Chapter 1: The Microscope - Introduction


Figure 1.1 Magnification vs. Resolution

Since its invention, the microscope has been a valuable tool in the development of scientific theory. Magnifying lenses have been known for as long as recorded history, but it was not until the advent of the modern compound light microscope that the device was used in biology. A compound microscope is composed of two elements; a primary magnifying lens and a secondary lens system, similar to a telescope. Light is caused to pass through an object and is then focused by the primary and secondary lens. If the beam of light is replaced by an electron beam, the microscope becomes a transmission electron microscope. If light is bounced off of the object instead of passing through, the light microscope becomes a dissecting scope. If electrons are bounced off of the object in a scanned pattern, the instrument becomes a scanning electron microscope.

The function of any microscope is to enhance resolution. The microscope is used to create an enlarged view of an object such that we can observe details not otherwise possible with the human eye. Because of the enlargement, resolution is often confused with magnification, which refers to the size of an image. In general, the greater the magnification, the greater the resolution, but this is not always true. There are several practical limitations of lens design which can result in increased magnification without increased resolution.

Figure 1.1 illustrates this point.

If an image of a cell is magnified from 10X to 45X, the image gets larger, but not necessarily any clearer. The image on the left is magnified with no increase in resolution. The image on the right is magnified the same, but with increasing resolution. Note that by the time the image is magnified 10X (from 10X to 100X), the image on the left is completely unusable. The image on the right, however, presents more detailed information. Without resolution, no matter how much the image is magnified, the amount of observable detail is fixed, and regardless of how much you increase the size of the image, no more detail can be seen. At this point, you will have reached the limit of resolution or the resolving power of the lens. This property of the lens is fixed by the design and construction of the lens. To change the resolution, a different lens is often the only answer.

The reason for a dichotomy between magnificatioin and resolution is the ability of the human eye to see two objects. It is necesarry that two objects be about 0.1 mm apart when held 10" from the face in order for us to detect them as two objects. If they are closer than 0.1 mm, we will perceive them as a single object. If two objects are 0.01 mm apart, we can not detect them unless we magnify an image of them by 10X. What has happened is that we have effectively altered our resolution ability from 0.1 mm to 0.01 mm through the use of a magnifying lens. We would say that our limit of resolution has changed from 0.1 mm to 0.01 mm, or inversely, our resolving power (resolution) has increased by a factor of 10.

Unfortunately, a lens can magnify an image without increasing the resolution. Several artifacts can be inherent in the lens design which causes the objects to become blurry at the edges. Thus, even though they can be made to appear 0.1 mm apart, the edges are so blurry that we lose the ability to see them as two objects. Think of a standard eye chart: you can see the increased size of a letter, but may be unable to tell what letter is projected.

Figure 1.1 illustrates what can be seen with increased magnification and resolution. If we were to look only at the left side of the figure, we could get the impression that the cell is filled with a homogeneous fluid (cytoplasm). If, however, we look at the right side of the figure, it becomes apparent that the cytoplasm is actually composed of smaller particulate components (chloroplasts, ribosomes, membranes). As we increased the resolution of our microscopes we changed our concepts from protoplasm (the fluid of life) to cytoplasm (the fluid of the cell outside of the nucleus) to a highly ordered machine full of individual organelles.

It is readily apparent that while microscope lenses are usually discussed in terms of their magnification, the most important value is their resolution. All microscopes will come with a lens that can magnify 40 times the normal size, but only a quality lens will allow you to see more than you would with a good hand-held magnifying lens.

As mentioned, the value for resolution may be determined in one of two ways. It can be measured as the smallest distance between two points, which allows us to see the points as distinct. With this measurement, resolution increases as the distance decreases--that is, there is an inverse correlation between the limit of resolution and what you actually resolve.

Equation 1.1b

0.61 x
Limit of Resolution = -----------
N.A.

To change this to a direct correlation, one need only use the reciprocal of the limit of resolution. Resolution is the recriprocal of the limit of resolution. For measures of resolution then, as the value increases, resolution increases. Consequently, most microscopists today use resolution rather than limit of resolution to measure the quality of their lenses.

Equation 1.1a

N.A.
Resolution = ----------
0.61 X

The resolution of a lens is a property of its physical properties and of the wavelength of light that is passed through the lens. The physical properties are summed up in a value known as the numerical aperture while the wavelength is determined by the color of light.

