Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye, using a microscope or other magnification tool.

In classical light microscopy, this involves passing light transmitted through or reflected from the subject through a series of lenses, to be detected directly by the eye, imaged on a photographic plate or captured digitally.

As resolution depends on the wavelength of the light, electron microscopy has been developed since the 1930s that use electron beams instead of light. Because of the much lower wavelength of the electron beam, resolution is far higher. Though less common, X-ray microscopy has also been developed since the late 1940s. The resolution of X-ray microscopy lies between that of light microscopy and the electron microscopy.

Microscopy usually involves the diffraction, reflection, or refraction of radiation incident upon the subject of study. In some types of microscopy, the subject of study is imaged by scanning it point by point (e.g. confocal/multiphoton microscopy) with a very fine physical probe (scanning probe microscopes). Examples of scanning probe microscopes are the atomic force microscope, the Scanning tunneling microscope and the photonic force microscope.

The development of microscopy revolutionized biology and remains an essential tool in that science.

Types of transmitted-light microscopy

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There are many different types of microscopes.

Light microscopy: improving contrast

Light microscopy can distinguish objects separated by down to 0.2 micrometers. Several optical configurations exist, depending on the amount of contrast a specimen under study needs.

• Bright field
• Dark field
• Phase contrast
• Differential interference contrast (DIC)

Live cells in general lack sufficient contrast to be studied successfully. Internal structures of the cell are colourless and transparent, i.e., without enough contrast to see detail. In a normal (brightfield) light microscope, contrast can generally be enhanced by closing the condenser aperture; however this tends to reduce resolution.

The most common way to increase contrast is to stain the different structures with selective dyes, but this generally involves killing and fixing the material followed by staining. Staining can also introduce artifacts, apparent structural details that are caused by the processing of the specimen and are thus not a legitimate feature of the specimen.

To observe structures in living cells, less invasive methods are used to enhance the contrast. In general, these techniques make use of differences in the refractive index of cell structures. It is comparable to looking through a glass window: you don't see the glass but merely the dirt on the glass. There is however a difference as glass is a more dense material, and this creates a difference in phase of the light passing through. The human eye is not sensitive to this difference in phase but clever optical solutions have been thought out to change this difference in phase into a difference in amplitude (i.e., light intensity).

Very old is the use of sideways (oblique) illumination, by covering part of the light entrance to the condenser. This method will give the specimen a sense of relief. A more recent technique based on this method is Hoffmann's modulation contrast. This system is most often found on inverted microscopes for use in cell culture. Dark field illumination is another well known technique where a cone of light is being produced by the condenser that will not reach the objective. Minute particles will show up brightly on a dark background much like the dust that shows up in a beam of sunlight in an otherwise darkened room (Tyndall effect).

More sophisticated techniques will show differences in optical density in proportion. Phase contrast is a widely used technique that shows differences in refractive index as difference in contrast. It was developed by the Dutch physicist Frits Zernike in the 1930s (for which he was awarded the Nobel Prize in 1953). The nucleus in a cell for example will show up darkly against the surrounding cytoplasm. Contrast is excellent; however it is not for use with thick objects. Frequently, a halo is formed even around small objects, which obscures detail. The system consists of a circular annulus in the condenser which produces a cone of light. This cone is superimposed on a similar sized ring within the phase-objective. Every objective has a different size ring, so for every objective another condenser setting has to be chosen. The ring in the objective has special optical properties: it first of all reduces the direct light in intensity, but more importantly, it creates an artificial phase difference of about a quarter wavelength. As the physical properties of this direct light have changed, interference with the diffracted light occurs, resulting in the phase contrast image.

Superior and much more expensive is the use of interference contrast. Differences in optical density will show up as differences in relief. A nucleus within a cell will actually show up as a globule in the most often used differential interference contrast system according to Nomarski. However, it has to be kept in mind that this is an optical effect, and the relief does not necessarily resemble the true shape! Contrast is very good and the condenser aperture can be used fully open, thereby reducing the depth of field and maximising resolution. The system consists of a special prism (Wolaston prism) in the condenser that splits light in an ordinary and an extraordinary beam. The spatial difference between the two beams is minimal (less than the maximum resolution of the objective). After passage through the specimen, the beams are reunited by a similar prism in the objective. In a homogeneous specimen, there is no difference between the two beams, and no contrast is being generated. However, near a refractive boundary (say a nucleus within the cytoplasm), the difference between the ordinary and the extraordinary beam will generate a relief in the image. Differential interference contrast uses polarised light to work properly. Two polarising filters have to be fitted in the light path, one below the condenser (the polarizer), and the other above the objective (the analyser).

Fluorescence microscopy

When certain compounds are illuminated with high energy light, they then emit light of a different, lower frequency. This effect is known as Fluorescence. Often specimens show their own characteristic autofluorescence image, based on their chemical makeup.

