Lab 2 Introduction to Microscopy_PhaseContrast_Darkfield (1)

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Feb 20, 2024

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1 LABORATORY EXERCISE #2: Introduction to Microscopy: Phase Contrast and Darkfield Background information This laboratory period will be used to introduce students to phase contrast and dark-field microscopy. Students will compare the effectiveness and limitations of bright-field, phase contrast and dark-field microscopy when observing prokaryotic cells, pigmented eukaryotic cells, and non-pigmented eukaryotic cells both in pure culture and in pond water. Today's lab activities 1. Introduction to phase contrast microscopy (Part A) 2. Introduction to dark-field microscopy (Part B) 2. Preparation of wet mounts and microscopy comparisons (Part C) A. Introduction to the phase contrast microscope No matter how good the resolution of a light microscope or how great its magnification, an object cannot be visualized unless it has sufficient contrast with the background. As an analogy, on an overcast day it may be impossible to see a white snowshoe hare on a snowfield at 200 yards, though it would be clearly visible on bare ground. With a good pair of binoculars one can magnify the snowfield and increase the resolution several fold, but the hare remains largely invisible because contrast is still lacking. Thus, in the ordinary bright-field microscope, the image of the microbe depends on having enough contrast generated between the cell and the surroundings by the absorbance and scattering of light. Learning objectives • Describe how phase contrast and dark-field microcopy work and how they differ from bright-field microscopy. • Set up your microscope for phase contrast and dark-field observations. • Prepare a wet mount. • Compare and contrast the types of microscopy (bright field, dark field, phase contrast), when to use them, and characteristics of the image they make. • Explain how light absorption, diffraction, and interference contribute to observing specimens usingdifferent types of microscopy. • Propose steps you could take on the microscope to improve the image you see.

2 However, due to their very small size, many microbes are virtually as transparent as the surrounding fluid, and are therefore almost invisible unless stained. In microbiology and especially in bacteriology, bright-field illumination is used principally for the observation of stained preparations. Since the procedures involved in staining microbial cells often lead to distortions of size, morphology and arrangement, this method is generally not acceptable for describing the living cell. Concept check: Using dyes to stain bacterial cells make them easier to view because the dyes increase the _____________ between the cells and the background. Why is this not a good technique for describing true cell size and shape? In wide use now for observing live specimens is the phase contrast microscope, for which the Dutch physicist Zernicke received a Nobel Prize in the 1950s. This instrument enhances contrast between the cell and surrounding fluid. By comparison to the bright-field scope, contrast in the phase contrast microscope depends not just on the specimen absorbing or scattering incident light but also upon its refraction of some light. The greater the difference in refractive indices between an object and its surroundings, the greater the amount of light refracted and thus the greater the achievable contrast. With bacteria, the difference is not large, but is enough to enhance contrast significantly. Concept check: In contrast to bright-field microscopy, phase contrast microscopy depends on __________ of light. What happens when the refractive index of a material is different from the surrounding water? Figure 1. Schematic of a bacterial cell in a wet mount and 3 paths of light rays A phase contrast microscope differs structurally from a bright-field scope in two main ways: the phase scope requires that each objective lens have a phase plate cemented in its barrel and annuli (annular rings/phase annulus) located in the substage condenser. These are described below. In order to gain an appreciation for how phase contrast microscopy is achieved, a simple hypothetical model may be employed (Figure 1). Assume a small microbe is suspended in water on a slide and covered by a coverslip. Further, assume three hypothetical light paths (1, 2, & 3) which are described below.

3 Light Path 1 = Light goes through the surrounding water and will eventually focus at the phase plate in the objective lens and will appear to you eye as the bright background against which the relatively darker microbes are seen. Light Path 2 = Light passes through the microbe and will be focused on the retina as an image of the microbe which will be visible only if diminished in intensity relative to the background. In other words, some of the light passing through the specimen must be absorbed, or scattered at such an angle that the objective lens cannot capture it. Thus, less total light comes through the microbe than through the fluid around it and the organism appears darker than its surroundings. Bacteria are small so they do not absorb much light. Light Path 3 = A small portion of the light which traverses the microbe is refracted (bent), since it has a refractive index slightly different from that of the remainder of the microbe. Although the light is retarded by about ¼ of a wavelength relative to Light Path 2 rays, which go directly through the organism, it can be focused by the objective along with Light Path 2 rays but creates minimal interference with the Light Path 2 rays. This is further explained below. Enhanced contrast is achieved by subtracting Light Path 3 rays from Light Path 2 rays. This is accomplished by inserting into the optical system an annular ring (annulus) in the condenser and a phase plate at the second principal focus of the objective lens. The annular ring is an opaque disc with a transparent ring (Figure 2). Figure 2. (a) An annular ring in top view; (b) side view of the annular ring sectioned through its center. When inserted into the light path, the annulus causes the specimen to be illuminated by a hollow cone of light. Be aware that there is a solid disc of light at the plane of the specimen if the substage condenser is properly aligned. After passing through the specimen and into the objective, the light then passes through the phase plate (located at the second principal focus of the lens) which is transparent across its entire width, but which has two different areas (Figure 3). The conjugate area is a ring-shaped area through which all un-deviated light (rays from Light Paths 1 & 2) passes without being altered. The complementary area is the rest of the plate, and is constructed such that the light rays passing through it will be retarded by ¼ of a wavelength. Most refracted light, i.e., rays from Light Path 3, goes through this area.

