The Light Microscope and Its Limitations
All students of science are familiar with the light microscope (Figure 1A.1), the instrument that made cell biology possible, beginning with Hooke’s pioneering microscopic studies.
Generations of biologists followed Hooke with steadily improved instruments. But as biology has looked deeper into the details of life, the light microscope has reached its limits.
To understand why these limits exist, consider the resolution of a microscope. The resolution (r) is quantitatively defined as the minimum distance between two objects that can just be dis-tinguished as separate. It is given by the equation
(1A.1) Here is the wavelength of the radiation used, n is the refractive index of the medium between the sample and the objective lens, and is the angular aperture of the objective lens. The quantity sin is basically a measure of the radiation-gathering power of the lens system. Resolution depends primarily on wavelength because the objects must be comparable in size to the wavelength in order to perturb the waves sufficiently to convey information.
The angular apertures of the best light microscopes are about 70°, so even if deep blue light of wavelength 450 nm is used and the medium between the sample and the objective lens is air
we get
(1A.2)
This value represents the practical limit of resolution for light microscopy. A bit more resolution can be gained by going into the near ultraviolet, but absorption of this light by cellular materials
r = 0.61 * 450
1.0 sin 70⬚ ⵒ 300 nm = 0.3 m m (n = 1),
aa l
r= 0.61l n sin a
limits its usefulness. Photographic images can be enlarged, but there is no sense in magnifying an image beyond the point where its resolution is just what the eye can resolve. Because our eyes can resolve images about 0.3–0.6 mm apart, the best light micro-scopes have a useful maximum magnifying power of about 1000–2000 (magnifying by 2000 gives 0.6 mm). Further magnification of the image does not help—the fuzziness just gets bigger. To make a major advance, it was necessary to use radiation of much shorter wavelength, radiation that we cannot see but that can produce a photographic image. Thus the electron microscope was born in the 1930s.
Transmission Electron Microscopy
There are several types of electron microscopes. The first type to be used was the transmission electron microscope (TEM), so called because it detects electrons that have been transmitted through a sample. The transmission electron microscope is com-pared with the light microscope in Figure 1A.1. An electron beam is emitted from a tungsten filament and accelerated by an electric field. Magnetic lenses focus the beam, as glass lenses focus a beam of light in the conventional microscope. The key to the higher resolution is that electrons, like the photons of light, have both a particle-like and a wavelike nature. A photon or an electron moving with an energy E is characterized by a wavelength where
h is Planck’s constant and
(1A.3)
c is the speed of light When electrons are acceler-ated by 50,000–100,000 volts between the cathode and the anode, their wavelengths are much shorter than that of visible light—in
(3 * 108m/s) . l = hc
E (6.626 * 1034J⭈s)
0.3mm
Biochemistry is an experimental science, and the remarkable recent advances in biochemistry are due in large part to the development of powerful new laboratory techniques. Some of these techniques are generating significant information far more rapidly than it can be integrated and understood without recourse to new approaches in information technology.
MICROSCOPY AT MANY LEVELS
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(a) Optical microscope (b) Transmission electron microscope Light source
Condenser lens Specimen Objective lens Ocular lens Eye
Final image on photographic plate or screen
Binoculars Eye
Intermediate image Projector lens Objective lens Specimen Condenser lens Electron source
FIGURE 1A.1
Structure of the optical microscope and the trans-mission electron microscope.The two images are not to the same scale; the electron microscope is much larger than a conventional light microscope.
fact, less than 1 nm. This wavelength would predict a resolution of better than 1 nm for a transmission electron microscope.
Practical considerations give an operational limit of about 2 nm for most instruments. Still, this resolution is about 100 times finer than even the best optical microscope can accomplish; a good transmission electron microscope can usefully magnify to over 100,000 times.
Clear as this advantage may be, transmission electron microscopy has some disadvantages. The electron beam requires that a high vacuum be maintained throughout the instrument, including the sample chamber. This, in turn, means that only completely dried samples can be examined. Although methods for sample fixation and drying have been devised, there is always the possibility of inducing changes in samples as a result of their dehydration. Living structures, of course, cannot be examined.
Some of the methods used to prepare samples for transmission electron microscopy are shown in Figure 1A.2. The electron energies in most transmission microscopes do not allow penetration of thick samples Thus, cell samples must be fixed, stained, and sliced very thin, by using an ultramicrotome (Figure 1A.2a).
Particles like viruses and large molecules can be deposited directly on a thin film supported by a copper grid. But the contrast between such a particle and the background is not sufficient, so the sample is usually negatively stained (Figure 1A.2b) or shadowed (Figure 1A.2c). Other techniques such as freeze fracturing and freeze etching are discussed in later chapters.
