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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Workshop 2.1 – Microscopy & Data Analysis Description and learning objectives In this workshop chapter you will learn how to use one of the most important tools in biology, the microscope.As the vast majority of species are microscopic, proper use of the microscope is critical in studying biology. You will also learn how to prepare images based on your observations, measure specimens and write concise descriptions of the specimens you observe; all of these are important for data presentation in the following workshops and in other courses. Specifically, you will:

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learn proper microscope setup and use. learn the relationship between magnification and resolution. capture images of specimens; present and describe microscope specimens in scientific format.

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use comparative scientific methods to describe results from microscopy experiments. perform statistical analysis on data sets of different species.

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Workshop Readings • Campbell Biology Chapter 6: Concept 6.1 Microscopy • Appendix 1: Word Processing • Appendix 2: Data Presentation and Analysis • Appendix 3: Scientific Reports and Referencing • Appendix 4: Electronic Submission of Assignments Videos and Internet Materials • Bionetwork virtual microscope: http://www.ncbionetwork.org/iet/microscope/ • Bionetwork microscopy for beginners: Click here. • How to calculate magnification: https://www.youtube.com/watch?v=FdaLMkoHF2o • Calculating magnification: https://www.youtube.com/watch?v=brb-Qy7KCYc • Calculating magnification (scale bars): https://www.youtube.com/watch?v=JBJdxRRmGeU • Using an ocular micrometer to measure size: https://www.youtube.com/watch?v=EIFVHbsRzts • Depth of focus (depth of field): https://www.youtube.com/watch?v=kJZh9wY37UM • • • 1

What is statistics: https://www.youtube.com/watch?v=sxQaBpKfDRk&list=PL8dPuuaLjXtNM_YbUAhblSAdWRnmBUcr&index=2 Running a t-test in Excel: https://www.rwu.edu/sites/default/files/downloads/fcas/mns/running_a_t-test_in_excel.pdf Hypothesis testing and p-values: https://www.youtube.com/watch?v=bf3egy7TQ2Q

Refer to the section Enabling Flash at the end of this document.

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BIOL 1103

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Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Null Hypothesis Statistical Testing: https://www.youtube.com/watch?v=YSwmpAmLV2s

Marked work associated with this Workshop session • •

Refer to Table A in the General Laboratory Introduction for the value of this assignment towards your final lab mark. Complete the Lab 2 Workshop Assignment and submit it electronically via Brightspace before the deadline. IMPORTANT: This is the workshop with the greatest associated workload (most manual chapters) this term. It is strongly recommended that you DO NOT leave it until the last minute!

NOTE: Although you may discuss these questions communally, you must write and submit all your work individually. Write all answers in your own words and do not copy from the course materials, internet pages or from each other. DO NOT plagiarize!

Introduction In this workshop you will focus on the first two elements of the scientific method: you will make observations using the comparative scientific approach and ask questions based on your observations of different types of cells. These observations will be made using one of the most important technical instruments that allowed microorganisms to be viewed and investigated – the microscope. You will analyze and interpret your observations in order to obtain answers to biologically-relevant questions. This will begin your journey to becoming a good scientist and an effective scientific communicator. Light Microscopy Much of the diversity in nature is too small to be seen with the unaided eye. It was not until the mid1600s that cells were first seen by Robert Hooke and Antonie van Leeuwenhoek using the earliest microscopes. Today, there are a number of different types of microscopes. One of the easiest to use is the compound light microscope which uses visible light to illuminate the specimen and has two or more lenses2 to bend and focus the light to produce a magnified image of the specimen that is viewed through the eyepiece. For example, compare the observable detail for fruit flies (Drosophila melanogaster) with and without magnification (Figure 1). Figure 1: Fruit fly (Drosophila melanogaster) images without magnification (left) and magnified 60X (right). 2 The word ‘lens’ is derived from the Latin word for lentil as the shape of early devices used to magnify objects were shaped like lentil beans.

