cells in their natural state with high contrast and high resolution. This tool works best with a thin specimen and is not ideal for a thick specimen. Phase-contrast images have a characteristic grey background with light and dark features found across the sample. One disadvantage of phase-contrast microscopy is halo formation called halo-light ring.

Interference Microscopy

Interference microscopy is a variation of phase-contrast microscopy that uses a prism to split a light beam in two.

Learning Objectives

Describe the principles and different types of interference microscopy

Key Takeaways

Key Points

  • Interference microscopy is superior to phase-contrast microscopy in its ability to eliminate halos and extra light.
  • In differential interference contrast microscopy (DIC), the optical path difference is determined by the product of the refractive index difference (between the specimen and its surrounding medium) and the thickness traversed by a light beam between two points on the optical path.
  • Images produced by DIC have a distinctive shadow-cast appearance.

Key Terms

  • photobleaching: The destruction of a photochemical fluorescence by high-intensity light
  • fluorochrome: Any of various fluorescent dyes used to stain biological material before microscopic examination

Stereo Light Source

Interference microscopy uses a prism to split light into two slightly diverging beams that then pass through the specimen. It is thus based on measuring the differences in refractive index upon recombining the two beams. Interference occurs when a light beam is retarded or advanced relative to the other.

There are three types of interference microscopy: classical, differential contrast, and fluorescence contrast. Since its introduction in the late 1960s differential interference contrast microscopy (DIC) has been popular in biomedical research because it produces high-resolution images of fine structures by enhancing the contrasted interfaces. The image produced is of a thin optical section and appears three-dimensional, with a shadow around it. This creates a contrast across the specimen that is bright on one side and darker on the other.

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Path of light in differential interference contrast microscopy (DIC): Two parallel light beams pass through the specimen and combine to produce an image.

The Interference Microscope

The microscope is a bright field light microscope with the addition of the following elements: a polarizer between the light source and the condenser, a DIC beam-splitting prism, a DIC beam-combining prism, and an analyzer. Manipulating the prism changes the beam separation, which alters the contrast of the image. When the two beams pass through the same material across the specimen they produce no interference. When the two beams pass through different material across the specimen such as on the edges, they produce alteration when combined.

Fluorescence differential interference contrast (FLIC) microscopy was developed by combining fluorescence microscopy with DIC to minimize the effects of photobleaching on fluorochromes bound to the stained specimen. The same microscope is equipped to simulataneously image a specimen using DIC and fluorescence illumination.

Fluorescence Microscopy

Fluorescence microscopy is used to study specimens that are chemically manipulated to emit light.

Learning Objectives

Describe the techniques, advantages, and disadvantages of fluorescence microscopy

Key Takeaways

Key Points

  • In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near-ultraviolet) light source.
  • Microorganisms appear as bright objects against a dark background.
  • Fluorescence microscopy is used primarily in a procedure called fluorescent- antibody (FA) technique, or immunofluorescence.

Key Terms

  • autofluorescence: Self-induced fluorescence
  • halogen: any element of group 7, i.e. fluorine, chlorine, bromine, iodine and astatine, which form a salt by direct union with a metal

Fluoroscopy

The fluorescent microscope uses a high-pressure mercury, halogen, or xenon vapor lamp that emits a shorter wavelength than that emitted by traditional brightfield microscopy. These light sources produce ultraviolet light. When ultraviolet light hits an object, it excites the electrons of the object, and they give off light in various shades of color. Since ultraviolet light is used a larger amount of information can be gathered; thus, the resolution of the object increases.

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Fluorescent cells.: Fixed endothelial cells stained with fluorescent dyes. Nuclei are stained blue with DAPI, microtubules are stained green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC.

Fluorescent-Antibody Technique and Dyes

This laboratory technique employs fluorescent dyes chemically linked to antibodies to help identify unknown microorganisms. This method uses the specificity of an antibody to its antigen to deliver a fluorescent dye to a target molecule. A filter is used to block the heat generated from the lamp and to match the fluorescent dye labeling the specimen. An additional barrier filter between the objective and the detector can filter out the remaining excitation light from fluorescent light.

Fluorescent dyes—molecules that absorb light of one wavelength and then re-emit it at a longer visible wavelength—can be used alone or in combination to gain specificity of the stained structure being visualized. The light emitted from the fluorophore is magnified through traditional objectives and ocular lenses. Staining organisms with these special dyes reduces the non-specific autofluorescence that some organisms can emit. Cells or organisms stained with fluorochromes appear colored against a dark background when fixed on a glass slide. Fluorescence microscopy does not allow examination of live microorganisms as it requires them to be fixed and permeabilized for the antibody to penetrate inside the cells.

