"How We See the Invisible World" Microbiology Textbook PDF

CHAPTER 2 How We See the Invisible World FIGURE 2.1 Different types of microscopy are used to visualize different structures. Brightfield microscopy (left) renders a darker image on a lighter background, producing a clear image of these Bacillus anthracis cells in cerebrospinal fluid (the rod-shape...

CHAPTER 2 How We See the Invisible World FIGURE 2.1 Different types of microscopy are used to visualize different structures. Brightfield microscopy (left) renders a darker image on a lighter background, producing a clear image of these Bacillus anthracis cells in cerebrospinal fluid (the rod-shaped bacterial cells are surrounded by larger white blood cells). Darkfield microscopy (right) increases contrast, rendering a brighter image on a darker background, as demonstrated by this image of the bacterium Borrelia burgdorferi, which causes Lyme disease. (credit left: modification of work by Centers for Disease Control and Prevention; credit right: modification of work by American Society for Microbiology) CHAPTER OUTLINE 2.1 The Properties of Light 2.2 Peering Into the Invisible World 2.3 Instruments of Microscopy 2.4 Staining Microscopic Specimens INTRODUCTION When we look at a rainbow, its colors span the full spectrum of light that the human eye can detect and differentiate. Each hue represents a different frequency of visible light, processed by our eyes and brains and rendered as red, orange, yellow, green, or one of the many other familiar colors that have always been a part of the human experience. But only recently have humans developed an understanding of the properties of light that allow us to see images in color. Over the past several centuries, we have learned to manipulate light to peer into previously invisible worlds–those too small or too far away to be seen by the naked eye. Through a microscope, we can examine microbial cells, using various techniques to manipulate color, size, and contrast in ways that help us identify species and diagnose disease. Figure 2.1 illustrates how we can apply the properties of light to visualize and magnify images; but these stunning micrographs are just two examples of the numerous types of images we are now able to produce with different microscopic technologies. This chapter explores how various types of microscopes manipulate light in order to provide a window into the world of microorganisms. By understanding how various kinds of microscopes work, we can produce highly detailed images of microbes that can be useful for both research and clinical applications. 2.1 The Properties of Light LEARNING OBJECTIVES By the end of this section, you will be able to: Identify and define the characteristics of electromagnetic radiation (EMR) used in microscopy Explain how lenses are used in microscopy to manipulate visible and ultraviolet (UV) light 36 2 How We See the Invisible World CLINICAL FOCUS Part 1 Cindy, a 17-year-old counselor at a summer sports camp, scraped her knee playing basketball 2 weeks ago. At the time, she thought it was only a minor abrasion that would heal, like many others before it. Instead, the wound began to look like an insect bite and has continued to become increasingly painful and swollen. The camp nurse examines the lesion and observes a large amount of pus oozing from the surface. Concerned that Cindy may have developed a potentially aggressive infection, she swabs the wound to collect a sample from the infection site. Then she cleans out the pus and dresses the wound, instructing Cindy to keep the area clean and to come back the next day. When Cindy leaves, the nurse sends the sample to the closest medical lab to be analyzed under a microscope. What are some things we can learn about these bacteria by looking at them under a microscope? Jump to the next Clinical Focus box. Visible light consists of electromagnetic waves that behave like other waves. Hence, many of the properties of light that are relevant to microscopy can be understood in terms of light’s behavior as a wave. An important property of light waves is the wavelength, or the distance between one peak of a wave and the next peak. The height of each peak (or depth of each trough) is called the amplitude. In contrast, the frequency of the wave is the rate of vibration of the wave, or the number of wavelengths within a specified time period (Figure 2.2). FIGURE 2.2 (a) The amplitude is the height of a wave, whereas the wavelength is the distance between one peak and the next. (b) These waves have different frequencies, or rates of vibration. The wave at the top has the lowest frequency, since it has the fewest peaks per unit time. The wave at the bottom has the highest frequency. Interactions of Light Light waves interact with materials by being reflected, absorbed, or transmitted. Reflection occurs when a wave bounces off of a material. For