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4 Figure 3. (a) A top view of the phase plate; (b) side view showing conjugate and complementary areas. ***Remember, in the hypothetical model, the Light Path 3 rays are retarded ¼ wavelength by the initial refraction of the specimen. When they go through the complementary area of the phase plate, they are retarded an additional ¼ wavelength for a total of ½ wavelength difference in phase. They now interfere destructively with rays in Light Path 2 to produce a reduction in the amount of light which goes to form the image of the specimen. The ray diagrams below summarize what is happening to the rays of the three hypothetical light paths. Figure 4. After passing though the specimen, rays 3 have been retarded ¼ wavelength; rays 2 are diminished in amplitude relative to rays 1 because they pass through the specimen but some of the light is absorbed. After passing through the phase plate, rays 3 are retarded an additional ¼ wavelength creating destructive interference . Concept check: Refer to Figs 1 and 4. Match each wave pattern in Fig. 4 with the three paths of light in Fig. 1. Why does the amplitude (wave height) decrease for path 2? Why is wave 3 shifted? Explain destructive interference. Figure 5. Simplified diagram of the light paths in a phase contrast microscope.

5 B. Dark-field microscopy Dark-field microscopy uses oblique illumination to enhance contrast. This is achieved using a opaque disc (often called a light stop or patch stop) in the substage condenser. Only light that is reflected by the specimen enters the objective lens. As a result, objects will appear light against a dark background. Figure 6. A comparison of light paths in bright-field (left) and dark-field (right) microscopy. Concept check: In terms of light paths, why is a specimen light against a dark background when dark-field microscopy is used? C. Preparation of wet mounts and microscopy comparisons 1. Alignment of the phase system a. Prepare a wet mount of pond water (or other water source provided) on the stage and focus with the 10X objective in place. b. Set the annular-stop turret to PH1. c. Make sure the condenser is adjusted to the appropriate height as described in Lab 1. d. Pull out one of the ocular lenses to observe the phase plate/ annular ring combination. e. If the dark ring of the phase plate does not superimpose over the bright ring of light (annulus), tell your laboratory instructor. f. Switch to bright-field (BF) and dark-field (DF) using the annular-stop turret and observe how the image changes. You will need to adjust the light with the aperture diaphragm in order to view the specimens when switching types of microscopy. g. Set the annular-stop turret to PH2 and rotate the 40X objective into place. h. Check to see if the condenser is properly aligned as described in Lab 1. i. Switch to bright-field and dark-field and observe how the image changes. You may have to adjust the condenser height slightly when using dark-field. Record an image at 400 X total magnification for each type of microscopy in your notebook. j. Switch back to phase contrast and readjust the condenser height. Place a drop of oil on the coverslip. Set the annular stop-turret to PH3 and rotate the 100X objective into place. DO NOT rotate the 40X objective into place if you lose focus. The 40X lens CANNOT go into

6 immersion oil. Record the image in your notebook. *Note that dark-field microscopy does not work with the 100X objective. k. Make wet mounts of Volvox (pigmented colonial algae, eukaryotic), Saccharomyces cerevisiae (non-pigmented yeast, fungi, eukaryotic), and your unknown bacterial strain (non-pigmented bacteria, prokaryotic). Look for actively dividing S. cerevisiae (divides by budding) and E. coli (divides by binary fission). Record the following in your notebook: 1. Volvox under 100X total magnification using each type of microscopy. 2. Volvox under 400X total magnification using either bright-field or phase contrast microscopy. 3. Saccharomyces cerevisiae under 400X total magnification using each type of microscopy. 4. Saccharomyces cerevisiae under 1000X total magnification using phase contrast. 5. Your unknown bacterial strain under 1000X total magnification using bright-field and phase contrast microscopy. D. Clean up When you have finished: • Follow instructions in Lab 1 to put away your microscope properly. Make sure your instructor checks that your microscope is clean before you put it away. • Discard slides and cover slips in the DC Gold container . • Toss transfer pipets and toothpicks in the small red biohazard . Toss paper wrappers in the normal trash . DO NOT put transfer pipets into the DC Gold! • Empty small red biohazard containers into the large red biohazard bin . • Wipe down labels on culture tubes with 95% ethanol. Place tubes in a rack on the Discard cart. • Wipe down your bench with 70% ethanol: Spray surface with ethanol, spread with a Kim wipe, and allow to air-dry. Toss Kim wipe in the normal trash. Return ethanol spray bottle to its secondary container in the back of the sink. • Follow the general lab clean-up instructions to exit the lab. E. Pre-lab questions 1. Fill in the table: Type of microscopy What we can see Background color (bright or dark?) Specimen color (bright, dark, or colored?) Brightfield Phase contrast Darkfield 2. How can you switch between different kinds of microscopy (brightfield, phase contrast, and darkfield) when you are using your microscope?

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7 F. Applying concepts 1. What type of microscopy (brightfield, darkfield, or phase contrast) would best be used to view the following specimens? Explain your responses. a. A plant cell b. A colorless microscopic worm, C. elegans. c. An unpigmented bacterial cell. 2 . What kind of light rays (for example, undiffracted, diffracted, absorbed, or oblique), or what properties of light (absorption, diffraction, or interference), are most important for creating contrast between the specimen and the background in the following types of microscopy? Explain your responses. a. Brightfield microscopy b. Darkfield microscopy c. Phase contrast microscopy

Lab 2 Introduction to Microscopy_PhaseContrast_Darkfield (1)

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