Confocal Microscopy

The key to the confocal approach is the use of spatial filtering techniques to eliminate out-of-focus light from biological samples.

Learning Objectives

Compare and contrast confocal and fluorescence microscopy

Key Takeaways

Key Points

  • Confocal microscopy requires immunoflurescence staining of biological samples.
  • Confocal microscopy serves to control depth of field, eliminate background, and collect optical sections.
  • The use of confocal microscopy has expanded to study both fixed and live cells with the ability to quantify targets.

Key Terms

  • photomultiplier tube: A vacuum tube that detects ultraviolet, visible, and near infrared light and multiplies it 100 million times.
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Confocal Microscopy: Tetrahymena cell, visualized using GFP-labeled anti-beta tubulin antibodies under confocal microscopy.

Confocal microscopy is a non-invasive fluorescent imaging technique that uses lasers of various colors to scan across a specimen with the aid of scanning mirrors. The point of illumination is brought to focus in the specimen by the objective lens. The scanning process uses a device that is under computer control. The sequences of points of light from the specimen are detected by a photomultiplier tube through a pinhole. The output is built into an image and transferred onto a digital computer screen for further analysis. The technique employs optical sectioning to take serial slices of the image. The slices are then stacked (Z-stack) to reconstruct the three-dimensional image of the biological sample. Optical sectioning is useful in determining cellular localization of targets. The biological sample to be studied is stained with antibodies chemically bound to fluorescent dyes similar to the method employed in fluorescence microscopy. Unlike in conventional fluorescence microscopy where the fluorescence is emitted along the entire illuminated cone creating a hazy image, in confocal microscopy the pinhole is added to allow passing of light that comes from a specific focal point on the sample and not the other. The light detected creates an image that is in focus with the original sample. Confocal microscopy has multiple applications in microbiology such as the study of biofilms and antibiotic-resistant strains of bacteria. Development of modern confocal microscopes has been accelerated by new advances in computer and storage technology, laser systems, detectors, interference filters, and fluorophores for highly specific targets.

Electron Microscopy

Electron microscopy uses magnetic coils to direct a beam of electrons from a tungsten filament through a specimen and onto a monitor.

Learning Objectives

Describe the technique employed for electron microscopy, distinguishing between different types

Key Takeaways

Key Points

  • A beam of electrons, instead of light, is used with an electron microscope.
  • Electron microscopes have a greater magnification because the wavelengths of electrons are much smaller than those of visible light (0.005nm as opposed to 500nm respectively–one hundred thousand times smaller).
  • There are two types of electron microscopes, scanning and transmission.
  • The best compound light microscopes can magnify 2000x, electron microscopes can magnify up to 100,000x.

Key Terms

  • electron beam: a stream of electrons observed in vacuum tubes.

Electron Microscopy

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Electron microscope: A modern electron microscope

Electron microscopy uses a beam of electrons as an energy source. An electron beam has an exceptionally short wavelength and can hit most objects in its path, increasing the resolution of the final image captured. The electron beam is designed to travel in a vacuum to limit interference by air molecules. Magnets are used to focus the electrons on the object viewed.

There are two types of electron microscopes. The more traditional form is the transmission electron microscope (TEM). To use this instrument, ultra-thin slices of microorganisms or viruses are placed on a wire grid and then stained with gold or palladium before viewing, to create contrast. The densely coated parts of the specimen deflect the electron beam and both dark and light areas show up on the image. TEM can project images in a much higher resolution—up to the atomic level of thinner objects.

The second and most contemporary form is the scanning electron microscope (SEM). It allows the visualization of microorganisms in three dimensions as the electrons are reflected when passed over the specimen. The same gold or palladium staining is employed.

Electron microscopy has multiple applications. It is ideal to:

  • explore the in vivo molecular mechanisms of disease;
  • visualize the three dimensional architecture of tissues and cells;
  • determine the conformation of flexible protein structures and complexes;
  • observe individual viruses and macromolecular complexes in their natural biological context.

Sample preparation can be critical to generate a successful image because the choice of reagents and methods used to process a sample largely depends on the nature of the sample and the analysis required.

Scanned-Probe Microscopy

Scanned-probe microscopy uses a fine probe rather than a light-beam or electrons to scan the surface of a specimen and produce a 3D image.

Learning Objectives

Describe the different types of scanning probe techniques and their advantages over other types of microscopy

Key Takeaways

Key Points

  • Scanned-probe microscopy has enabled researchers to create images of surfaces at the nanometer scale with a probe.
  • The probe has an extremely sharp tip that interacts with the surface of the specimen.
  • There are several variations of scanned-probe microscopy of which atomic force microscopy, scanning tunneling microscopy, and near-field scanning optical microscopy are most commonly used.

