Light microscopy

Try this simple experiment. Fill a glass with water, then partly immerse a pencil and observe from one side; what do you see? The apparent 'bending' of the pencil is due to rays of light being slowed down as they enter the water, because air and water have different refractive indices. Light rays are similarly retarded as they enter glass and all optical instruments are based on this phenomenon. The compound light microscope consists of three sets of lenses (Figure 1.3):

• the condenser focuses light onto the specimen to give optimum illumination

• the objective provides a magnified and inverted image of the specimen

• the eyepiece adds further magnification

The refractive index of a substance is the ratio between the velocity of light as it passes through that substance and its velocity in a vacuum. It is a measure of how much the substance slows down and therefore refracts the light.

EYEPIECE TUBE MAIN FOCUS KNOB

OBJECTIVES STAGE

SUBSTAGE CONDENSER

Figure 1.3 The compound light microscope. Modern microscopes have a built-in light source. The light is focused onto the specimen by the condenser lens, and then passes into the body of the microscope via the objective lens. Rotating the objective nosepiece allows different magnifications to be selected. The amount of light entering the microscope is controlled by an iris diaphragm. Light microscopy allows meaningful magnification of up to around 1000 x

EYEPIECE TUBE MAIN FOCUS KNOB

OBJECTIVES STAGE

SUBSTAGE CONDENSER

BASE WITH BUILT-IN ILLUMINATION

Figure 1.3 The compound light microscope. Modern microscopes have a built-in light source. The light is focused onto the specimen by the condenser lens, and then passes into the body of the microscope via the objective lens. Rotating the objective nosepiece allows different magnifications to be selected. The amount of light entering the microscope is controlled by an iris diaphragm. Light microscopy allows meaningful magnification of up to around 1000 x

f

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Figure 1.4 Light rays parallel to the axis of a convex lens pass through the focal point. The distance from the centre of the lens to the focal point is called the focal length (f) of the lens

Figure 1.4 Light rays parallel to the axis of a convex lens pass through the focal point. The distance from the centre of the lens to the focal point is called the focal length (f) of the lens

Most microscopes have three or four different objectives, giving a range of magnifications, typically from 10x to 100x. The total magnification is obtained by multiplying this by the eyepiece value (usually 10x), thus giving a maximum magnification of 1000x.

In order to appreciate how this magnification is achieved, we need to understand the behaviour of light passing through a convex lens:

• rays parallel to the axis of the lens are brought to a focus at the focal point of the lens (Figure 1.4)

• similarly, rays entering the lens from the focal point emerge parallel to the axis

• rays passing through the centre of the lens from any angle are undeviated.

Because the condenser is not involved in magnification, it need not concern us here. Consider now what happens when light passes through an objective lens from an object AB situated slightly beyond its focal point (Figure 1.5a). Starting at the tip of the object, a ray parallel to the axis will leave the lens and pass through the focal point; a ray leaving the same point and passing through the centre of the lens will be undeviated. The point at which the two rays converge is an image of the original point formed by the lens. The same thing happens at an infinite number of points along the object's length, resulting in a primary image of the specimen, A'B'. What can we say about this image, compared to the original specimen AB? It is magnified and it is inverted (i.e. it appears upside down).

The primary image now serves as an object for a second lens, the eyepiece, and is magnified further (Figure 1.5b); this time the object is situated within the focal length. Using the same principles as before, we can construct a ray diagram, but this time we find that the two lines drawn from a point do not converge on the other side of the lens, but actually get further apart. The point at which the lines do eventually converge is actually 'further back' than the original object! What does this mean? The secondary image only appears to be coming from A" B", and isn't actually there. An image such as this is

A real image is one that can be projected onto a flat surface such as a screen. A virtual image does not exist in space and cannot be projected in this way. A familiar example is the image seen in a mirror.

Figure 1.5 The objective lens and eyepiece lens combine to produce a magnified image of the specimen. (a) Light rays from the specimen AB pass through the objective lens to give a magnified, inverted and real primary image. (b) The eyepiece lens magnifies this further to produce a virtual image of the specimen

Figure 1.5 The objective lens and eyepiece lens combine to produce a magnified image of the specimen. (a) Light rays from the specimen AB pass through the objective lens to give a magnified, inverted and real primary image. (b) The eyepiece lens magnifies this further to produce a virtual image of the specimen called a virtual image. Today's reader, familiar with the concept of virtual reality, will probably find it easier to come to terms with this than have students of earlier generations! The primary image A'B', on the other hand, is a real image; if a screen was placed at that position, the image would be projected onto it. If we compare A"B" with A'B', we can see that it has been further magnified, but not further inverted, so it is still upside down compared with the original. One of the most difficult things to get used to when you first use a microscope is that everything appears 'wrong way around'. The rays of light emerging from the eyepiece lens are focussed by the lens of the observer's eye to form a real image on the retina of the viewer's eye.

Figure 1.6 Magnification must be accompanied by improved resolution. Compared to (a), the image in (b) is magnified, but also provides improved detail; there are two objects, not just one. Further magnification, as seen in (c), provides no further information (empty magnification)

So, a combination of two lens systems allows us to see a considerably magnified image of our specimen. To continue magnifying an image beyond a certain point, however, serves little purpose, if it is not accompanied by an increase in detail (Figure 1.6). This is termed empty magnification. The resolution (resolving power, d) of a microscope is its capacity for discerning detail. More specifically, it is the ability to distinguish between two points a short distance apart, and is determined by the equation:

Immersion oil is used to improve the resolution of a light microscope at high power. It has the same refractive index as glass and is placed between the high power objective and the glass slide. With no layer of air, more light from the specimen enters the objective lens instead of be-

ing refracted outside of it, resulting in a sharper image.

where X is the wavelength of the light source, n is the refractive index of the air or liquid between the objective lens and the specimen and 0 is the aperture angle (a measure of the light-gathering ability of the lens).

The expression n sin0 is called the numerical aperture and for high quality lenses has a value of around 1.4. The lowest wavelength of light visible to the human eye is approximately 400 nm, so the maximum resolving power for a light microscope is approximately d

= 0.17^m that is, it cannot distinguish between two points closer together than about 0.2 /m. For comparison, the naked eye is unable to resolve two points more than about 0.2 mm apart.

For us to be able to discern detail in a specimen, it must have contrast; most biological specimens, however, are more or less colourless, so unless a structure is appreciably denser than its surroundings, it will not stand out. This is why preparations are commonly subjected to staining procedures prior to viewing. The introduction of coloured dyes, which bind to certain structures, enables the viewer to discern more detail.

Since staining procedures involve the addition and washing off of liquid stains, the sample must clearly be immobilised or fixed to the slide if it is not to end up down the sink. The commonest way of doing this is to make a heat-fixed smear; this kills and attaches the cells to the glass microscope slide. A thin aqueous suspension of the cells is spread across the slide, allowed to dry, then passed (sample side up!) through a flame a few times. Excessive heating must be avoided, as it would distort the natural structure of the cells.

Using simple stains, such as methylene blue, we can see the size and shape of bacterial cells, for example, and their arrangement, while the binding properties of differential stains react with specific structures, helping us to differentiate between bacterial types. Probably the most widely used bacterial stain is the Gram stain (see Box 1.2), which for more than 100 years has been an invaluable first step in the identification of unknown bacteria.

A nanometre (nm) is 1 millionth of a millimetre. There are 1000 nm in one micron (/m), which is therefore one thousandth of a millimetre. 1 mm = 10-3 m 1 /m = 10-6 m 1 nm = 10-9 m

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