example, a red piece of cloth may reflect red light to our eyes while absorbing other colors of light. Absorbance occurs when a material captures the energy of a light wave. In the case of glow-in-the- dark plastics, the energy from light can be absorbed and then later re-emitted as another form of phosphorescence. Transmission occurs when a wave travels through a material, like light through glass (the process of transmission is called transmittance). When a material allows a large proportion of light to be transmitted, it may do so because it is thinner, or more transparent (having more transparency and less opacity). Figure 2.3 illustrates the difference between transparency and opacity. FIGURE 2.3 (a) A Petri dish is made of transparent plastic or glass, which allows transmission of a high proportion of light. This transparency allows us to see through the sides of the dish to view the contents. (b) This slice of an iron meteorite is opaque (i.e., it has Access for free at openstax.org  2.1 The Properties of Light 37 opacity). Light is not transmitted through the material, making it impossible to see the part of the hand covered by the object. (credit a: modification of work by Umberto Salvagnin; credit b: modification of work by “Waifer X”/Flickr) Light waves can also interact with each other by interference, creating complex patterns of motion. Dropping two pebbles into a puddle causes the waves on the puddle’s surface to interact, creating complex interference patterns. Light waves can interact in the same way. In addition to interfering with each other, light waves can also interact with small objects or openings by bending or scattering. This is called diffraction. Diffraction is larger when the object is smaller relative to the wavelength of the light (the distance between two consecutive peaks of a light wave). Often, when waves diffract in different directions around an obstacle or opening, they will interfere with each other. CHECK YOUR UNDERSTANDING If a light wave has a long wavelength, is it likely to have a low or high frequency? If an object is transparent, does it reflect, absorb, or transmit light? Lenses and Refraction In the context of microscopy, refraction is perhaps the most important behavior exhibited by light waves. Refraction occurs when light waves change direction as they enter a new medium (Figure 2.4). Different transparent materials transmit light at different speeds; thus, light can change speed when passing from one material to another. This change in speed usually also causes a change in direction (refraction), with the degree of change dependent on the angle of the incoming light. FIGURE 2.4 (a) Refraction occurs when light passes from one medium, such as air, to another, such as glass, changing the direction of the light rays. (b) As shown in this diagram, light rays passing from one medium to another may be either refracted or reflected. The extent to which a material slows transmission speed relative to empty space is called the refractive index of that material. Large differences between the refractive indices of two materials will result in a large amount of refraction when light passes from one material to the other. For example, light moves much more slowly through water than through air, so light entering water from air can change direction greatly. We say that the water has a higher refractive index than air (Figure 2.5). FIGURE 2.5 This straight pole appears to bend at an angle as it enters the water. This optical illusion is due to the large difference between the refractive indices of air and water. 38 2 How We See the Invisible World When light crosses a boundary into a material with a higher refractive index, its direction turns to be closer to perpendicular to the boundary (i.e., more toward a normal to that boundary; see Figure 2.5). This is the principle behind lenses. We can think of a lens as an object with a curved boundary (or a collection of prisms) that collects all of the light that strikes it and refracts it so that it all meets at a single point called the image point (focus). A convex lens can be used to magnify because it can focus at closer range than the human eye, producing a larger image. Concave lenses and mirrors can also be used in microscopes to redirect the light path. Figure 2.6 shows the focal point (the image point when light entering the lens is parallel) and the focal length (the distance to the focal point) for convex and concave lenses. FIGURE 2.6 (a) A lens is like a collection of prisms, such as the one shown here. (b) When light passes through a convex lens, it is refracted toward a focal point on the other side of the lens. The focal length is the distance to the focal point. (c) Light passing through a concave lens is refracted away from a focal point in front of the lens. The human eye contains a lens that enables us to see images. This