Key Terms

  • micrometer: An SI/MKS unit of measure, the length of one one-millionth of a meter. Symbols: µm, um, rm

3-D Images

Scanned-probe microscopy (SPM) produces highly magnified and three-dimensional-shaped images of specimens in real time. SPM employs a delicate probe to scan the surface of the specimen, eliminating the limitations that are found in electron and light microscopy. SPM covers several related technologies for imaging and measuring surfaces on a fine scale, down to the level of molecules and groups of atoms.

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Scanning tunneling microscopy: Schematic diagram of a scanning tunneling microscope.

A scan may cover a distance of over 100 micrometers in the x and y directions and 4 micrometers in the z direction. SPM technologies share the concept of scanning a sharp probe tip with a small radius of curvature across the object surface. The tip is mounted on a flexible cantilever, allowing the tip to follow the surface profile. When the tip moves in proximity to the investigated object, forces of interaction between the tip and the surface influence the movement of the cantilever. Selective sensors detect these movements. Various interactions can be studied depending on the mechanics of the probe.

There are three common scanning probe techniques: atomic force microscopy (AFM) measures the interaction force between the tip and surface. The tip may be dragged across the surface, or may vibrate as it moves. The interaction force will depend on the nature of the sample, the probe tip and the distance between them. Scanning tunneling microscopy (STM) measures a weak electrical current flowing between tip and sample as they are held apart. Near-field scanning optical microscopy (NSOM) scans a very small light source very close to the sample. Detection of this light energy forms the image.

X-Ray Diffraction Analysis

X-ray diffraction is a method that characterizes the structural composition of matter and using mathematical models.

Learning Objectives

Summarize the methods used for x-ray diffraction analysis and the contributions they have made to science

Key Takeaways

Key Points

  • X-ray diffraction utilizes x-ray beams targeted to hit crystallized matter and generates a diffraction pattern.
  • Data collected using this method undergo a systematic analytical process that employes mathematical models and computer algorithms to obtain the final 3D atom model of a matter.
  • X-ray diffraction analysis identifies composition and chemical bonds between atoms of crystal, liquid, powder, or amorphous samples.

Key Terms

  • Bragg’s equation: Gives the angles for coherent and incoherent scattering from a crystal lattice.
  • crystallography: The experimental science of determining the arrangement of atoms in solids.

X-ray diffraction (XRD) is a tool for characterizing the arrangement of atoms in crystals and the distances between crystal faces. The technique reveals detailed information about the chemical composition, crystallography, and microstructure of all types of natural and manufactured materials, which is key in understanding the properties of the material being studied.

Since many materials can form crystals—such as salts, metals, minerals, semiconductors, as well as various inorganic, organic, and biological molecules —X-ray crystallography has been fundamental in the development of many scientific fields. The method determined the size of atoms, the lengths and types of chemical bonds, and the atomic-scale differences among various materials, especially minerals and alloys. The method also revealed the structure and function of many biological molecules, including vitamins, drugs, proteins, and nucleic acids such as DNA.

Samples are commonly analyzed in a crystal form. X-ray diffraction is caused by constructive interference of x-ray waves that reflect off internal crystal planes. A thin film or layer of powder is fixed in the path of monochromatic x-rays. A detector measures x-rays from the sample over a range of angles. The powder consists of tiny crystals randomly oriented. At certain angles of the sensor, populations of crystals have the correct angle so that Bragg’s equation is satisfied for one of the crystal planes, resulting in a spike in X-rays.

The output graph displays x-ray intensity over 2 theta, the angle of the detector. The data generated with this technique requires extensive mathematical analysis that is now made easier by available computer algorithms. The analysis consists of indexing, merging, and phasing variations in electron density. It begins with the identification of molecules using the international center for diffraction database (ICDD). This is an organization dedicated to collecting, editing, publishing, and distributing powder diffraction data for the identification of crystalline materials. Further analysis involves structure refinement and quantitative phase using the general structure analysis system (GSAS), which ultimately leads to the identification of the amorphous or crystalline phase of a matter and helps construct its three dimensional atomic model.

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Structure determination by X-ray crystallography: X-ray diffraction analysis workflow. In an X-ray diffraction measurement, a crystal is mounted on a goniometer and gradually rotated while being bombarded with X-rays, producing a diffraction pattern of regularly spaced spots known as reflections. The two-dimensional images taken at different rotations are converted into a three-dimensional model of the density of electrons within the crystal using the mathematical method of Fourier transforms, combined with chemical data known for the sample.