Equation 1.2

N.A. = X

The numerical aperture of a lens is dependent upon two parameters, the angle of the incidence of light onto the lens, and the refractive index of the glass of which the lens is composed. The angle of incidence is also known as the cone angle and 1/2 of this value is designated by the symbol . Half the cone angle is used to calculate the angle the light subtends relative to the light axis. The cone angle and thus can be altered by inclusion of a substage condenser. If the condenser is moveable, the cone angle can be varied; the closer the substage condenser is to the object, the greater is the cone angle. This is a relatively inexpensive means of effecting the resolution of the microscope and thus nearly all microscopes are equipped with substage condensers.


Figure 1.2 Cone Angle and Numerical Aperture

The refractive properties of a lens are summed up in a measurement known as the refractive index (R.I. or ). The refractive index is a function of the bending of light from air through glass and back again. In a microscope, the glass of the lens is specially formulated to increase its refactive index. Once manufactured, however, this property can not be changed. The media around the lens can be altered, however, by removing air from between the objective and the slide, and replacing it with immersion oil. 1

Putting all of this to practical use, it is apparent that resolution can be increased in three ways. The easiest method is to increase the angle of light incidence, by altering the position and/or design of the substage condenser. Second, the refractive index can be maximized by using specially manufactured lenses, and by controlling the medium through which the light travels, i.e. using immersion oil with lenses designed for this purpose. The third method is to decrease the wavelength of light used. For practical purposes, the wavelength has a larger effect on resolution than either changes in the angle of incidence or the refractive index. For maximum resolution, all three properties must be optimized.

For routine bright field microscopy, it is more convenient to work in the visible light range, and the shortest wavelength of visible light is blue. Thus, even inexpensive microscopes have incorporated a blue filter into their design, which is often referred to as a daylight filter. As a rule, the cheaper the microscope the thicker and darker this filter. 2 More expensive and higher quality lenses manipulate the light source to enhance the quality of the light and to correct for lens aberrations inherent in their design.

Resolution can be enhanced by reducing the wavelength to the ultraviolet range and yet again by levels of magnitude to the wavelengths electrons have in motion. The use of electrons as the light source gives rise to the electron microscope. UV light can not be seen directly by the human eye (it will injure the retina of the eye) nor can we see electron beams. Thus, these forms of microscopy rely on photography, or upon fluorescent screens.

Visible light ranges in wavelength from the long red waves ( = 760 nm) to the short blue/violet waves ( = 400 nm). Ultraviolet waves can be as short as 230 nm. The wavelength of an electron beam depends upon its acceleration voltage, with the wavelength being given by Planck's law. For an electron of charge e, accelerated by a potential difference of V, is given by the formula:

Equation 1.3

h
= --------
(2m_eVe)^1/2

This is made simpler by the approximation:

Equation 1.4

= (1.5/V)^1/2 nm

For an electron microscope with 40,000 volts accelerating voltage, the wavelength of the electron would be 0.006 nm ((1.5/40,000)^1/2). Note that UV light increases the resolution by a factor of 2 over visible light, while the electron microscope has the potential to increase it by a factor of 10^4 to 10^5 over visible light.

Maximum resolution is not attainable, however, unless the lenses are corrected for problems of lens design. Modern microscope lenses are not single lenses, but highly complex collections of lenses assembled to enhance the refractive index while minimizing chromatic and spherical distortions of the image.

Aberrations

Chromatic and Spherical distortions ("aberrations") are inherent in the design of a lens. Because the lens is a sphere, it projects an image that is spherical, while optical theory is based on images that are flat. Moreover, because different wavelengths of light are refracted differently, the spherical image is even further distorted into multiple images, as each wavelength of light forms a separate image.

A lens that is corrected to yield flat fields rather than curved is known as a plan lens, while one corrected for flat field and color aberrations is termed a plan achromat lens. If the lens is corrected for chromatic aberrations for red and blue, while correcting spherically for green, the lens is an achromat lens. Increased resolution and increased cost of the microscope are primary factors in correction of these aberrations. Other than adding colored filters to create monochrome light, there is little or no alteration possible once the system has been built. Thus, we will continue to discuss only those parameters that can be controlled by the user.

Angle of Incidence


Figure 1.3 Optical path through a light microscope

Refer to Figure 1.3 for the location of typical microscope components.