lens focuses the light reflecting off of objects in front of the eye onto the surface of the retina, which is like a screen in the back of the eye. Artificial lenses placed in front of the eye (contact lenses, glasses, or microscopic lenses) focus light before it is focused (again) by the lens of the eye, manipulating the image that ends up on the retina (e.g., by making it appear larger). Images are commonly manipulated by controlling the distances between the object, the lens, and the screen, as well as the curvature of the lens. For example, for a given amount of curvature, when an object is closer to the lens, the focal points are farther from the lens. As a result, it is often necessary to manipulate these distances to create a focused image on a screen. Similarly, more curvature creates image points closer to the lens and a larger image when the image is in focus. This property is often described in terms of the focal distance, or distance to the focal point. CHECK YOUR UNDERSTANDING Explain how a lens focuses light at the image point. Name some factors that affect the focal length of a lens. Electromagnetic Spectrum and Color Visible light is just one form of electromagnetic radiation (EMR), a type of energy that is all around us. Other forms of EMR include microwaves, X-rays, and radio waves, among others. The different types of EMR fall on the electromagnetic spectrum, which is defined in terms of wavelength and frequency. The spectrum of visible light occupies a relatively small range of frequencies between infrared and ultraviolet light (Figure 2.7). Access for free at openstax.org  2.1 The Properties of Light 39 FIGURE 2.7 The electromagnetic spectrum ranges from high-frequency gamma rays to low-frequency radio waves. Visible light is the relatively small range of electromagnetic frequencies that can be sensed by the human eye. On the electromagnetic spectrum, visible light falls between ultraviolet and infrared light. (credit: modification of work by Johannes Ahlmann) Whereas wavelength represents the distance between adjacent peaks of a light wave, frequency, in a simplified definition, represents the rate of oscillation. Waves with higher frequencies have shorter wavelengths and, therefore, have more oscillations per unit time than lower-frequency waves. Higher-frequency waves also contain more energy than lower-frequency waves. This energy is delivered as elementary particles called photons. Higher- frequency waves deliver more energetic photons than lower-frequency waves. Photons with different energies interact differently with the retina. In the spectrum of visible light, each color corresponds to a particular frequency and wavelength (Figure 2.7).The lowest frequency of visible light appears as the color red, whereas the highest appears as the color violet. When the retina receives visible light of many different frequencies, we perceive this as white light. However, white light can be separated into its component colors using refraction. If we pass white light through a prism, different colors will be refracted in different directions, creating a rainbow-like spectrum on a screen behind the prism. This separation of colors is called dispersion, and it occurs because, for a given material, the refractive index is different for different frequencies of light. Certain materials can refract nonvisible forms of EMR and, in effect, transform them into visible light. Certain fluorescent dyes, for instance, absorb ultraviolet or blue light and then use the energy to emit photons of a different color, giving off light rather than simply vibrating. This occurs because the energy absorption causes electrons to jump to higher energy states, after which they then almost immediately fall back down to their ground states, emitting specific amounts of energy as photons. Not all of the energy is emitted in a given photon, so the emitted photons will be of lower energy and, thus, of lower frequency than the absorbed ones. Thus, a dye such as Texas red may be excited by blue light, but emit red light; or a dye such as fluorescein isothiocyanate (FITC) may absorb (invisible) high-energy ultraviolet light and emit green light (Figure 2.8). In some materials, the photons may be emitted following a delay after absorption; in this case, the process is called phosphorescence. Glow-in-the-dark plastic works by using phosphorescent material. 