Contrast Techniques in Light Microscopy

S. Bradbury and P.J. Evennett

• Publisher: Garland Science
• Year: 2020
• ISBN:
  9781000144604
• Edition: 1
• Topic: Medicine
• Subtopic: Medical Microbiology & Parasitology

Contrast in an image is essential to distinguish features from one another and from the background. This practical handbook describes the ways in which light interacts with the specimen in the microscope.

1Introduction

In many mammals, such as the dog, information derived from the sense of smell predominates, whilst in others, such as the mole, both touch and smell are important. Humans, however, gain most of the information about our surroundings from our eyes, so we live in a markedly different perceptual world from most other mammals; for us the eye is our most important sense organ. Complex as it is, the human eye has only limited sensitivity. It responds to that part of the electromagnetic spectrum, between wavelengths of approximately 400–700 nm, which we term light’. If all these different wavelengths are present, the signals are interpreted by the brain as white light, whilst if the range is restricted we have the sensation of colour. Short wavelengths represent violet and blue, whilst progressively longer wavelengths give the impression of green, yellow, orange and red. Throughout the whole range of wavelengths the eye is sensitive to changes in brightness, being most sensitive in the green and declining dramatically at the extreme ends of its range. Other properties of light, such as changes in the phase of the waves and changes in the state of their polarization, are not detected by our eyes. It follows that, if we wish to ensure that our eyes respond to any changes imposed by a microscope specimen on the light which illuminates it, then we must make sure that such changes are presented in the final image as changes in brightness and/or colour.

1.1 Why use a microscope?

The eye is not only limited by its response to wavelength and complete lack of response to polarization or phase changes, but also by the extent to which it can resolve fine detail. If a film is used in a camera to record the same scene then a much better resolution of detail may be obtained. It is hard to put a figure on the actual resolution of the eye in terms of point-to-point separation, since this is very variable between individuals of different ages and in differing states of health. If we ignore these considerations the point resolution of the eye is still dependent on several factors. Some are related to the geometry (i.e. the shape) of the object, whilst others involve considerations of the brightness and/or colour of the object. A detailed consideration of the function of the eye in microscopy has been published by Baker (1966) and more recently by Inoué (1986). The quality of the retinal image is also important, since if there is distortion or lack of focusing by the refractive media of the eye (the cornea and lens) then it is obvious that fine detail will be lost. The anatomical structure of the retina must also be borne in mind, since the reception of the image and its conversion into nervous signals is carried out by individual retinal cells of finite size. In the case of fine detail this is accomplished by the cones of the macula (the area of greatest sensitivity in the centre of the retina). Clearly, for two separate points to be perceived as separate, their image on the retina must cover at least two separate cones. Finally, it is important not to ignore physiological and psychological factors which play a part; for example, attention, tiredness and degree of familiarity with the object. Taking all these factors into account, under good conditions at a reference distance of 250 mm, it is usually assumed that objects with a point-to-point separation of 0.1 mm will be seen as separate.

There are many instances when finer detail than this must be studied. In order to do this we need optical assistance in the form of a simple magnifier or, alternatively, a compound microscope. This latter instrument will, under favourable conditions, extend our capability to see details which are separated by about 0.25 μm. The provision of resolution (ensuring that the fine detail is present in the image) is the principal reason for using a microscope. Having resolved the detail it is usually essential to ensure that the image has sufficient magnification for this detail to be accepted by the eye and brain of an observer, the sensitive area of a TV camera or a photographic emulsion. Such magnification is easy to achieve by combining suitable lenses and will not therefore be considered further here, since it is well covered in other handbooks in this series.

Although it is not relevant to this handbook, it should not be forgotten that the light microscope also provides a valuable tool for gaining qualitative and quantitative information about the physical nature of the specimen. When the instrument is used for this purpose, it is often necessary to sacrifice some of the contrast and resolution of which it is capable when adjusted for maximal resolution of structural detail. For example, in many histochemical procedures, where a chemical reaction is performed on the specimen itself whilst this is on the slide, the presence or absence of a certain colour or reaction product is what is sought. The actual structural detail of the specimen may be very poorly preserved indeed and may be hard to make out.

