Microscopy

From Encyclopedia Jr, free information reference for Kids

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. It is often used more specifically as a technique of using a microscope.

Microscopy has evolved with the development of the microscopes with which to work with. Hence there are three main branches of microscopy; optical, electron and scanning probe microscopy.

Optical and electron microscopy involves the diffraction, reflection, or refraction of radiation incident upon the subject of study, and the subsequent collection of this scattered radiation in order to build up an image. This process may be carried out by wide field irradiation of the sample (for example standard light microscopy and transmission electron microscopy) or by scanning of a fine beam over the sample (for example confocal microscopy and scanning electron microscopy. Scanning probe microscopy involves the interaction of a scanning probe with the surface or object of interest.

The development of microscopy revolutionized biology and remains an essential tool in that science, along with many others.

Contents 1 Optical Microscopy 1.1 The Optical Microscope 1.2 Limitations of Optical Microscopy 1.3 Optical Microscopy Techniques 1.3.1 Bright Field Optical Microscopy 1.3.2 Oblique Illumination 1.3.3 Dark Field Optical Microscopy 1.3.4 Phase Contrast Optical Microscopy 1.3.5 Differential Interference Contrast Microscopy 1.3.6 Fluorescence microscopy 1.3.7 Confocal laser scanning microscopy 1.3.8 Deconvolution microscopy 1.4 Use of the optical microscope 1.4.1 X-Ray Microscopy 1.4.2 Electron microscopy 1.5 Optical Microscope Enhancements 2 Scanning Probe Microscopy 2.1 Ultrasonic force microscopy 3 References 4 External links

[edit] Optical Microscopy

[edit] The Optical Microscope

Main article: optical microscope

Optical (or light) microscopy involves passing visible light transmitted through or reflected from the subject through a single lens [see the Brian J,. Ford page on the simple microscope] or a series of lenses. The image can be detected directly by the eye, imaged on a photographic plate or captured digitally. The single lens with its attachments, or the system of lenses and imaging equipment, along with the appropriate lighting equipment, sample stage and support, makes up the light microscope.

[edit] Limitations of Optical Microscopy

Limitations of standard optical microscopy (bright field microscopy) lie in three areas;

• The technique can only image dark or strongly refracting objects effectively.
• Diffraction limits resolution to approximately 0.2 micrometre (see: microscope).
• Out of focus light from points outside the focal plane reduces image clarity.

Live cells in particular generally lack sufficient contrast to be studied successfully, internal structures of the cell are colourless and transparent. The most common way to increase contrast is to stain the different structures with selective dyes, but this involves killing and fixing the sample. Staining may 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.

These limitations have, to some extent, all been overcome by specific microscopy techniques which can non-invasively increase the contrast of the image. 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 (bright field microscopy) 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 (light intensity).

[edit] Optical Microscopy Techniques

[edit] Bright Field Optical Microscopy

Main article: Bright field microscopy

Bright field microscopy is the simplest of all the light microscopy techniques. Sample illumination is via transmitted white light, ie. illuminated from below and observed from above.

Limitations:

• Very low contrast of most biological samples.
• Low apparent resolution due the blur of out of focus material.

Advantages:

• Simplicity of setup with only basic equipment required.
• No sample preparation required, allowing viewing of live cells.

Simple enhancements to this technique may involve:

• Reducing the size of the light source via the condenser aperture, although this reduces resolution.
• Use of coloured or polarising filters on the light source to highlight features not visible under white light. This is especially useful with mineral samples.

[edit] Oblique Illumination

This uses sideways (oblique) illumination; either by covering part of the light source to give asymmetric lighting, or even an external light source being shone sideways in the sample. This gives the image a 3D appearance and can highlight otherwise invisible features. 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.

Limitations:

• Still has low contrast of many biological samples.
• Low apparent resolution due the blur of out of focus objects.

Advantages:

• May highlight otherwise invisible structures.
• Simplicity of setup with only basic equipment required.
• No sample preparation required, allowing viewing of live cells.