40 2 How We See the Invisible World FIGURE 2.8 The fluorescent dyes absorbed by these bovine pulmonary artery endothelial cells emit brilliant colors when excited by ultraviolet light under a fluorescence microscope. Various cell structures absorb different dyes. The nuclei are stained blue with 4’,6-diamidino-2-phenylindole (DAPI); microtubles are marked green by an antibody bound to FITC; and actin filaments are labeled red with phalloidin bound to tetramethylrhodamine (TRITC). (credit: National Institutes of Health) CHECK YOUR UNDERSTANDING Which has a higher frequency: red light or green light? Explain why dispersion occurs when white light passes through a prism. Why do fluorescent dyes emit a different color of light than they absorb? Magnification, Resolution, and Contrast Microscopes magnify images and use the properties of light to create useful images of small objects. Magnification is defined as the ability of a lens to enlarge the image of an object when compared to the real object. For example, a magnification of 10⨯ means that the image appears 10 times the size of the object as viewed with the naked eye. Greater magnification typically improves our ability to see details of small objects, but magnification alone is not sufficient to make the most useful images. It is often useful to enhance the resolution of objects: the ability to tell that two separate points or objects are separate. A low-resolution image appears fuzzy, whereas a high-resolution image appears sharp. Two factors affect resolution. The first is wavelength. Shorter wavelengths are able to resolve smaller objects; thus, an electron microscope has a much higher resolution than a light microscope, since it uses an electron beam with a very short wavelength, as opposed to the long-wavelength visible light used by a light microscope. The second factor that affects resolution is numerical aperture, which is a measure of a lens’s ability to gather light. The higher the numerical aperture, the better the resolution. LINK TO LEARNING Read this article (https://www.openstax.org/l/22aperture) to learn more about factors that can increase or decrease the numerical aperture of a lens. Even when a microscope has high resolution, it can be difficult to distinguish small structures in many specimens because microorganisms are relatively transparent. It is often necessary to increase contrast to detect different structures in a specimen. Various types of microscopes use different features of light or electrons to increase contrast—visible differences between the parts of a specimen (see Instruments of Microscopy). Additionally, dyes that bind to some structures but not others can be used to improve the contrast between images of relatively transparent objects (see Staining Microscopic Specimens). CHECK YOUR UNDERSTANDING Explain the difference between magnification and resolution. Explain the difference between resolution and contrast. Name two factors that affect resolution. Access for free at openstax.org  2.2 Peering Into the Invisible World 41 2.2 Peering Into the Invisible World LEARNING OBJECTIVES By the end of this section, you will be able to: Describe historical developments and individual contributions that led to the invention and development of the microscope Compare and contrast the features of simple and compound microscopes Some of the fundamental characteristics and functions of microscopes can be understood in the context of the history of their use. Italian scholar Girolamo Fracastoro is regarded as the first person to formally postulate that disease was spread by tiny invisible seminaria, or “seeds of the contagion.” In his book De Contagione (1546), he proposed that these seeds could attach themselves to certain objects (which he called fomes [cloth]) that supported their transfer from person to person. However, since the technology for seeing such tiny objects did not yet exist, the existence of the seminaria remained hypothetical for a little over a century—an invisible world waiting to be revealed. Early Microscopes Antonie van Leeuwenhoek, sometimes hailed as “the Father of Microbiology,” is typically credited as the first person to have created microscopes powerful enough to view microbes (Figure 2.9). Born in the city of Delft in the Dutch Republic, van Leeuwenhoek began his career selling fabrics. However, he later became interested in lens making (perhaps to look at threads) and his innovative techniques produced microscopes that allowed him to observe microorganisms as no one had before. In 1674, he described his observations of single-celled organisms, whose existence was previously unknown, in a series of letters to the Royal Society of London. His report was initially met with skepticism, but his claims were soon verified and he became something of a celebrity in the scientific community. FIGURE 2.9 (a) Antonie van Leeuwenhoek (1632–1723) is credited as being the first person to observe microbes, including bacteria, which he called “animalcules” and “wee little beasties.” (b) Even though van Leeuwenhoek’s microscopes were simple microscopes (as seen in this replica), they were more powerful and provided better resolution than the compound microscopes of his day. (c) Though more famous for developing the telescope, Galileo Galilei (1564–1642) was also one of the pioneers of microscopy. (credit b: modification of work by “Wellcome Images”/Wikimedia Commons) While van Leeuwenhoek is credited with the discovery of microorganisms, others before him had contributed to the development of the microscope. These included eyeglass makers in the Netherlands in the late 1500s, as well as the Italian astronomer Galileo Galilei, who used a compound microscope to examine insect parts (Figure 2.9). Whereas van Leeuwenhoek used a simple microscope, in which light is passed through just one lens, Galileo’s compound microscope was more sophisticated, passing light through two sets of lenses. Van Leeuwenhoek’s contemporary, the Englishman Robert Hooke (1635–1703), also made important contributions to microscopy, publishing in his book Micrographia (1665) many observations using compound microscopes. Viewing a thin sample of cork through his microscope, he was the first to observe the structures that we now know as cells (Figure 2.10). Hooke described these structures as resembling “Honey-comb,” and as “small Boxes or Bladders of Air,” noting that each “Cavern, Bubble, or Cell” is distinct from the others (in Latin, “cell” literally means “small room”). They likely appeared to Hooke to be filled with air because the cork cells were dead, with only the rigid cell walls providing the structure. 42 2 How We See the Invisible World FIGURE 2.10 Robert Hooke used his (a) compound microscope to view (b) cork cells. Both of these engravings are from his seminal work Micrographia, published in 1665. CHECK YOUR UNDERSTANDING Explain the difference between simple and compound microscopes. Compare and contrast the contributions of van Leeuwenhoek, Hooke, and Galileo to early microscopy. MICRO CONNECTIONS Who Invented the Microscope? While Antonie van Leeuwenhoek and Robert Hooke generally receive much of the credit for early advances in microscopy, neither can claim to be the inventor of the microscope. Some argue that this designation should belong to Hans and Zaccharias Janssen, Dutch spectacle-makers who may have invented the telescope, the simple microscope, and the compound microscope during the late 1500s or early 1600s (Figure 2.11). Unfortunately, little is known for sure about the Janssens, not even the exact dates of their births and deaths. The Janssens were secretive about their work and never published. It is also possible that the Janssens did not invent anything at all; their neighbor, Hans Lippershey, also developed microscopes and telescopes during the same time frame, and he is often credited with inventing the telescope. The historical records from the time are as fuzzy and imprecise as the images viewed through those early lenses, and any archived records have been lost over the centuries. By contrast, van Leeuwenhoek and Hooke can thank ample documentation of their work for their respective legacies. Like Janssen, van Leeuwenhoek began his work in obscurity, leaving behind few records. However, his friend, the prominent physician Reinier de Graaf, wrote a letter to the editor of the Philosophical Transactions of the Royal Society of London calling attention to van Leeuwenhoek’s powerful microscopes. From 1673 onward, van Leeuwenhoek began regularly submitting letters to the Royal Society detailing his observations. In 1674, his report describing single-celled organisms produced controversy in the scientific community, but his observations were soon confirmed when the society sent a delegation to investigate his findings. He subsequently enjoyed considerable celebrity, at one point even entertaining a visit by the czar of Russia. Similarly, Robert Hooke had his observations using microscopes published by the Royal Society in a book called Micrographia in 1665. The book became a bestseller and greatly increased interest in microscopy throughout much of Europe. Access for free at openstax.org  2.3 Instruments of Microscopy 43 FIGURE 2.11 Zaccharias Janssen, along with his father Hans, may have invented the telescope, the simple microscope, and the compound microscope during the late 1500s or early 1600s. The historical evidence is inconclusive. 2.3 Instruments of Microscopy LEARNING OBJECTIVES By the end of this section, you will be able to: Identify and describe the parts of a brightfield microscope Calculate total magnification for a compound microscope Describe the distinguishing features and typical uses for various types of light microscopes, electron microscopes, and scanning probe microscopes The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses short- wavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope. Light Microscopy Many types of microscopes fall under the category of light microscopes, which use light to visualize images. Examples of light microscopes include brightfield microscopes, darkfield microscopes, phase-contrast microscopes, differential interference contrast microscopes, fluorescence microscopes, confocal scanning laser