1.2 The need for contrast enhancement

Once we have resolution, and the image has been enlarged sufficiently for comfortable viewing, then the essential requirement is to achieve sufficient contrast between the object and its background for the observer or the recording system to register a difference between the two. This is often summarized by saying that we needresolution, visibility (i.e. contrast), and magnification, in that order, magnification being the least important.

Contrast results from the interactions of the specimen with light; some of the effects which the specimen has on light will be detailed in Chapter 2 and include the change of its direction by refraction or scattering, changes in phase, or change in the direction of vibration of the light waves. These, however, are not normally accepted by the eye, by film or by TV cameras. It is, therefore, the function of contrast techniques to convert the results of such phenomena into variations in amplitude and/or colour. A good overview of contrast has been given by Sanderson (1994).

Contrast, then, means the degree to which the object is separated from its background in terms of colour and/or brightness. Because of the characteristics of the eye, in bright light this separation may be as little as 2%, but in poor light the difference (often called the contrast threshold) may need to be increased to 5%, or even more, before the object can be seen clearly. This contrast threshold is also related to the size of the object. If this is very small, for instance approximately 100 μm, then the contrast threshold must be increased even further; in such circumstances there may need to be at least a 20% difference from the background before the object becomes visible. The contrast difference between an object and its background is, therefore, essential for seeing fine detail and it is desirable that this difference be made as large as possible, whether we look at the object with our naked eyes, or with an optical device.

There are several useful analogies which may help the reader to appreciate the concept of contrast. One familiar saying is to liken something which is hard to see as, ‘like looking for a black cat in a coal cellar’. Here an object (the cat) has very little difference in colour between it and its location (the cellar), which latter is also often supposed to be poorly lit. The same lack of contrast might apply if one thought about a soldier, wearing white camouflage, standing in an Arctic landscape in the midst of a snowstorm. Yet another example where contrast is lacking is when a transparent object (e.g. a contact lens) is immersed in a clear fluid, such as water in a tumbler. The necessity for the enhancement of contrast is equally a problem when the microscope is used for the study of opaque materials with epi-illumination. The subject of epi-illumination is well covered in the books by Galopin and Henry (1972) and Ineson (1989).

The ability to see resolved structural detail, therefore, requires that we have contrast in our images. Biologists had long ago realized this problem and developed a technique for coping with it. As Hartley (1977) puts it: “Biological microscopists long ago became accustomed to dealing with invisible specimens by staining them, and this practice gained such a hold that the instinctive action of a biologist given a live specimen was to kill and fix it as an essential preliminary, after which the use of dyes could be made to provide the visibility, either in a whole mount or in sections.”

There must be sufficient contrast in a specimen so that the observer may see the structural detail resolved by the objective. In many cases the provision of this contrast is far more difficult than the subsequent study and description of the specimen. Some specimens may be naturally coloured, or colour may have been added as a result of some specific staining or histochemical technique. Alternatively, the specimens may be almost colourless and transparent. If the object is coloured, wholly or selectively, then contrast may be manipulated by means of colour filters. Other contrast methods rely on the use of oblique light, either from a single direction or from two or more directions, alterations in the illuminating aperture or, with opaque specimens, treatments of the surface such as polishing, etching, etc.

The above requirements are true, whether the specimen under observation is translucent (i.e. thin enough for the light to pass through it, so-called transmitted-light microscopy), or whether the specimen is opaque to light and is illuminated from the same side as that from which it is viewed (epi-illumination or reflected-light microscopy). The same requirements of resolution, magnification and visibility also apply to the situation in which the specimen itself emits light (fluorescence), as a result of excitation with short wave radiation.

