Ex1_Microscopy_S24[100].pdf

Cell Biology Laboratory S24 Exercise 1 Microscopy As per the principles of cell theory, cells are considered to be the fundamental building block of all biological life. These tiny, self-contained units possess all the necessary characteristics of life, making them critical to our understanding of t...

Cell Biology Laboratory S24 Exercise 1 Microscopy As per the principles of cell theory, cells are considered to be the fundamental building block of all biological life. These tiny, self-contained units possess all the necessary characteristics of life, making them critical to our understanding of the processes that govern living organisms. Given their pivotal role in the organization of life, it's not hard to see why the study of cells is indispensable to the study of life itself. However, since cells are too small to be seen with the naked eye, we must rely on microscopes to observe and study them in detail. Microscopy is the technical field of using microscopes to view samples or specimens. Two types of microscopes are named according to the source of illumination used: light microscopes and electron microscopes. Light microscopy employs visible light to detect small objects. It is probably the most wellknown and well-used research tool in biology. Examples of light microscopy include bright, dark, phasecontrast, and fluorescence microscopy. Fluorescence microscopy is a technique used by biologists to observe live samples in real time. This technique involves using fluorescent molecules to tag and light up target cells or cellular components, mostly proteins. However, visible light cannot distinguish between objects that are closer than 200 nanometers to each other. Therefore, fluorescence microscopy alone is insufficient to reveal the detailed structures of tiny functional components in cells known as organelles (Fleming, 2019). In contrast, electron microscopy techniques such as transmission electron and scanning electron microscopies can achieve much higher resolutions but require a vacuum and cannot be used on live samples. Although the specifics of each microscopy technique vary widely, they all involve ways of seeing the cell in greater detail than is possible by the naked eye. Scientists have their own preferences, but the goal remains the same - to gain a better understanding of the cell's structures and functions. In modern science, cell biologists have a wide range of microscopy tools at their disposal. Over the past decade, scientists have been continuously improving microscopes, resulting in remarkable advancements. For instance, the 2014 Nobel Prize in Chemistry was given to the creators of super-resolution fluorescence microscopy, while the 2017 prize recognized the development of cryo-electron microscopy (cryo-EM) (Fleming, 2019). This technique can locate individual atoms within a protein, a resolution that was previously achievable only by X-ray crystallography, which required samples to be in crystal form. The laboratory is equipped with compound, light microscopes that transmit light through the specimen on the stage and two lenses before reaching the user. The objectives of this laboratory exercise are to calculate magnification and limit of resolution using the Abbe equation, identify different cell structures, estimate the size of a specimen or cell structure, and identify various human cell types and their functions. You will receive stained prepared slides of different human cells to aid in achieving these objectives. Additionally, an article will be provided to you on the day of the lab exercise to learn and differentiate different types of microscopy and to fundamentally address how to choose the right microscope for your experiment. Procedures Materials, I and II: compound microscope, calculator, Kimwipe®, lens paper, 70% ethanol and dropper With your background in your previous courses, Principles of Biology and General Microbiology you are expected (1) to know the fundamentals of the microscope and (2) how to care for and use the microscope. However, below is a review, as the following are important when one uses a microscope. Page 1 of 5 I. Using the Microscope (a review) To ensure proper use of microscopes, there are a few important guidelines to follow. Firstly, when moving the microscope, it's essential to use both hands - one to hold the arm and the other to support the base. Secondly, only use lens paper to clean the lenses. Other materials, such as tissues, paper towels, or clothing, can potentially scratch the lenses. Thirdly, when starting and finishing use, the microscope should always be on the lowest power (scanning objective) setting. Before returning it to its designated slot, ensure the power is off, there are no slides on the stage, and the dust cover is in place. Finally, avoid using the course focus knob on high power and use only the fine focus knob for optimal image clarity. The microscope not only magnifies and resolves an object, but it also provides contrast to distinguish detail between adjacent objects. To optimize resolution and contrast, you need to understand that resolution is the ability to distinguish two closely spaced points on your specimen, and it is best when the iris diaphragm is wide open. On the other hand, contrast is the magnitude of the difference between light and dark objects, and it increases when you close the aperture of the iris diaphragm. You need to find the right balance between resolution and contrast to get the best image. You can do this by slowly opening and closing the iris diaphragm to see its effect on your image. To change the magnification of your microscope, always start with the lowest power objective (4x, scanning) to get oriented and locate an area of interest. Then, switch to higher power to examine interesting regions more closely. To change the magnification, simply rotate the nosepiece to bring one of the other objectives into the light path. When you're finished, follow these steps in order: turn down the illumination, turn off the power, switch back to the 4X objective, remove your slide, unplug the power cord, and wrap it around the base of the scope. Lower the stage to hold the cord in place and return your scope to the cabinet. II. Magnification and Resolution Background Information: When using a typical set of objectives, the magnifications available are 10x (low power), 40x (high power), and 100x (oil immersion). Most eyepieces have a magnification between 8x and 12.5x. The resolution and numerical aperture of the microscope allows for the exposure of subunits within the sample. The microscope's resolving power is determined by the limit of resolution (l.r.), which specifies the smallest distance at which two neighboring points can still be observed as separate entities. The l.r. of the naked eye is approximately 0.1mm, whereas a high-quality microscope has an l.r. of about 0.2 µm, providing a 500-fold improvement. The limit of resolution for a microscope is calculated by using the Abbe equation: l.r. = 0.61  N.A. where  signifies the wavelength of light used to view the object and N.A. signifies the numerical aperture, a measure of the cone angle of light entering the objective lens. It is evident from the Abbe equation that the limit of resolution is indirectly proportional to N.A. As a result, when N.A. increases, the resolving power of the microscope also increases. This increase in resolving power is due to the larger numerical aperture, which decreases the diffraction pattern, i.e., the fringe of dark and light rings around object borders. As the fringes become too wide, it becomes challenging to distinguish individual objects. The most superior quality objectives have an N.A. of 1.40. The lowest possible limit of resolution that can be achieved with a light microscope is when a 1.40-N.A. objective is used in conjunction with a violet filter (= 400 nm). The limit of resolution is 0.61 (400 nm) /1.40, which equals 0.17 m. By the end of the lab period, you should be able to understand and apply the meaning of the limit of resolution. 2|Page Take note of the varying magnification of each objective and of the ocular. Compute the total magnification and l.r. of each objective and enter the data on Table 1. Take note of the N.A. of each objective and compute the best limit of resolution attainable with each. Assume that a green filter ( =550 nm) is used.  Record these data on Table 1 of your lab data sheet (provided to you on the day of the lab exercise). Table 1. Calculated values for magnification and limit of resolution Magnification Total Magnification N.A. l.r. (µM) Low Power High-dry Oil-immersion Ocular ____ ____ ____ To become proficient in locating a specimen, focusing clearly, and adjusting the light for the best contrast, it is important to practice adjusting your microscope. You should begin each use of the microscope by cleaning the lenses, including the ocular, objective, and condenser lenses. Use lens paper moistened with a drop or two of 70% ethanol to effectively and safely clean and disinfect. For the objective and condenser lenses, use a drop or two of distilled water to moisten the lens paper. When cleaning the lens surface, wipe from the center to the periphery in a circular motion. This will help keep your microscope lenses clean and in good condition. https://www.olympus-lifescience.com/ It's important to keep surfaces clean, especially when they are touched frequently. Viruses can survive on surfaces like metal, glass, or plastic for hours to days, which can put lab technicians and research staff at risk. To keep everyone safe and healthy, it's important to regularly clean and disinfect your microscope. You can use disinfecting wipes available in the lab to clean the surfaces of the microscope that you will touch or have touched, even if you are wearing gloves. However, avoid using organic solvents except ethanol as they may damage plastic parts. III. The Organization of Cells In this exercise, you will examine the features common to all eukaryotic cells that are indicative of their common ancestry. Cells are not the same. Some organisms are unicellular (single-celled), with all living functions (respiration, digestion, reproduction, and excretion) handled by that one cell. Examples include Amoeba and Paramecium. Others form random, temporary aggregates, or clusters, of cells. On the other hand, multicellular organisms have large numbers of cells with specialized structures and functions, and no one cell can exist successfully by itself. Multicellular organisms are composed of groups of specialized cells, called tissues that together perform particular functions for the organisms. Tissues, in turn, may be 3|Page grouped to form organs, and organs may be grouped into organ systems. In this lab study, you will examine and identify some of the cells that compose the basic tissue types of plants and animals. Materials: compound microscope, microscope slides, coverslips, transfer pipettes, and Elodea or Red Onion, forceps; compound microscope, a box of prepared microscope slides The major characteristics of a typical plant cell are readily seen in the leaf cells of Elodea, a common aquatic plant. Elodea is an aquatic plant commonly grown in freshwater aquaria. The cell structures may be difficult to see because of the three-dimensional cell shape and a large central vacuole. (a) Prepare a wet mount and examine one of the youngest (smallest) leaves from a sprig of Elodea under the compound microscope. Use a drop of iodine to make the nucleus more visible. (b) Prepare a wet mount and examine one of the youngest (smallest) leaves from a sprig of Elodea under the compound microscope. Use a drop of Janus and 3 drops of 7% sucrose to make the mitochondria more visible. Show your results to your lab instructor! https://courses.lumenlearning.com/suny-biolabs1/chapter/the-microscope-and-cells A. Identify the following structures: o The cell wall is the rigid outer framework surrounding the cell. This structure gives the cell a definite shape and support. It is not found in animal cells. o Cytoplasm consists all of the contents of the cell, excluding the o Protoplasm is the cytoplasm plus the nucleus. o The central vacuole is a membrane-bound sac within the cytoplasm that is filled with water and dissolved substances. This structure serves to store metabolic wastes and gives the cell support by means of turgor pressure. Animal cells also have vacuoles, but they are not as large and conspicuous as those found in plants. o Chloroplasts are the green, spherical organelles often seen moving within the cytoplasm. These organelles carry the pigment chlorophyll that is involved in photosynthesis. As the microscope light heats up the cells, cytoplasm and chloroplasts may begin to move around the central vacuole in a process called cytoplasmic streaming, or cyclosis. o The nucleus is the usually spherical, transparent organelle within the cytoplasm. The structure controls cell metabolism and division. B. Estimating the Size of Objects To determine the size of the object you are viewing, you must know the distance across the field of view (the diameter of the total circular area you see when looking through the microscope). Millimeters (mm) measure distances across the field of view on scanning power, whereas micrometers (μm) are used for greater magnification. The fields of view and approximate distances across for scanning, low, and high power are as follows: 4|Page https://courses.lumenlearning.com/suny-biolabs1/chapter/the-microscope-and-cells  In your data sheet (this will be provided to you on the day of the lab exercise), Create a figure/figure1. Paste images of Elodea cells at all three magnifications. 2. Identify (use arrows) all organelles, cytoplasm, and cell membrane 3. Determine the average length of the cell at each magnification; a table has been provided for you. Remember to figure number/s and figure title/s. C. Identifying Different Cell Types Use the handout “reference ex1-C” and the slides provided to you and do your microscope observations.  Fill in the table given in your datasheet. IV. Meet the Microscopes  You will be using “BIOIMA, N., & OL, T. (2019). MEET THE MICROSCOPES. Nature, 575, S91.”, and answer several questions (refer to the data sheet provided to you) References: Fleming, N. 2019. Meet the Microscopes. Nature 575: S91-S93 Herzik Jr., MA. 2020. Cryo-electron microscopy reaches atomic resolution. Nature 587: 37-40. Thorn, K. 2016. A quick guide to light microscopy in cell biology. Mol Biol Cell. 27(2): 219–222. https://courses.lumenlearning.com/suny-biolabs1/chapter/the-microscope-and-cells/ accessed: 01/18/2023 5|Page