While the angle can be altered, there is a theoretical limit to this angle which would still allow light to pass into a lens. For any given lens, there should be an ideal or maximum position of the substage condenser which would present light to the lens at the appropriate angle, which in turn would allow a maximum light intensity, while maintaining as large as possible. Practically, for most student microscopes at anything above the lowest power (2.5-4X), this is usually in the uppermost position of condenser travel. Good microscopes allow you to see the condenser diaphragm in the field of light and allow precise adjustment of the condenser to its ideal location. Most student microscopes do not allow this, although they will often allow movement of the condenser in a vertical direction, without the ability to adjust the alignment. Since iris diaphragms are inexpensive, virtually all condensers are equipped with these. Iris diaphragms are used to correct for spherical aberrations of the lenses and should be adjusted for each objective. They should not be used to control light intensity, unless resolution is unimportant to the user.

Alignment

Proper use of a microscope demands that the optics and light source be aligned on the optical axis. All of the corrections for aberrations depend on proper alignment of the microscope components. There are two general techniques used for proper alignment of the microscope. The first, and perhaps best, is known as critical illumination. In this process an image of the light source (bulb filament) is projected into the plane of the object, thus superimposing the light source onto the object. It has a distinct disadvantage, however, in that it calls for a flat even light source, not really possible with a tungsten filament bulb.

The second alignment procedure is known as Koehler illumination; after a pioneer in light optics, August Koehler. In this procedure, an image of the field diaphragm is projected onto the object plane. This procedure requires a field condenser lens equipped with a moveable (centerable) iris or diaphragm. Koehler illumination is the most commonly used alignment procedure.

Bright Field, Dark Field, Phase Contrast

All microscopes actually allow visualization of objects through minute shifts in the wavelength phase as the light passes through the object. Further image forming can be had through the use of color, or through a complete negative image of the object. If the normal phase shift is increased (usually by 1/4 wavelength), then the microscope becomes a phase contrast microscope. Phase contrast microscopes can be designed to have medium phase or dark phase renditions, by altering the degree of additional shift to the wavelength from 1/4 to 1/2 wavelengths, respectively.

If the beam of light is shifted in phase by a variable amount, the system becomes a differential interference contrast microscope. The most commonly used system of interference microscopy is known as a Normarski Interference Microscope, named for the optical theoretician, George Nomarski. Once used nearly exclusively by parasitologists, this type of microscopy has increased in use because of the work currently done on the nematode C. elegans; interference microscopes are superb for both observation and measuring thickness of embryos within specimens with little or no contrast.

If the light image is reversed, then the microscope becomes a dark field microscope. All standard bright field microscopes can be readily converted to dark field by inserting a round opaque disk beneath the condenser. Dark field microscopy was first utilized to examine trans-filterable infectious agents, later to be termed viruses, and to determine that they were particulate in nature. Small objects, even those below the limits of resolution, can be detected easily with dark field, as the object appears to emit light on a dark field. Look at the sky for a comparison. It is fairly easy to see stars in a dark sky, but impossible during the day. The same is true for dark field vs bright field microscopy.

Finally, if the normal light microscope is functionally turned upside down, the microscope becomes an inverted microscope. This is particularly useful in tissue culture since it allows observation of cells through the bottom of a culture vessel, without opening the container, and without the air interface normally present between the objective and the surface of the culture. By adding phase contrast optics to the inverted microscope, it is possible to monitor tissue cultures directly, without the aid of stains or other enhancements.

The Electron Microscope

The transmission electron microscope (TEM) is the workhorse of histology primarily because of its resolving power (3-10 Å), and its similarity to traditional light microscopy and histotechnique. The scanning electron microscope (SEM) is becoming increasingly popular with cell biologists because of its remarkable ability for quantifiable mapping of surface detail, along with improved resolution (30-100 Å ) and its ability to show 3D structure.

The transmission electron microscope is identical in concept to the modern binocular light microscope. It is composed of a light source (in this case an electron source), a substage condenser to focus the electrons on the specimen, and an objective and ocular lens system. In the electron microscope, the ocular lens is replaced with a projection lens, since it projects an image onto a fluorescent screen or a photographic plate. Since the electrons do not pass through glass, they are focused by electro-magnetic fields. Instead of rotating a nose-piece with different fixed lenses, the EM merely changes the current and voltage applied to the electromagnetic lenses.

The size of an electron microscope is dependent upon two factors. Primary is the need for a good vacuum through which the electrons must pass (it takes less than 1 cm of air to completely stop an electron beam). Peripheral pumps and elaborate valves controls are needed to create the vacuum. A substantial electrical potential (voltage) is also needed to accelerate the electrons out of the source. The source is usually a tungsten filament, very much like a light bulb, but with 40-150 Killivolts of accelerating voltage applied to an anode to accelerate the electrons down the microscope column. 3 Modern electronics have produced transformers which are reasonably small but capable of generating 60,000 volts. Transformers used to be room sized (and a large room at that). The million-volt electron microscopes have transformers as large as buildings - in fact, the building housing such a microscope is for the most part a cover for the transformer. While electron microscopes work with high voltage, they use only milliamps of current through the electron gun.