1.3 Assessment of the image

In addition to the ways in which the specimen affects light (detailed in Chapter 2) it is useful to consider briefly those factors which are used by the eye and brain system of the observer to assess and interpret the image. With a microscope, the user is not looking at the object itself, but at an image of the object. Dictionaries define an image as “an artificial imitation of an object, a replica”. In art there is a range of kinds of image, more or less ‘faithful’ to the object, produced by different techniques (sculpture, oil-painting, photography) and in different styles (realistic, abstract), conveying different messages about the object at the artist’s choice. Similarly, in microscopy, we may produce many different images of our objects according to the information we wish the image to contain. We must accept that the image is an artefact, and that it does not necessarily represent ‘what the object looks like’, unlike normal photographs. The microscopical image is a pictorial representation of some of the information about the object, and is usually the result of a series of manipulations of the specimen, the illumination and the imaging system.

The eye/brain of an observer is very good at the recognition of shapes. We have no difficulty in recognizing and differentiating between triangles with differing angles. Squares are easy to separate from rectangles and circles from ellipses. Even objects with irregular shapes are recognizable and similarities assessed in a qualitative, subjective manner. Similarly, contours are dealt with by our perception, and terms such as ‘rough’, ‘smooth’, ‘scalloped’ and so on are easily applied to their edges. Difficulties do arise, however, when we try to communicate these to others. What exactly is meant by ‘irregular’ or ‘rough’ when used in the description of the profile of a microscopic specimen? In recent years the development of TV-based image analysers using powerful microcomputers has made it much easier to approach such problems in an objective manner. Mathematical measurements of areas, perimeters, Feret (or calliper) diameters are now possible, together with many more sophisticated analyses, such as measures of curvature of lines or surfaces and fractal dimensions for assessment of surface roughness. Photometric techniques are available for the measurement of optical densities and it is possible to state with certainty the amount of absorption which is occurring in the specimen, rather than have to make a subjective assessment, as had hitherto been the case. These disciplines, which form what may be termed quantitative microscopy, are rapidly developing. We now have the ability to obtain data from which precise objective answers may be obtained to problems which were formerly only subjectively decided by individual observers. With the availability of accurate measures of shape and optical density, the powerful techniques of multi-variate statistical analysis may be applied to microscopical specimens and valid evaluations of changes in experimental situations are thus possible.

Recent developments in the use of computers for image analysis have now extended precise measurements to include colour, by assessing parameters such as hue, saturation and luminance. Here again, as with shapes and contour, the brain of the individual observer (assuming there is no defect in his or her colour vision) is very good at recognizing often quite small differences in colour, but without instrumental help we are poor at making precise measurements of colour differences.

The final parameter which is often invoked in the description of an object is texture. This gives us many of our visual clues which we use when classifying images, and again, it has proved hard to quantify. It has been defined for image analysis purposes as, “the repetition of a pattern which may vary randomly, in varying degrees, from one position to the next. The repetition frequency may be fine or coarse, the patterns can be simple dots or intricate wavy structures, and they can be anisotropic”. Many of the newer generation of computer-based image analysers now incorporate algorithms for the effective assessment of texture in microscopic images.

The complexity and uncertainty of our knowledge of the criteria required for adequate image description is emphasised by the differing parts played in the assessment of an image by the sharpness or definition (related to the outline or contour of objects) and the image detail (often not related to the outline). The presence of detail in an image seems to be of much less importance, however, since if the contrast threshold for the eye is satisfied (as it must be for detail to be perceived at all), then it is noticeable that the observer can often tolerate a very large degree of ‘downgrading’ of the image in terms of loss of detail. The brain seems to accept this as natural and in many cases it ‘fills-in’ the missing detail from previous experience. This phenomenon is shown well in some of the illustrations in the books by Frisby (1980) and Gregory (1970). On the other hand, tolerance for loss of sharpness is ...