Scanning Electron Microscopy
A quite different technique is called scanning electron microscopy (SEM). A schematic diagram of a scanning electron microscope is shown in Figure 1A.3. Here, the electron beam is scanned back and forth across the sample, in a pattern generated by the scan generator and beam deflector, and secondary elec-trons emitted from the point at which the beam impinges on the
(7100 nm).
sample surface are picked up by a detector. The image is then dis-played on a video screen, whose surface is scanned in register with the scanning of the sample. SEM does not have the resolu-tion of TEM, but it is excellent for obtaining extremely clear views of the surfaces of minute objects, as you can see in Figure 1A.4. Preparation for SEM studies does not require sec-tioning, but the specimen must be fixed and dried to be stable at high vacuum and is usually coated with a thin layer of gold to aid in emission of secondary electrons.
Another technique should be mentioned: scanning transmis-sion electron microscopy (STEM). In this method the electron beam is scanned over the specimen, as in SEM, but it is detected in transmission. The method has the advantage that unstained, unfixed specimens can sometimes be used. Furthermore, the absorption of electrons of different energies provides information about the composition of different portions of the sample.
Laser Scanning Confocal Microscopy
Aside from the problems of resolution mentioned on page 20, there exists another fundamental limitation to conventional light microscopy for studying internal structure in cells and other biological samples. At high resolution the depth of focus of a light microscope is about Superposition of images of material in this thick slice will obscure detail. To get around this problem, the confocal microscope was developed. As shown in Figure 1A.5, a light beam (preferably from a laser) is focused into a very small volume at the desired level within the sample. Reflected or fluorescent light from this spot is brought back to a detector, through a pinhole that excludes light scattered from other regions. The position of the illuminated spot is scanned back and forth through the sample, always at the same level. The image that is electronically built up in this way represents a very thin, highly resolved “slice” through the sample. It can also be repeated at different levels to build up a three-dimensional image.
3mm.
(~0.3mm)
22
CHAPTER 1 THE SCOPE OF BIOCHEMISTRY0.1mm
0.1mm The sample is
fixed in aldehyde and stained with OsO4 to enhance contrast.
(a) Sectioning and staining with OsO4 1
Particles are collected on copper grid covered with a thin plastic film.
(b) Negative staining 1
Particles are deposited on a mica plate.
(c) Shadowing
1 Metal is deposited from
an angle while in a vac-cuum, forming a replica of the specimen.
2 The specimen is
dissolved away, and the metal rep-lica is placed on a grid for examination.
3 A drop of heavy
metal staining solution is placed on the grid.
2 The heavy metal forms
a layer around the particle, which then ap-pears more transparent than the background.
3 The sample is
embedded in a block of plastic.
2 Thin sections
are cut on an ultramicrotome.
3 Sections are laid
on copper grids for examination.
Blade
Sample Microtome arm
4
Stained skeletal muscle 5
Negatively stained muscle protein fibers 4
Shadowed muscle protein fibers 4
FIGURE 1A.2
Three methods of preparing samples for transmission electron microscopy.
Electron Microscopy in Biology, Vol. II, T. Pollard and P. Maupin; J. D. Griffith, ed. © 1982 John Wiley & Sons, Inc. Reproduced with permission from John Wiley
& Sons, Inc.
MICROSCOPY AT MANY LEVELS
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The method is most powerful when fluorescence detection is used, for then specifically labeled structures or substances can be precisely located within a cell. Because the method is relatively nondestructive, it can be used to follow dynamic processes in liv-ing cells. It has been employed, for example, to pinpoint the places within a cell nucleus where active replication of DNA is taking place. The rapid development of more versatile and dis-criminating fluorescent probes is making confocal microscopy a major technique in cellular biochemistry (See Figure 1A.6).
Scanning Tunneling and Atomic Force Microscopy
Recently, a remarkable new kind of microscope has been devel-oped. Scanning tunneling microscopy uses a very fine, electri-cally charged metal tip, which is scanned across the sample. As electrons leak (tunnel) between the tip and the surface support-ing the sample, the resistance they encounter varies accordsupport-ing to the height of microscopic objects lying on the surface. The result-ing fluctuations in current produce a video display of the surface with a resolution comparable to that of an electron microscope.
In atomic force microscopy (Figure 1A.7), an extremely sharp tip is either dragged or tapped back and forth across the sample, and its up-and-down motion is detected by the deflection of a laser beam reflected off the cantilever which holds the tip. This
Electron gun
Electron beam
Beam deflector: scans beam across sample
Deflector circuitry (scan generator) correlates scan on sample with sweep on video screen
Video screen:
displays image Screen
deflector Scintillation
detector:
detects scattered electrons Secondary
electrons Specimen
Primary electrons Objective lens
Condenser lens
FIGURE 1A.3
The principle of the scanning electron microscope.