microscopes, and two-photon microscopes. These various types of light microscopes can be used to complement each other in diagnostics and research. Brightfield Microscopes The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 2.12; in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the 44 2 How We See the Invisible World objective magnification: For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be FIGURE 2.12 Components of a typical brightfield microscope. The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage (a platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses. When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator, which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than- maximal light levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator. A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses. Access for free at openstax.org  2.3 Instruments of Microscopy 45 At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (Figure 2.13). A variety of oils can be used for different types of light. FIGURE 2.13 (a) Oil immersion lenses like this one are used to improve resolution. (b) Because immersion oil and glass have very similar refractive indices, there is a minimal amount of refraction before the light reaches the lens. Without immersion oil, light scatters as it passes through the air above the slide, degrading the resolution of the image. MICRO CONNECTIONS Microscope Maintenance: Best Practices Even a very powerful microscope cannot deliver high-resolution images if it is not properly cleaned and maintained. Since lenses are carefully designed and manufactured to refract light with a high degree of precision, even a slightly dirty or scratched lens will refract light in unintended ways, degrading the image of the specimen. In addition, microscopes are rather delicate instruments, and great care must be taken to avoid damaging parts and surfaces. Among other things, proper care of a microscope includes the following: cleaning the lenses with lens paper not allowing lenses to contact the slide (e.g., by rapidly changing the focus) protecting the bulb (if there is one) from breakage not pushing an objective into a slide not using the coarse focusing knob when using the 40⨯ or greater objective lenses only using immersion oil with a specialized oil objective, usually the 100⨯ objective cleaning oil from immersion lenses after using the microscope cleaning any oil accidentally transferred from other lenses covering the microscope or placing it in a cabinet when not in use LINK TO LEARNING Visit the online resource linked below for simulations and demonstrations involving the use of microscopes. Keep in mind that execution of specific techniques and procedures can vary depending on the specific instrument you are using. Thus, it is important to learn and practice with an actual microscope in a laboratory setting under expert supervision. 46 2 How We See the Invisible World University of Delaware’s Virtual Microscope (https://www.openstax.org/l/22virtualsim) Darkfield Microscopy A darkfield microscope is a brightfield microscope that has a small but significant modification to the condenser. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. The resulting image typically shows bright objects on a dark background (Figure 2.14). FIGURE 2.14 An opaque light stop inserted into a brightfield microscope is used to produce a darkfield image. The light stop blocks light traveling directly from the illuminator to the objective lens, allowing only light reflected or refracted off the specimen to reach the eye. Darkfield microscopy can often create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains. For example, thin spirochetes like Treponema pallidum, the causative agent of syphilis, can be best viewed using a darkfield microscope (Figure 2.15). FIGURE 2.15 Use of a darkfield microscope allows us to view living, unstained samples of the spirochete Treponema pallidum. Similar to a photographic negative, the spirochetes appear bright against a dark background. (credit: Centers for Disease Control and Prevention) CHECK YOUR UNDERSTANDING Identify the key differences between brightfield and darkfield microscopy. Access for free at openstax.org  2.3 Instruments of Microscopy 47 CLINICAL FOCUS Part 2 Wound infections like Cindy’s can be caused by many different types of bacteria, some of which can spread rapidly with serious complications. Identifying the specific cause is very important to select a medication that can kill or stop the growth of the bacteria. After calling a local doctor about Cindy’s case, the camp nurse sends the sample from the wound to the closest medical laboratory. Unfortunately, since the camp is in a remote area, the nearest lab is small and poorly equipped. A more modern lab would likely use other methods to culture, grow, and identify the bacteria, but in this