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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Electron Microscopy The more powerful electron microscope uses electrons to create the image of the specimen and electromagnets to bend and focus the electrons to produce a magnified image which is viewed on a digital screen. In scanning electron microscopes (SEMs), the image of the object is produced by sending a high-energy beam of electrons to scan the surface of an object. These electrons are deflected by atoms of gold or other heavy metals embedded in the surface to produce a signal that represents the surface topography of the object (Figure 2). Transmission electron microscopy (TEM) is similar to

Figure 2: SEM of an insect eye (Carleton University).

light microscopy, where the interactions of electrons and the surface of the object are captured on a fluorescent screen. As resolution depends on the wavelength of light, the effective resolution limit for the light microscope is about 0.2 μm. As electrons have a much smaller wavelength than light, and have similar properties as light in a vacuum, they can be used to visualize objects in the same way as a light microscope. The effective resolution for an electron microscope is about 0.05 nanometres (nm) or 5 x 10-5 μm. If you are interested, there is a tutorial on virtual scanning electron microscopy (click here) that will allow you toadjust the settings (e.g., magnification, brightness, contrast & focus) for various specimens. Factors Affecting the Image There are three main parameters of microscopy that affect our ability to see objects using a microscope: magnification, resolving power, and contrast. Magnification is the ratio of the size of an image (drawing or photograph) to its actual size and is determined by the lenses found in the eyepieces and objectives of the microscope.

Most light microscopes have a 10X lens in the eyepiece and a 4X, 10X, 40X, and 100X lens in each of the objectives (the 4X objective is a scanning lens that is rarely used). The final magnification of the object, as seen through the eyepiece of the microscope, is the product of the two lenses (i.e., 1000X using the 100X objective). This type of microscope is called a compound microscope as two sources of magnification are used. A magnification of 1000X is the maximum effective magnification for most light microscopes; beyond this magnification, resolution decreases.

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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Resolution is the minimum distance between two points that still allows the two points to be distinguished as separate. The resolution of the human eye is about 0.2 mm or 200 µm (micrometers). Objects closer together than 0.2 mm cannot be resolved as separate objects. The resolution of the human eye can be improved by using a microscope where the resolution depends on both the wavelength of light (λ) used to illuminate the specimen and the numerical aperture (NA) of the objective lens of the microscope.

The resolving power (resolution) improves as the wavelength of light decreases. Most microscopes are fitted with a blue filter which will produce light with a wavelength of 500 nm (nanometers). An electron microscope can resolve much smaller structures than a light microscope as the electrons used in electron microscopy have a wavelength of only 0.0025 nm. The resolving power of a microscope also improves as the NA increases. The NA is a measure of the light gathering capabilities of a lens and increases in value from the 10X to the 100X objective lenses. As a result, the resolution also increases with the higher magnification objectives. Contrast is the last parameter of microscopy that affects your ability to see structures with the microscope. Contrast can be defined as the accentuation of differences in the sample. In light microscopy, a desired contrast can be achieved by adjusting the iris diaphragm or using a dye to stain the specimen. This allows details of the specimen to be viewed due to differences in density or colour. Other Factors The specimen viewed in a microscope is always on a glass slide, which is illuminated by light from the light source below the stage. As the light passes through air, the glass slide and air again the light is refracted (bent) rather than continuing directly to your eye. Refraction occurs when light passes between materials of different densities. For example, consider the image of a green straw in water (Figure 3). When light crosses the boundary between water and air, it is refracted. The light reflecting the image of the portion of the straw above the water goes directly to the viewers’ eye. Light from the image of the straw portion below the water refracts as it enters the air and appears to come from a different direction. The refracted light is lost and the image becomes difficult to see. Refraction can be reduced by eliminating the air between the glass side and objective lens with a material of the same density. For high magnification lenses (e.g., 100X) this material Figure 3: Refraction of light. is an oil referred to as immersion oil (as the objective lens is immersed in the oil drop). The numerical 4

BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

aperture of a microscope lens is affected by the refraction index of the material between the specimen and the lens. For a more detailed explanation of refraction, and how different materials refract light to different degrees, see the Refractive Index page at MicroscopyU by Nikon. As light moves from air into a glass microscope slide supporting the specimen and then out again towards the objective lens, it is bent or refracted due to the difference in the refractive index (RF) of air and glass. The degree to which light is refracted affects the ability of the objective lens to capture the light. Because of this phenomenon, the only way to maintain good resolution when using the 100X Figure 4: Refraction due to air and immersion oil. objective is to use immersion oil, which has a RF equal to that of the glass slide, to replace the air between the slide and the lens. This prevents refraction of the light as it passes out of the slide enabling the objective lens to collect more light (Figure 4). Depth-of-field (DOF) is the vertical distance that remains in focus at any one time. DOF decreases as magnification increases such that the horizontal section (optical section) of the specimen that you see is greatest at low magnification and least at high magnification. If you were focussed on the black line in Figure 5, the DOF would be between the blue dashed lines; any part of the specimen outside the DOF would be unclear. However, note that the focus is on the black line and parts of the specimen away from this focal plan may be out of focus. For example, if you were to observe a prepared slide with three threads of different colours (blue, red & yellow) in the microscope the intersection point where the three threads cross is beyond the DOF and thus only one thread is in focus (Figure 6). Which thread is in focus in Figure 6? Only the fibres

Figure 5: Depth of field.

of the yellow thread can be resolved; the yellow thread is therefore the one in the focal plane. If the focus is changed to view the detail of another thread, then the other two coloured threads would be out of focus.

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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

As magnification changes, the working distance of the objective lens changes, as does the field diameter (field ofview) (Figure 7). As the light intensity is reduced at higher magnifications, the light intensity is generally set below full power at lower magnification and increased to full at higher magnification. Since the field diameter or field of view is smaller with increased magnification, it is important that your specimen is centred in the field as when you increase the magnification (i.e., by switching to a higher power objective lens), you may be unable to locate the specimen as it is outside the field of view. Parts of the microscope (Figure 9)

Figure 6: Three thread slide image.

Ocular lens or ocular (eyepiece) collects and magnifies the image initially formed by the objective lens. The engraved value (e.g., 10X) indicates magnification; the w.f. means wide field of vision. Binocular microscopes have two eyepieces. The distance between the two ocular lenses can be adjusted to fit the distance between your eyes. The left ocular can be focusedto adjust for astigmatism by rotating it.

Compound microscopes, like the one you will use, have at least two lens systems in the optical train. The first lens system is in the objective and produces a real, inverted and magnified image of the object. This primary image is then magnified further by a second lens system in the eyepiece. As a result, the image appears backwards when you are viewing it – moving the specimen (i.e. the slide) to the right using the stage control knobs will cause the image to move to the left. Viewing head supports the ocular lenses. The head is held in place by the viewing head clamp screw. When this screw is loosened you can rotate the head without moving the microscope on some models. Figure 7: Comparison of field diameters, light intensity and working distance for the objective lenses of a Leica microscope.

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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Coarse focus adjustment knob allows you to bring the specimen into focus when working with the 10X objective by moving the stage up or down to change the distance between the specimen and the objective lens. Because the movement of the stage is very rapid, the course focus knob should be used only with the 10X objective, never with the 40X or 100 X objectives (this is important in order to avoid breaking the slide and/or damaging the objective). The course focus knob should NOT be used when placing a slide on, or removing a slide from, the stage. Fine focus adjustment knob is used for the final focusing of the image when using the 10X objective and for fine focusing after changing to the 40X or 100X objectives. Revolving nosepiece supports the objective lenses. By rotating the nosepiece, you can change objective lenses and thus change magnification. Always grasp the ring, not the lenses, otherwise you will loosen the lenses and focusing will be impossible and the lenses may fall out. Objectives contain magnifying lenses producing the first image of the object viewed. Most microscopes have three or four objectives, each providing a different degree of magnification: 10X, 40X and 100X. Each objective has an engraved set of numbers consisting of the magnifying power (e.g., 10X) and the numerical aperture (e.g., 0.25 for the 10X lens) (Figure 5). The 100X objective lens is an oil immersion lens as indicated by O.I. (oil immersion) on the Figure 8: Engravings on a 10X objective lens. lens barrel. In order to obtain the expected resolution, you must use immersion oil when using this objective. However, immersion oil cannot be used with the lower powered (10X and 40X) objectives. The objective lenses of your microscope are parfocal, meaning that once the image has been focused using a lower power objective, only minor adjustments with the fine focus should be required to bring it into focus upon switching to the next higher power objective. Mechanical stage is a moveable platform with spring-loaded clips to hold, and move, the slide. Always place the slide between the spring-loaded clips NOT under or on top of them. The mechanical stage control knobs are located below the stage and move the slide left and right or forward and backwards. Condenser is a set of lenses that focuses the light through the specimen into the objective lens. Ensure the condenser is all the way up each time you begin a microscope session. A misaligned condenser will result in an unclear image. The condenser is held in place by the condenser clamp screw (do not loosen this screw or the condenser may fall out). The condenser focusing knob, located below the stage, is used to raise or lower the condenser. It is important to remember not to use this control to adjust light intensity; changing the condenser position causes improper specimen illumination.

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BIOL 1103

Workshop 2.1 – Microscopy & Data Analysis

Fall 2021

Condenser (iris) diaphragm lever (ring) is located on the condenser and is moved horizontally to adjust the amount of light passing through the specimen. It should be fully open. It is frequently used to heighten the image contrast. Some microscopes may also have a filter holder that holds a blue filter. Do not use condenser lever control to adjust light intensity; use the light intensity or brightness control knob. The source of light, the lamp, varies with different microscopes. The light should be fairly bright (although not so bright that you are unable to see) for good resolution; dim light distorts the image. NOTE: Only use the light intensity control to adjust light intensity. Do not adjust the iris diaphragm. Regardless of how thin a specimen is, it is still three-dimensional and, therefore, has depth. When using the microscope only a thin slice of a specimen (called the optical section) will be in focus at any one time. The thickness of that slice is the called the depth of field. As the depth of field decreases with higher magnification objectives, less of the specimen will be in focus. However, if you carefully focus up and down with the fine focus knob you can mentally construct a three-dimensional picture from the series of optical sections.

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BIOL 1103

Workshop 3 – Microscopy & Data Analysis

Figure 9: Parts of Olympus (left) and Leica (right) compound light microscopes.

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Fall 2020

BIOL 1103

Workshop 2.1 – Microscopy

Summer 2021

Calculating Specimen Size and Magnification To determine the actual size of the specimen you are viewing, you need a microscopic ‘ruler’ called an ocular micrometer. This is a glass disc in a special ocular lens containing a scale that is divided into small units like a ruler (Figure 10). This “ruler” is superimposed over the image of the specimen you see using the microscope. You can rotate the ocular micrometer to position the scale across or alongside the specimen you wish to measure in the field of view and then determine the number of ‘ocular units’ of the specimen you are viewing. One ocular unit is the distance between the smallest divisions of the micrometer scale. Figure 10: Eyepiece ocular micrometer. Ocular micrometers are calibrated to determine the actual size of each ocular unit for each objective lens by placing a slide with a scale of known length on the stage and superimposing the ocular micrometer scale over this segment to determine unit measurements (Figure 11). In this case the stage micrometer has a length of 1 mm and each unit of the eyepiece micrometer is 0.01 mm. Knowing this conversion factor allows you to convert ocular units into real Figure 11: Eyepiece ocular micrometer (top) and stage...