FIGURE 1A.4
A scanning electron micrograph showing phagocytosis. A macrophage is engulf-ing several sausage-shaped E. coli cells in this image, which is magnified 4300X.
Eye of Science/Science Photo Library.
24
CHAPTER 1 THE SCOPE OF BIOCHEMISTRYImage plane
Incident light
Light from specimen Pinhole
Photomultiplier tube
Laser
Dichroic mirror
Scanner
Objective
Specimen x
y
FIGURE 1A.5
Diagram illustrating the principle of laser scanning confocal microscopy. A laser beam is passed through an x–y scanner, collimated to a small spot by the objective lens, and scanned across the specimen. Fluoresced light is collected by the objective and directed by a dichroic mirror (a mirror that reflects the fluorescent light but not the shorter-wavelength laser light) to a pinhole aperture placed in the conjugate image plane. Light originating from the specimen plane of focus passes through the pinhole to a photomultiplier detector. Light from above or below the specimen focal plane strikes the walls of the aperture and is not transmitted.
Reprinted from Optical Microscopy: Emerging Methods and Applications, Brian Herman and John J. Lemasters, eds., pp. 339–354. © 1993, with permission from Elsevier.
(a) (b) FIGURE 1A.6
Image of a thick slice of hippocampus from a mouse brain visual-ized either by conventional optical microscopy (a) or by laser scanning confocal microscopy (b). The preparation was treated with fluorescent-tagged antibodies to glial fibrillary acidic protein (red) and neurofilaments H (green), and with a fluorescent DNA-binding dye, Hoechst 33342, to label nuclei (blue).
Michael Davidson, The Florida State University/Molecular Expressions™.
MICROSCOPY AT MANY LEVELS
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motion is greatly amplified to give a contour map of the object.
Both of these techniques have the enormous advantage over electron microscopy that wet, even immersed, samples can be studied, And the resolution allows visualization of single macro-molecules.
References
Claxton, N. S., T. J. Fellers, and M. W. Davidson (2006) Laser Scanning Confocal Microscopy. http://www.olympusfluoview.com/theory/
LSCMIntro.pdf. A 37-page web archive that describes the theory and applications of this technique.
Corle, T. R., and G. S. Kino (eds.) (1998) Confocal Scanning Optical Microscopy and Related Imaging Systems. Academic Press, San Diego.
Egerton, R. F. (2005) Physical principles of electron microscopy: An introduction to TEM, SEM, and AEM. Springer, New York.
Engel, A. (1991) Biological application of scanning probe microscopy. Annu.
Rev. Biophys. & Biophys. Chem. 20:79–108.
Herman, B., and J. J. Lemasters (eds.) (1996) Optical Microscopy: Emerging Methods and Applications. Academic Press, San Diego. A collection of short papers on a wide variety of new microscopic methods.
Laser
Piezoelectric crystal Mirror
Photodiode
Lens
Cantilever Glass or mica sheet carries sample
FIGURE 1A.7
The principle of the atomic force microscope.Power to the piezoelectric crystal is regulated to move the sample up and down and keep the tip at con-stant height as the sample is scanned.
The macromolecules that participate
in the structural and functional matrix of life are immense structures held together by strong, covalent bonds. Yet covalent bonding alone cannot begin to describe the complexity of molecular structure in biology. Much weaker interactions are responsible for most of the ele-gant cellular architecture visible in the electron micrographs of Chapter 1. These are the noncovalent interactions, also called noncovalent forces or noncovalent bonds, between ions, molecules, and parts of molecules.Consider the macromolecules we discussed in Chapter 1. The linear sequence of the nucleotide residues in a strand of DNA is maintained by covalent bonds.
But DNA also has a highly specific three-dimensional structure, which is stabi-lized by noncovalent interactions between different parts of the molecule.
Similarly, every kind of protein is made up of amino acids linked by covalent peptide bonds; but each protein is also folded into a specific molecular conforma-tion that is stabilized by noncovalent interacconforma-tions. Proteins interact with macro-molecules, such as other proteins or nucleic acids or lipids, to form still higher lev-els of organization, ultimately leading to cells, tissues, and whole organisms. All of this complexity is accounted for by a myriad of noncovalent interactions within and between macromolecules.
What makes noncovalent interactions so important in biology and biochem-istry? The key is seen in Figure 2.1, which compares noncovalent and covalent bond energies. The covalent bonds most important in biology (such as C C and C H) have bond energies in the range of 300–400 kJ/mol. Biologically important noncovalent bonds are 10 to 100 times weaker. It is their very weakness that makes noncovalent bonds so essential, for it allows them to be continually broken and re-formed in the dynamic molecular interplay that is life. This interplay depends
i i