Microscopes have been used for centuries in some form, and are now more widely used than ever. Today, many labs rely on a number of microscopes, each used for a specific purpose.
There are a number of ways of classifying microscopes, but commonly they are defined according to the way in which they interact with the sample to generate the image. The three main categories are optical microscopes, electron microscopes, and scanning probe microscopes.
The first decision when purchasing a microscope should be between these overall categories, each of which is more suited to different applications.
Optical microscopes are the most common type of microscope found in a standard laboratory. They use optical lenses in order to magnify the image generated by the passage of a wave through the sample or reflected by the sample.
Standard optical microscopes
Typical magnification of a standard optical microscope, assuming visible range light, is up to 1500x, with a theoretical resolution limit of around 0.2 Î¼m. Resolution is limited by the wavelength of the radiation used to image the sample. The use of shorter wavelengths of light, such as the ultraviolet, is one way to improve the spatial resolution of an optical microscope.
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A standard optical microscope is adequate for many laboratory requirements. Once the decision has been made to purchase a standard laboratory microscope, the appropriate specifications need to be determined.
Compound versus stereo
Compound microscopes offer higher magnification power than stereo microscopes. A compound microscope would be required to study cells, for example, explaining why this type of microscope is standard in many laboratories.
However, in order to study samples on a larger than cellular scale, a stereo microscope provide the advantage of scanning in three dimensions. This can be useful in many applications, including during dissection of tissues, for example.
Monocular, binocular or trinocular
A monocular microscope is the most affordable type of microscope and is favored by students. However, this type is rarely used in a professional laboratory.
A binocular microscope is generally preferred as it minimizes eye fatigue and muscle strain and offers improved contrast and color definition.
Trinocular microscopes have an additional port or access to which a digital camera may be attached. This type of microscope has the advantage of being able to save images electronically, allowing for easy sharing and access of images.
The way in which a sample is illuminated is an important consideration in optical microscopy, with some microscopes offering a range of options. The most common method of illumination is direct sunlight; however for some purposes a higher intensity light source is required from lamps such as strong halogen or metal-halide lamps. More sophisticated options, which may be provided as accessories for specialized purposes, include phase contrast, dark field and simple polarized light.
Most microscopes have coarse and fine adjustments to facilitate effective focusing.
Many microscopes also have an adjustable focus-stop to prevent damage to glass microslides and objective lenses.
Innovative optical microscopes
Technology in the field of optical microscopy is advancing rapidly. There are numerous types of microscope that are based on the optical principle, but which offer significant advances over the standard model. These innovative microscopes can be used for greater magnification and for other more specialized purposes for which a standard optical microscope is not sufficient.
Scanning confocal microscope
Scanning confocal microscopy is a technique used to increase optical resolution and contrast by eliminating out-of-focus light in specimens that are thicker than the focal plane. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects.
The image quality of the interior of a sample is greatly improved over simple microscopy because data from multiple layers within the specimen are not superimposed.
Spatially modulated illumination (SMI) microscope
SMI microscopy is a type of wide-field fluorescence microscopy involving interferometric illumination, which delivers structural information in fluorescently labeled cells. Vertico-SMI is currently the fastest light microscope for the 3D analysis of complete cells in the nanometer range.
This technology allows entire cells to be imaged at the nano scale, typically within minutes. Wide field exposures signify that the entire object is illuminated and detected simultaneously.
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Sarfus is a recent optical technique based on the use of non-reflecting substrates for cross-polarized reflected light microscopy. The sensitivity achieved with Sarfus is much greater than that with a standard optical microscope, allowing direct visualization of 0.3 nm thickness films and single nano-objects of the order of 2Â nm in diameter.
The digital microscope works on the same principle as the traditional optical microscope, but instead of directly viewing the object, a charge-coupled device (CCD) is used to record the image, which can then be displayed on screen.
Phase contrast microscope
Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. This microscope first enabled the study of the cell cycle.
An electron microscope uses a beam of highly energetic electrons to investigate objects on a very fine scale. Many electron microscopes have superior magnification to optical microscopes due to the fact that electrons have shorter wavelengths than light. Indeed, electron microscopes routinely achieve magnification of almost 1,000,000x.
However, electron microscopes can be expensive to operate, and require both high voltages and a water supply to cool the lenses and pumps. In addition, electron microscopes should be housed carefully in stable, dedicated buildings or underground as vibrations and magnetic fields can interfere with their readings.
Scanning electron microscope (SEM)
A scanning electron microscope images the surface of a sample by scanning it with a high-energy beam of electrons in a raster scan pattern. The electrons interact with the atoms of the sample releasing signals that contain information about the sample's surface topography, composition and conductivity.
Transmission electron microscopy (TEM)
In transmission electron microscopy, a beam of electrons interacts with a specimen as they are transmitted through. An image is formed from the interaction of the electrons with the specimen. TEMs are capable of imaging at a significantly higher resolution than optical microscopes, due to the small wavelength of electrons.
TEM is an important method of analysis in a range of scientific fields, including cancer research, virology, materials science as well as pollution and semiconductor research.
Scanning probe microscopes
The term "scanning probe microscopy" covers several related technologies for imaging right down to the level of molecules and groups of atoms.Â
Scanning probe microscopes analyze a single point in the sample then scan the probe over a rectangular sample region to build up an image. As these microscopes do not use electromagnetic or electron radiation for imaging they are not subject to the same resolution limit as the optical and electron microscopes.
The three most common scanning probe techniques are atomic force microscope (AFM), scanning tunneling microscopy (STM) and near-field scanning optical microscopy (NSOM).
Atomic force microscopy (AFM)
Atomic force microscopy is a very high-resolution type of scanning probe microscopy, offering a resolution below 1 nm. This represents an increase of more than 1000 times compared with the optical diffraction limit. AFM is one of the most important tools for imaging, measuring, and manipulating matter at the nancoscale.
Scanning tunneling microscope (STM)
A scanning tunneling microscope is a powerful instrument for imaging surfaces at the atomic level. 0.1 nm lateral resolution and 0.01Â nm depth resolution can be achieved, allowing the routine imaging and manipulation of individual atoms.
STM can be used in a wide variety of conditions, including ultra high vacuum, air, water, and various other liquids or gasses, as well as at enormous temperatures ranges.
Near-field scanning optical microscopy (SNOM)
Near-field scanning optical microscopy is a technique for nanostructure investigation that overcomes the far field resolution limit by exploiting the properties of evanescent waves by positioning the detector very close to the specimen surface.
In SNOM, the resolution of the image is limited by the size of the detector aperture and not by the wavelength of the illuminating light; allowing lateral resolution of up to 20 nm and vertical resolution of 2-5 nm have been demonstrated.
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