case, the technician decides to make a wet mount from the specimen and view it under a brightfield microscope. In a wet mount, a small drop of water is added to the slide, and a cover slip is placed over the specimen to keep it in place before it is positioned under the objective lens. Under the brightfield microscope, the technician can barely see the bacteria cells because they are nearly transparent against the bright background. To increase contrast, the technician inserts an opaque light stop above the illuminator. The resulting darkfield image clearly shows that the bacteria cells are spherical and grouped in clusters, like grapes. Why is it important to identify the shape and growth patterns of cells in a specimen? What other types of microscopy could be used effectively to view this specimen? Jump to the next Clinical Focus box. Go back to the previous Clinical Focus box. Phase-Contrast Microscopes Phase-contrast microscopes use refraction and interference caused by structures in a specimen to create high- contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. To create altered wavelength paths, an annular stop is used in the condenser. The annular stop produces a hollow cone of light that is focused on the specimen before reaching the objective lens. The objective contains a phase plate containing a phase ring. As a result, light traveling directly from the illuminator passes through the phase ring while light refracted or reflected by the specimen passes through the plate. This causes waves traveling through the ring to be about one-half of a wavelength out of phase with those passing through the plate. Because waves have peaks and troughs, they can add together (if in phase together) or cancel each other out (if out of phase). When the wavelengths are out of phase, wave troughs will cancel out wave peaks, which is called destructive interference. Structures that refract light then appear dark against a bright background of only unrefracted light. More generally, structures that differ in features such as refractive index will differ in levels of darkness (Figure 2.16). 48 2 How We See the Invisible World FIGURE 2.16 This diagram of a phase-contrast microscope illustrates phase differences between light passing through the object and background. These differences are produced by passing the rays through different parts of a phase plate. The light rays are superimposed in the image plane, producing contrast due to their interference. Because it increases contrast without requiring stains, phase-contrast microscopy is often used to observe live specimens. Certain structures, such as organelles in eukaryotic cells and endospores in prokaryotic cells, are especially well visualized with phase-contrast microscopy (Figure 2.17). FIGURE 2.17 This figure compares a brightfield image (left) with a phase-contrast image (right) of the same unstained simple squamous epithelial cells. The cells are in the center and bottom right of each photograph (the irregular item above the cells is acellular debris). Notice that the unstained cells in the brightfield image are almost invisible against the background, whereas the cells in the phase-contrast image appear to glow against the background, revealing far more detail. Differential Interference Contrast Microscopes Differential interference contrast (DIC) microscopes (also known as Nomarski optics) are similar to phase- contrast microscopes in that they use interference patterns to enhance contrast between different features of a specimen. In a DIC microscope, two beams of light are created in which the direction of wave movement (polarization) differs. Once the beams pass through either the specimen or specimen-free space, they are recombined and effects of the specimens cause differences in the interference patterns generated by the combining of the beams. This results in high-contrast images of living organisms with a three-dimensional appearance. These microscopes are especially useful in distinguishing structures within live, unstained specimens. (Figure 2.18) Access for free at openstax.org  2.3 Instruments of Microscopy 49 FIGURE 2.18 A DIC image of Fonsecaea pedrosoi grown on modified Leonian’s agar. This fungus causes chromoblastomycosis, a chronic skin infection common in tropical and subtropical climates. CHECK YOUR UNDERSTANDING What are some advantages of phase-contrast and DIC microscopy? Fluorescence Microscopes A fluorescence microscope uses fluorescent chromophores called fluorochromes, which are capable of absorbing energy from a light source and then emitting this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) as well as fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI) and acridine orange. The microscope transmits an excitation light, generally a form of EMR with a short wavelength, such as ultraviolet or blue light, toward the specimen; the chromophores