[edit] Dark Field Optical Microscopy

Main article: Dark field microscopy

Dark field microscopy uses a carefully aligned light source to minimise the quantity of directly transmitted light (ie. unscattered light) entering the image, and only collected light scattered by the sample. This is done by confining the illumination to a ring of light.

Limitations:

• Low light intensity in final image of many biological samples.
• Low apparent resolution due the blur of out of focus objects.

Advantages:

• Clearly shows even transparent objects in the sample.
• Simplicity of setup with only basic equipment required.
• No sample preparation required, allowing viewing of live cells.

Rheinberg illumination is a special variant of dark field illumination and is named after its inventor, Julius Rheinberg. In this variant transparent colored filters are inserted just before the condenser so that light rays at high aperture are differently colored than those at low aperture. E.g. the background to the specimen may be blue whilst the object appears self-luminous yellow. Other color combinations are possible but their effectiveness is quite variable.

[edit] Phase Contrast Optical Microscopy

Main articles: Phase-contrast imaging and Phase contrast microscope

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.

[edit] Differential Interference Contrast Microscopy

Main article: Differential interference contrast microscopy

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 Georges 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 (Nomarski prism, Wollaston 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).

[edit] Fluorescence microscopy

Main article: 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 led 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.

[edit] Confocal laser scanning microscopy

Main article: 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.

[edit] 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.

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

[edit] Use of the optical microscope

Most modern instruments provide simple solutions for micro-photography and image recording electronically. However such capabilities are not always present and the more experienced microscopist will, in many cases, still prefer a hand drawn image rather than a photograph. This is because a microscopist with knowledge of the subject can accurately convert a three dimensional image into a precise two dimensional drawing . In a photgraph or other image capture system however, only one thin plane is ever in good focus.

In order to create careful and accurate micrographs requires a microscopical technique using a monocular eyepiece. It is essential that both eyes are open and that the eye that is not observing down the microscope is instead concentrated on a sheet of paper on the bench besides the microscope. With practice, and without moving the head or eyes, it is possoble to accurately record the observed details by tracing round the observed shapes by simultaneously "seeing" the pencil point in tne microscopical image.

Practising this technique also establishes good general microscopical technique. It is always least tiring to observe with the microscope focussed so that the image is seen at infinity and with both eyes open at all times.

[edit] X-Ray Microscopy

Main article: X-ray microscopy

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.

[edit] 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.

[edit] Optical Microscope Enhancements

stereomicroscope

[edit] Scanning Probe Microscopy

Examples of scanning probe microscopes are the atomic force microscope, the Scanning tunneling microscope and the photonic force microscope.

[edit] Ultrasonic force microscopy

Ultrasonic Force Microscopy (UFM) has been developed in order to improve the details and image contrast on "flat" areas of interest where the AFM images are limited in contrast. The combination of AFM-UFM allows a near field acoustic microscopic image to be generated. The AFM tip is used to detect the ultrasonic waves and overcomes the limitation of wavelength that occurs in acoustic microscopy. By using the elastic changes under the AFM tip, an image of much greater detail than the AFM topography can be generated.

Ultrasonic force microscopy allows the local mapping of elasticity in atomic force microscopy by the application of ultrasonic vibration to the cantilever or sample. In an attempt to analyse the results of ultrasonic force microscopy in a quantitative fashion, a force-distance curve measurement is done with ultrasonic vibration applied to the cantilever base, and the results are compared with a model of the cantilever dynamics and tip-sample interaction based on the finite-difference technique.

[edit] References

1. Advanced Light Microscopy vol. 1 Principles and Basic Properties by Maksymilian Pluta, Elsevier (1988)
2. Advanced Light Microscopy vol. 2 Specialised Methods by Maksymilian Pluta, Elsevier (1989)
3. Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle, BIOS Scientific Publishers (1998)
4. Video Microscopy by Shinya Inoue, Plenum Press (1986)
5. Microscopy and Imaging Literature

[edit] External links

• Microscopy in Detail - A resource with many illustrations elaborating the most common microscopy techniques
• Images formed by simple microscopes - examples of observations with single-lens microscopes.
• 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
• Quantitative Microscopy
• Royal Microscopical Society (RMS)
• Microscopy Society of America (MSA)
• European Microscopy Society (EMS)