This method is of critical importance in the modern life sciences, as it can be extremely sensitive, allowing the detection of single molecules.. Many different fluorescent dyes can be used to stain different structures or chemical compounds. One particularly powerful method is the combination of antibodies coupled to a fluorochrome as in immunostaining. Examples of commonly used fluorochromes are fluorescein or rhodamine. The antibodies can be made tailored specifically for a chemical compound. For example, one strategy often in use is the artificial production of proteins, based on the genetic code (DNA). These proteins can then be used to immunize rabbits, which then form antibodies which bind to the protein. The antibodies are then coupled chemically to a fluorochrome and then used to trace the proteins in the cells under study.

In recent work, highly efficient fluorescent proteins such as the green fluorescent protein (GFP) have been specifically fused on a DNA level to the protein of interest. This combined fluorescent protein is not toxic and hardly ever impedes the original task of the protein under study. Genetically modified cells or organisms directly express the fluorescently tagged proteins, which enables the study of the function of the original protein in vivo.

Since fluorescence emission differs in wavelength (color) from the excitation light, a fluorescent image ideally only shows the structure of interest that was labelled with the fluorescent dye. This high specificity lead to the widespread use of fluorescence light microscopy in biomedical research. Different fluorescent dyes can be used to stain different biological structures, which can then be detected simultaneously, while still being specific due to the individual color of the dye.

To block the excitation light from reaching the observed or the detector, filter sets of high quality are needed. These typically consist of an excitation filter selecting the range of excitation wavelengths, a dichroic mirror, and an emission filter blocking the excitation light. Most fluorescence microscopes are operated in the Epi-illumination mode (illumination and detection from one side of the sample) to further decrease the amount of excitation light entering the detector.

See also total internal reflection fluorescence microscope.

Confocal laser scanning microscopy

main article see Confocal laser scanning microscopy

Generates the image by a completely different way than the normal visual bright field microscope. It gives slightly higher resolution, but most importantly it provides optical sectioning without disturbing out-of-focus light degrading the image. Therefore it provides sharper images of 3D objects. This is often used in conjunction with fluorescence microscopy.

Deconvolution microscopy

Fluorescence microscopy is extremely powerful due to its ability to show specifically labelled structures within a complex environment but also because of its inherent ability to provide three dimensional information of biological structures. Unfortunately this information is blurred by the fact, that upon illumination all fluorescently labelled structures emit light no matter if they are in focus or not. This means, that an image of a certain structure is always blurred by the contribution of light from structures which are out of focus. This phenomenon becomes apparent as a loss of contrast especially when using objectives with a high resolving power, typically oil immersion objectives with a high numerical aperture.

Fortunately though, this phenomenon is not caused by random processes such as light scattering but can be relatively well defined by the optical properties of the image formation in the microscope imaging system. If one considers a small fluorescent light source (essentially a bright spot), light coming from this spot spreads out the further out of focus one is. Under ideal conditions this produces a sort of "hourglass" shape of this point source in the third (axial) dimension. This shape is called the point spread function ("PSF") of the microscope imaging system. Since any fluorescence image is made up of a large number of such small fluorescent light sources the image is said to be "convolved by the point spread function".

Knowing this point spread function means, that it is possible to reverse this process to a certain extent by computer based methods commonly known as deconvolution. There are various algorithms available for 2D or 3D Deconvolution. They can be roughly classified in non restorative and restorative methods. While the non restorative methods can improve contrast by removing out of focus light from focal planes, only the restorative methods can actually reassign light to it proper place of origin. This can be an advantage over other types of 3D microscopy such as confocal microscopy, because light is not thrown away but reused. For 3D deconvolution one typically provides a series of images derived from different focal planes (called a Z-stack) plus the knowledge of the PSF which can be either derived experimentally or theoretically from knowing all contributing parameters of the microscope.

A very good introduction into deconvolution for microscopy can be found here: A working persons guide to deconvolution

Electron microscopy

For Light Microscopy the wavelength of the light limits the resolution to around 0.2 micrometers. In order to gain higher resolution, the use of an electron beam with a far smaller wavelength is used in Electron Microscopes.

• Transmission electron microscopy (TEM) is principally quite similar to the compound light microscope, by sending an electron beam through a very thin slice of the specimen. The resolution limit nowadays (2005) is around 0.05 nanometer.
• Scanning electron microscopy (SEM) visualizes details on the surfaces of cells and particles and gives a very nice 3D view. It gives results much like the stereo light microscope and akin to that its most useful magnification is in the lower range than that of the transmission electron microscope.

Ultrasonic force microscopy

External links

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• Microscopy in Detail - A resource with many illustrations elaborating the most common microscopy techniques
• Confocal microscopy theory and protocols
• Dark field microscopy page from FSU
• The Science of Spectroscopy - supported by NASA. Spectroscopy education wiki and films - introduction to light, its uses in NASA, space science, astronomy, medicine & health, environmental research, and consumer products.
• SVI-wiki on 3D microscopy and deconvolution

Microscopy | Laboratory techniques | Mikroskopie | microscopía | microscopie | microscopie