absorb the excitation light and emit visible light with longer wavelengths. The excitation light is then filtered out (in part because ultraviolet light is harmful to the eyes) so that only visible light passes through the ocular lens. This produces an image of the specimen in bright colors against a dark background. Fluorescence microscopes are especially useful in clinical microbiology. They can be used to identify pathogens, to find particular species within an environment, or to find the locations of particular molecules and structures within a cell. Approaches have also been developed to distinguish living from dead cells using fluorescence microscopy based upon whether they take up particular fluorochromes. Sometimes, multiple fluorochromes are used on the same specimen to show different structures or features. One of the most important applications of fluorescence microscopy is a technique called immunofluorescence, which is used to identify certain disease-causing microbes by observing whether antibodies bind to them. (Antibodies are protein molecules produced by the immune system that attach to specific pathogens to kill or inhibit them.) There are two approaches to this technique: direct immunofluorescence assay (DFA) and indirect immunofluorescence assay (IFA). In DFA, specific antibodies (e.g., those that the target the rabies virus) are stained with a fluorochrome. If the specimen contains the targeted pathogen, one can observe the antibodies binding to the pathogen under the fluorescent microscope. This is called a primary antibody stain because the stained antibodies attach directly to the pathogen. In IFA, secondary antibodies are stained with a fluorochrome rather than primary antibodies. Secondary antibodies do not attach directly to the pathogen, but they do bind to primary antibodies. When the unstained primary antibodies bind to the pathogen, the fluorescent secondary antibodies can be observed binding to the primary antibodies. Thus, the secondary antibodies are attached indirectly to the pathogen. Since multiple secondary antibodies can often attach to a primary antibody, IFA increases the number of fluorescent antibodies attached to the specimen, making it easier visualize features in the specimen (Figure 2.19). 50 2 How We See the Invisible World FIGURE 2.19 (a) A direct immunofluorescent stain is used to visualize Neisseria gonorrhoeae, the bacterium that causes gonorrhea. (b) An indirect immunofluorescent stain is used to visualize larvae of Schistosoma mansoni, a parasitic worm that causes schistosomiasis, an intestinal disease common in the tropics. (c) In direct immunofluorescence, the stain is absorbed by a primary antibody, which binds to the antigen. In indirect immunofluorescence, the stain is absorbed by a secondary antibody, which binds to a primary antibody that has bound to the antigen. (credit a: modification of work by Centers for Disease Control and Prevention; credit b: modification of work by Centers for Disease Control and Prevention) CHECK YOUR UNDERSTANDING Why must fluorochromes be used to examine a specimen under a fluorescence microscope? Confocal Microscopes Whereas other forms of light microscopy create an image that is maximally focused at a single distance from the observer (the depth, or z-plane), a confocal microscope uses a laser to scan multiple z-planes successively. This produces numerous two-dimensional, high-resolution images at various depths, which can be constructed into a three-dimensional image by a computer. As with fluorescence microscopes, fluorescent stains are generally used to increase contrast and resolution. Image clarity is further enhanced by a narrow aperture that eliminates any light that is not from the z-plane. Confocal microscopes are thus very useful for examining thick specimens such as biofilms, which can be examined alive and unfixed (Figure 2.20). FIGURE 2.20 Confocal microscopy can be used to visualize structures such as this roof-dwelling cyanobacterium biofilm. (credit: modification of work by American Society for Microbiology) Access for free at openstax.org  2.3 Instruments of Microscopy 51 LINK TO LEARNING Explore a rotating three-dimensional view (https://www.openstax.org/l/22biofilm3d) of a biofilm as observed under a confocal microscope. After navigating to the webpage, click the “play” button to launch the video. Two-Photon Microscopes While the original fluorescent and confocal microscopes allowed better visualization of unique features in specimens, there were still problems that prevented optimum visualization. The effective sensitivity of fluorescence microscopy when viewing thick specimens was generally limited by out-of-focus flare, which resulted in poor resolution. This limitation was greatly reduced in the confocal microscope through the use of a confocal pinhole to reject out-of-focus background fluorescence with thin (