With the need for mechanical stability that is imposed by resolution in the Angstrom range, electron microscopes are usually formidable instruments. Ideally they lie on their own shock mountings to isolate the instrument from building and ground vibrations. The size and cost of an electron microscope is due primarily to the need for stability and maintenance of the vacuum. With advances in vacuum technology (similar to the microchip revolution in electronics), and perhaps with the ability to manufacture devices in deep space, the size (and cost) of electron microscopes should decrease.

Another characteristic of electron microscopes is that they are usually designed upside-down, similar to an inverted light microscope. The electron source is on top, and the electrons travel down the tube, opposite to light rays traveling up a microscope tube. This is merely a design feature that allows the operator and technicians ease of access to its various components. The newer electron microscope is beginning to look like a desk with a TV monitor on it.

Until recently, the major advantage of an electron microscope also has been its major disadvantage. In theory, the transmission electron microscope should be capable of giving a resolution of several angstroms. This would give excellent molecular resolution of cell organelles. However, as the resolution increases, the field of view decreases and it becomes increasingly difficult to view the molecular detail within the cell. Electron microscopes designed to yield high resolution have to be compromised to view larger objects. Cell structures fall within the size range that was most problematic for viewing. For example, if we wished to resolve the architecture of an entire eucaryotic chromosome, not just the chromosome, but the cell itself was too large to be seen effectively in an electron microscope. Zooming in on paired chromosomes was impossible. Modern electron microscope design allows for this zooming, and the observation of whole tissues while retaining macromolecular resolution. 4

The Scanning Electron Microscope


Figure 1.4 Useful emission from electron bombardment


Figure 1.5 Secondary electron and x-ray images from SEM

The scanning electron microscope works by bouncing electrons off of the surface and forming an image from the reflected electrons. Actually, the electrons reaching the specimen (the 1 ° electrons) are normally not used (although they can form a transmitted image, similar to standard TEM), but they incite a second group of electrons (the 2 ° electrons) to be given off from the very surface of the object. Thus, if a beam of primary electrons is scanned across an object in a raster pattern (similar to a television scan), the object will give off secondary electrons in the same scanned pattern. These electrons are gathered by a positively charged detector, which is scanned in synchrony with the emission beam scan. Thus, the name scanning electron microscope, with the image formed by the collection of secondary electrons.

It is possible to focus the primary electrons in exactly the same manner as a TEM. Since the primary electrons can be focused independently of the secondary electrons, two images can be produced simultaneously. Thus, an image of a sectioned material can be superimposed on an image of its surface. The instrument then becomes a STEM, or Scanning-Transmission Electron Microscope. It has the same capabilities of a TEM, with the added benefits of an SEM.

SEM allows a good deal of analytical data to be collected in addition to the formed image. As the primary electrons bombard the surface of an object, they interact with the atoms of the surface to yield even more particles and radiations other than secondary electrons. Among these radiations are Auger electrons, and characteristic X-rays. The X-rays have unique, discreet energy values, characteristic of the atomic structure of the atom from which they emanated. If one collects these X-rays and analyzes their inherent energy, the process becomes Energy Dispersive X-ray Analysis. Combining the scan information from secondary and Auger electrons, together with the qualitative and quantitative X-ray information allows the complete molecular mapping of an object's surface.

Figure 1.4 presents a diagram of the principal emissions from an electron bombardment, while Figure 1.5 compares a secondary electron image to an image recreated from x-ray data.

Finally, the scanning microscope has one further advantage that is useful in cell structure analysis. As the electron beam scans the surface of an object, it can be designed to etch the surface. That is, it can be made to blow apart the outermost atomic layer. As with the emission of characteristic x-rays, the particles can be collected and analyzed with each pass of the electron beam. Thus, the outer layer can be analyzed on the first scan, and subsequently lower layers analyzed with each additional scan. Electrons are relatively small, and the etching can be enhanced by bombarding the surface with ions rather than electrons (the equivalent of bombarding with bowling balls rather than BB's). The resultant Secondary Emmisions-Ion Scanning data can finally be analyzed and the three- dimensional bit-mapped atomic image of an object reconstructed.

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu