Lab -Microscopy Part A PDF

Lab -Microscopy Part A LABORATORY IA INTRODUCTION TO MICROSCOPY PART A OBJECTIVES When you have completed this lab, you will be expected to:  List the major branches of microscopy and briefly describe their advantages/disadvantages.  Identify the various components of a compound light microscope...

Lab -Microscopy Part A LABORATORY IA INTRODUCTION TO MICROSCOPY PART A OBJECTIVES When you have completed this lab, you will be expected to:  List the major branches of microscopy and briefly describe their advantages/disadvantages.  Identify the various components of a compound light microscope and associate each component with a function in microscopy.  Prepare a slide of a specimen.  Use a compound light microscope to magnify a specimen and bring the image into fine focus.  Define field of view, magnification, resolution, working distance, field diameter, and depth of field.  Describe the relationship between magnification and working distance, magnification and field diameter, and magnification and depth of field.  Estimate the actual size of objects viewed through the microscope. EACH STUDENT must complete the Pre-Lab Exercises AND ALL the YELLOW highlighted sections in the Lab IN THEIR OWN WORDS before coming to the lab. This includes any videos shown in this document. Answers can be found by READING the lab document. You may have a PRE-LAB QUIZ. ALSO, BRING THIS DOCUMENT TO THE NEXT LAB, ALONG WITH LAB 1B. INTRODUCTION The microscope is a fundamental tool in biological research to view a magnified image of a microscopic specimen. All microscopes rely on one or more lenses to magnify images. Three important considerations in microscopy are the degree of magnification, degree of resolution, and whether the microscope can produce a 3-dimensional image or simply a 2-dimensional image. Magnification: Magnification is the ratio of an object’s image to its real size. Expressed a factor such as 40 times (40X). Resolution: Resolution is the ability to see fine details of structure. Limit of resolution refers to how far apart adjacent objects must be in order to be distinguished as separate entities. Expressed as a distance such as 500 nm. There are various types of microscopy. Two main branches of microscopy are light microscopy, which uses visible light to produce a magnified image, and electron microscopy, which uses a beam of electrons. One major advantage of light microscopy is that it can be used to view live specimens. In the case of electron microscopy, a live specimen is killed during the procedure. However, electron microscopes have much higher magnification and resolution powers. The most powerful electron microscopes have magnification powers of 100,000X and resolution powers of 0.1 nm whereas the most powerful light microscopes have magnification powers of 1,000X and resolution powers of 200 nm. Within each of these branches of microscopy there are several types of microscopes and techniques, each with certain advantages and disadvantages leading to specific applications. Biology I Laboratory Manual Page IA.1 Lab -Microscopy Part A Light Microscopy In Part A, we will be using the compound light microscope. In Part B we will be using a stereoscopic (dissecting) light microscope in addition to the compound light microscope. Our compound light microscope offers higher maximum levels of magnification than the stereoscopic microscope (400X compared to 50X). However, the stereoscopic microscope provides a 3-dimensional view of a specimen that the compound light microscope does not. The compound light microscope is the instrument of choice when a high magnification is required (e.g. viewing a cell) whereas the stereoscopic microscope would be a useful tool when low magnification and a 3-D image are required (e.g. dissecting a small insect). The Compound Light Microscope The compound light microscope (various views illustrated in Fig. 2) is designed to pass a beam of condensed visible light through a thin slice of specimen placed firmly on the sample stage, through a series of lenses that magnify the image before it enters the eyes. Each component carries out a different mechanical function and overall function in producing a clear, sharp magnified image of your specimen. When viewing a specimen under a compound light microscope such as cells, it is common to use stains. A stain is a colored chemical that will bind to certain parts of a cell and allow you to highlight structures compared to the background by enhancing contrast. In Part B, we will be using iodine to highlight the cell wall and nucleus of onion cells. We will also be using methylene blue to highlight the cytoplasm and nucleus of our own epithelial cells (see Fig. 1). Most stains are semi-specific for certain structures or chemicals. Fluorescent-based labels can also be used with the right light microscope. These fluorescent stains can be designed to bind to specific structures and molecules in specimens. They are important tools and widely used in biology. Figure 1. Epithelial cells stained with methylene blue. Biology I Laboratory Manual Page IA.2 Lab -Microscopy Part A 1. Identify the components of the microscope in Fig. 1 with the following labels, you may use table 1 below (p.4-5) to help you. (to be completed before coming to the lab) A. ________________ B. ________________ C. ________________ D. ________________ E. ________________ F. ________________ G. ________________ H. ________________ I. ________________ J. ________________ K. ________________ L. ________________ M. ________________ N. ________________ O. ________________ Figure 2. The Nikon YS100 compound light microscope. Biology I Laboratory Manual Page IA.3 Lab -Microscopy Part A 2. Use the letter labels in question 1 to match a microscope component to the following list of functions, you may use table 1 below (p.4-5) to help you. (to be completed before coming to the lab) a) Lens system that collects light from the light source and concentrates it onto the specimen. Can be controlled to optimize resolution and contrast of the image viewed and reduce glare. _____ b) Supplies the initial magnified image of the specimen. Magnification level can be varied. _____ c) Clips a slide into place. Can be controlled to move the specimen in the x-plane. _____ d) Magnifies the image at a fixed level of 10X and puts it in a form and position suitable for viewing. _____ e) Adjusts the height of the sample stage relative to the objective to focus the specimen. Used to bring the image into coarse focus. _____ Table 1. Components of a compound light microscope and their functions in microscopy Microscope Component Function Arm (Not labeled in Fig. 2) Supports the body tube and lenses. Use the arm to carry your microscope. Base (Not labeled in Fig. 2) Supports the entire microscope. Broad and heavy, the base gives the instrument stability. (this is also where the light is located. The light intensity knob (near the light source on/off switch) can be used to adjust the light intensity at the light source.) Light Source, Light Intensity Knob, and Provides illumination of the specimen. The intensity of On/Off switch the light can be adjusted using the light intensity knob near the light source on/off switch. Oculars (Eyepieces) Further magnifies the image (10X) from the objective and puts it in a form and position suitable for viewing. Can be adjusted to fit the users interpupillary distance and correct for vision differences (except astigmatism) in the users eyes. Revolving Nose Piece Allows the user to change objective lenses without damaging them. The objectives should not be pushed or pulled to change lenses. Objective Lenses Supplies the initial magnified image of the specimen. The magnification of each objective is written on its side. Biology I Laboratory Manual Page IA.4 Lab -Microscopy Part A Mechanical Stage (Slide Holder) Clips a slide into place and allows user to move slide in the x plane (left to right) using the stage control knob. A Vernier scale tracks movement of the specimen in the x plane. Sample Stage Provides a platform for the specimen and allows user to move the specimen in the y plane (front to back) using the stage control knob. A Vernier scale tracks movement of the specimen in the y plane. The sample stage can also be moved up and down to focus (see coarse and fine focusing knobs). Coarse and Fine Focus Adjustment Adjusts the height of the sample stage relative to the Knobs objective to focus the specimen. The Coarse Focus Knob is very sensitive (in terms of the change in height as the knob is rotated) and allows for the initial focusing (only used with the 4X objective.) The Fine Focus Knob is much less sensitive and allows for fine focusing when using any objective. Condenser Collects and concentrates light onto the specimen and projects image onto front lens of the objective in place. Can be controlled to optimize contrast and resolution of the image viewed and reduce glare. Controlled using the Condenser Adjustment Lever. Condenser Adjustment Lever Controls the distance of the condenser to the sample stage. Lever downCondenser up; Lever upCondenser down. As a rule of thumb, the lower the magnification, the higher the condenser should be (i.e. the lever should be down) positioned in the optical path. Iris Diaphragm & Lever Controls the amount of light entering and leaving the condenser. This control should not be used to adjust the light intensity. Can be used to optimize contrast and resolution of the image viewed and reduce glare. Controlled using the Iris Diaphragm Lever. Exercise 1: Identification of Compound Light Microscope Components Your instructor will ask you to get a microscope from the storage cabinets. Use two hands (one holding the base and the other holding the arm) to carry the microscope to your bench. Take a moment to observe how the microscope was put away and the note the number of your microscope. Do not remove the plastic tie used to wind-up the excess power cord. You will be asked to complete a sign-out sheet. This microscope will be the one you take for all subsequent labs that require a microscope. Biology I Laboratory Manual Page IA.5 Lab -Microscopy Part A Once you have your compound light microscope in front of you, try to identify the various parts listed in Table 1. Determine what the various controls are doing mechanically and think about how this plays a role in light microscopy. Exercise 2: Using your Compound Light Microscope to View a Specimen PART OF PRE-LAB: how to focus on a specimen, go to: https://www.youtube.com/watch?v=SUo2fHZaZCU In this exercise, you will learn how to correctly set-up your microscope, prepare a specimen on a slide, and produce a sharp magnified image of your specimen. This exercise will also allow you to observe the way in which the lenses of the compound light microscope operate. Exercise 2-1 Setting-up your microscope and finding your field of view: 1. Put the 4X objective in place. Rotate the 4X objective using the revolving nosepiece, ensuring that the objective clicks into place. DO NOT ROTATE OBJECTIVES BY GRABBING ONTO THE OBJECTIVES THEMSELVES. THIS WILL BEND AND DAMAGE THE OBJECTIVE. 2. Turn on the light source. Raise the condenser by pulling down on the condenser control level in the down position. 3. Look through the oculars and adjust the intensity of light to a comfortable level. 4. Adjust the distance between the oculars to accommodate your interpupillary distance (distance between the two eyes). Adjust the distance by using the baseplate of the oculars (as illustrated in Fig. 2) until you see only one circle of light when using both eyes. This circle of light is your field of view. While you are adjusting the microscope, try varying how close your eyes are to the ocular lenses. Figure 2. Adjusting the microscope for interpupillary distance. Biology I Laboratory Manual Page IA.6 Lab -Microscopy Part A Exercise 2-2 Preparing a specimen on a slide: 1. Cut the letter “e” out of the paper provided. 2. Using forceps, place it right side up on a clean microscope slide. 3. Place a drop of water near the letter on the slide. 4. Lower a coverslip carefully so that one end touches the slide near the letter at roughly a 45- degree angle. Drop the other end of the coverslip onto the specimen using your finger or a toothpick (illustrated in Fig. 3). Using a toothpick will avoid adding oil to the coverslip or contaminating your specimen. The water should spread evenly over the paper specimen. This procedure minimizes the number of air bubbles caught under the coverslip. If any bubbles obscure your view of the letter, gently tap the coverslip with the forceps to force them out. 5. If the slide only has a portion of the specimen submerged in water, you can add more water by placing a drop of water on the edge of the coverslip near the region without water. The water will be sucked under the coverslip by capillary action. This procedure can introduce air bubbles however. Ask your instructor for assistance to avoid introducing air bubbles. Figure 3. Preparing a specimen on a slide. Biology I Laboratory Manual Page IA.7 Lab -Microscopy Part A Exercise 2-3 Viewing your specimen at scanning (lowest) magnification: Scanning magnification is the lowest magnification. It allows a user to view the specimen and identify an area to further magnify. N.B. Whenever magnifying a specimen, you should start by creating a sharp image at the scanning magnification. Even if you intend to magnify at the highest level, you should always start with the scanning magnification first. In addition, if you are viewing an image at the highest magnification level and you lose your image for whatever reason, you should always return to the scanning magnification and start again. 1. Check that your microscope has the 4X objective in place and that the condenser is up close to the stage (lever down). Bring the sample stage down to its lowest position. 2. Place your slide on the sample stage and use the mechanical stage to clip in into place. Use the stage control knobs to move the specimen around in the X-Y plane. Place the specimen in the path of light coming from the condenser. 3. While looking through the oculars, raise the specimen using the coarse focus knob until you get a rough outline of the “e” shape. Use the fine focus knob to sharpen the image. 4. Use the stage control knobs to center the image of the “e” in the field of view (i.e. move the specimen in the x/y plane, x=horizontal, left to right; y=vertical, forward/up and backward/down). 5. Use the iris diaphragm lever to adjust resolution and contrast. As a result of the lens system inside a compound microscope, the orientation of the image in the field of view (as seen through the oculars) appears differently than the actual orientation of the specimen. 6. Draw the orientation of the actual specimen and the magnified image you see below. Actual Image 7. Move the specimen in the x-y plane and determine how the movement of the image compares to the movement of the specimen. 8. Complete the Lab Report section for this exercise. Biology I Laboratory Manual Page IA.8 Lab -Microscopy Part A Exercise 2-4 Further magnifying your specimen: 1. Put the 10X objective into place using the revolving nosepiece and bring the condenser down (lever up). 2. If you have not touched the focusing knobs since having a sharp image in Exercise B-3, the image should be in coarse focus. The microscope objectives are designed to have different lengths that will roughly be the appropriate distance between the specimen and lens for the specimen to be in focus. Do not use the coarse focus knob at this point! The distance between the specimen and the objective is too close and you may hit the objective lens against the specimen. Sharpen the image using the fine focus knob. Only use the coarse focus knob when the scanning (4X) objective is in place. If you are having trouble remembering which is the coarse and fine controls, the right hand side only has a fine focus control knob. 3. Use the iris diaphragm lever to adjust resolution and contrast. 4. Repeat the process for the 40X objective. N.B. As you magnify your specimen you are zooming into a smaller, smaller section of your specimen. If you are having trouble seeing black ink in this exercise as you magnify your specimen, you may have to move your specimen in the X-Y plane using the stage control knobs to make sure that the ink is in your field of view. 5. Complete the Lab Report section for this exercise. Exercise 2-5 Calculating total magnification: The total magnification is the ocular magnification, which stays constant (ex. 10X) multiplied by the objective magnification (ex. 4X on the lowest objective). Calculate the total magnification when each of the objectives is used and complete the Lab Report section for this exercise. Exercise 3: Relationships between Magnification and other Variables In this exercise, you will explore the relationship between magnification and other variables in microscopy. Exercise 3-1 Relationship between magnification and working distance: The working distance is the distance between the top of the specimen and the lens of the objective. In a compound microscope, the working distance changes inversely with the magnification of the objective used. 1. With the ruler provided, estimate the working distance for the smallest objective when your specimen is in place (see fig. below, p.10). Notice that the working distance changes as you use different objectives. 4X _________ mm Biology I Laboratory Manual Page IA.9 Lab -Microscopy Part A 2. Complete the Lab Report section for this exercise. Working Distance Exercise 3-2 Relationship between magnification and field diameter: The field diameter is the actual diameter of the area viewed on the slide. Although the diameter of your field of view does not change, the field diameter does as you increase the magnification. A quick and easy way to measure the field diameter is to simply use a ruler, which can be viewed when the working distance is large enough to accommodate the width of the ruler. This can only be done using the 4X objective. You can then use a formula to calculate the field diameter when using the other objectives. 1. Bring the sample stage down, remove your slide from the microscope. Do not dispose of your specimen, you will be using it again. Put it in a safe place. Put the 4X objective in place. Place the ruler (cut to the dimensions of a slide) on the microscope and clip it into the mechanical stage. DO NOT USE THE RULER FOR ANY OF THE OTHER OBJECTIVES!!!!! 2. Bring the ruler markings into fine focus. Move the ruler until one side is bisecting your field of view. 3. Move the ruler in the X-plane so that one of the graduation lines is at the edge of your field of view (as seen in the figure below, p.11). Count the number of millimeters you can see. Estimate the fraction of the last millimeter. _________ mm Biology I Laboratory Manual Page IA.10 Lab -Microscopy Part A 4. Use the following formula to calculate the field diameters at the higher magnifications. Formula: Total mag.1 x Field diameter1 = Total mag.2 x Field diameter2 5. Put your specimen back into the microscope and bring it into fine focus using the 4X objective. Use the field diameter PREVIOUSLY measured at the 4x magnification (#3 above) to estimate the diameter of the “e”. DO NOT USE THE RULER!! 6. Remove the slide and dispose of it in the designated disposal bin. 7. Complete the Lab Report section for this exercise. Exercise 3-3 Relationship between magnification and depth of field: One of the challenges in using the compound light microscope is to extract a three-dimensional shape from the two-dimensional images generated by this microscope. Veteran compound light microscope users constantly move the sample stage up and down using the fine focus adjustment knob when observing a specimen to determine which parts are on the top, middle, and bottom. By doing this, the observer can look at multiple slices of the specimen and build a 3-D image of the specimen. This highlights the relationship between magnification and depth of field and building a 3-D image of a specimen. In a slide with multiple levels/structures, using the highest magnification and, therefore, the thinnest focal plane (lowest depth of field), you will be able to slowly raise (using only the fine focus) the out-of-focus sample stage from a position where the whole sample is out of focus and determine which level/structure is on top (the first one that comes into sharp focus), which is in the middle (the second to come into sharp focus), and which is on the bottom (the third to come into sharp focus). Accomplishing this at the highest magnification is easiest because you are looking at the thinnest slice and will spend more time in each individual level as you slowly raise the sample stage using the fine focus knob. Biology I Laboratory Manual Page IA.11 Lab -Microscopy Part A Exercise 4: Putting Away your Microscope and Cleaning your Bench Area Since many people will be using these microscopes, it is good lab etiquette to put a microscope (or any common equipment) back clean and in a correct manner. In addition, these instruments contain many fragile components, so putting a microscope back properly will avoid damage. 1. Turn the light intensity knob to its lowest position and turn off the light source. Unplug the microscope. 2. Bring the sample stage all of the way down and remove your slide. 3. If your microscope has a slot in the revolving nosepiece where no objective is installed, put this blank slot into place. Otherwise, put the shortest (scanning, ex. 4x) objective into place. 4. Move the oculars close together and center the mechanical stage. 5. Look for any obvious areas to clean up (e.g. spilt stain on the sample stage) and use water and a tissue to clean up any messes. Use the lens cleaner and lens tissues provided to gently clean the objective and ocular lenses. 6. Put the wound up region of the power cord around one of the oculars and replace the plastic bag over the microscope. 7. With two hands, bring the microscope to the storage cabinets and put it back with objectives and oculars facing the BACK of the cabinet. Make sure to put the microscope back in the space dedicated for your specific microscope (match the numbers on the microscope and storage area in the cabinet, i.e. if the number on the back of your microscope says 10, put it in slot 10 of the cabinet). 8. Make sure you clean up your bench area and station. Put all materials back to where you got them from and clean your lab bench with water from the sinks at the side benches and paper towels. 9. Marks may be deducted for improper microscope storage!!! Biology I Laboratory Manual Page IA.12 Lab -Microscopy Part A NAMES:________________________________ LAB SECTION: ______ Lab 1: Introduction to Microscopy Part A Exercise 2-3 Viewing your specimen at scanning (low) magnification: 1. How does the orientation of the image compare to the actual orientation of the specimen on the sample stage? ________________________________________________________________________________ 2. When the slide is moved to the right, the image in the field of view moves: _______________. 3. When the slide is moved away from you, the image in the field of view moves: ________________. Exercise 2-4 ANSWER BEFORE COMING TO THE LAB Further magnifying your specimen: 1. In what position should the condenser be when viewing a specimen with the 10X and 40X objective? ________________________________________________________________________________ ________________________________________________________________________________ 2. Why shouldn’t you use the coarse focusing knob when using the 10X or 40X objective? ________________________________________________________________________________ ________________________________________________________________________________ Exercise 2-5 ANSWER BEFORE COMING TO THE LAB Calculating total magnification: Table Exercise 2-5. Total magnification when using the various objectives of our microscope. Objective Mag. Ocular Mag. Total Magnification 4X 10X 40X Exercise 3-1 ANSWER BEFORE COMING TO THE LAB Relationship between magnification and working distance: Describe the relationship between magnification and working distance. ________________________________________________________________________________ _______________________________________________________________________________ Biology I Laboratory Manual Page IA.13 Lab -Microscopy Part A Exercise 3-2 Relationship between magnification and field diameter: 1. Describe the relationship between magnification and field diameter. ________________________________________________________________________________ ________________________________________________________________________________ 2. Complete the following table. N.B. List field diameters in Micrometers (um). (1 mm = 1000 um) Table Exercise 3-2. Field diameter when using the various objectives of our microscope. Objective Field Diameter (um) 4X 10X 40X 3. Estimate the diameter of the “e” specimen ________ um Explain how you calculated this diameter. NOTE: DO NOT DIRECTLY MEASURE THE ‘E’ USING A RULER! ________________________________________________________________________________ Exercise 3-3 ANSWER BEFORE COMING TO THE LAB Relationship between magnification and depth of field: 1. Describe the relationship between magnification and depth of field. ________________________________________________________________________________ 2. Why is this exercise best done at the highest magnification? ________________________________________________________________________________ ________________________________________________________________________________ 3. How can you use a compound light microscope to build a 3-D image of a small specimen at high magnification? ________________________________________________________________________________ ________________________________________________________________________________ Biology I Laboratory Manual Page IA.14 Lab -Microscopy Part A NAME:________________________ Student #: ______________ LAB SECTION:______ Lab 1: Introduction to Microscopy Part A Pre-Lab Exercises: MUST BE COMPLETED BEFORE COMING TO LAB Exercise 1: microscopy 1. What are the two main branches of microscopy? __________________________ and __________________________ 2. What is the main advantage of each branch? ________________________________________________________________________________ ________________________________________________________________________________ 3. What type of microscope will we be using during this lab period? __________________________ 4. What is the major advantage of this type of microscope when compared to a stereoscopic light microscope? ________________________________________________________________________________ ________________________________________________________________________________ Exercise 2: Size Estimation 1. ESTIMATING THE SIZE OF MICROSCOPICALLY-SMALL OBJECTS The table below provides the approximate diameter of the visual field for each objective lens. Complete the chart converting millimeters into micrometers. Then use the chart to estimate the length of a line (below). Name of Magnification Magnification Total Field Field Diameter Objective Lens of Objective of Eyepiece Magnification Diameter mm μm Low power 4X 10X 40X 3.5mm 3500μm Medium power 10X 10X 100X 1.4mm μm High power 40X 10X 400X 0.35mm μm Oil immersion 100X 10X 1000X 0.14mm μm Biology I Laboratory Manual Page IA.15 Lab -Microscopy Part A Example: With the medium power objective (10X), the diameter of the visual field is 1400 μm. You can estimate the size of an object in the visual field by comparing it with the total diameter of the visual field. Since line AC is approximately half AB, AC=700μm (i.e. 1400 μm ÷ 2) Line AD is half the length of AC, therefore line AD in micrometers = ___________________ The object X in the medium power field below has a diameter approximately the same as (AC or AD?) ______________ Therefore X has an approximate diameter of ______________ micrometers. Biology I Laboratory Manual Page IA.16 Lab -Microscopy Part A Exercise 3: Putting away your microscope True (T) or False (F) 1. Microscopes can be put away in any available shelve. _____ 2. Never place a microscope back into the cabinet with a slide in place. _____ 3. Microscopes should be placed in the cabinet with the objective lenses facing out. _____ 4. Microscopes should be put away with the 40X objective in place. _____ 5. The sample stage should be brought all the way down before storage. _____ Biology I Laboratory Manual Page IA.17