Lab #1 ? Introduction to the Microscopy & Observation of Prokaryotic and Eukaryotic Cells Introduction Many of the cells and organisms that you will be studying are at the lower limits of visibility of light microscopes; therefore, it is extremely important that you attain critical lighting and focussing. It is also important to handle the microscope competently to avoid damaging either the microscope or the preparation you are studying. Even students who have previously used microscopes should read the instructions carefully. Guide Biolabo Using a web rowser, go to the following web site: http://salinella. bio. uottawa. ca/biolabo/ (you can try it from home). Under Microscopy you will find links to pages that describe both type of microscopes you will use this semester, as well as how to set up and use them. It is strongly recommended that you visit these pages prior to attending your first lab. Image J / Qcapture Although you can make all your observations by watching directly through the oculars, it also can be done on the computer screen using the digital camera attached to each microscope.
For that, you will use the Image J program together with a capture plugin called Qcapture. Visit the lab website to learn how to use Image J (link on the homepage). All observations can be made on your computer screen or in the oculars. Each method has its advantages and drawbacks; you will have to choose which one it more appropriate (or the one you prefer): Oculars Screen ? Greater resolution ? Wider field of view ? Can share observation with others ? More comfortable for users ? Take pictures while observing Lab1 ? Microscopy The Compound Microscope On the Guide Biolabo page click on the CX41 Compound Microscope link then on Parts and Function. This will bring up a labelled line diagram of your microscope. Familiarize yourself with the various components shown in this figure. Then, click on Setup and Bright field alignment in order to know how to use and handle the microscope. Now, locate your compound microscope in the cupboard below the sink of your workstation. Place it on the counter between the omputer and the end of the counter. Be sure that whenever you transport the microscope, it is always kept upright; the ocular lens will fall out if the scope is tilted or swung. Even though you don’t need the dissecting microscope right now, take it out of the cupboard and install it beside the compound microscope. Connect one firewire cable to each of the cameras installed on top of the microscopes. This way, everything is setup for further observations both on your computer screen and through the oculars. Parts of the compound microscope
The microscope consists of a system of lenses, a light source, and a geared mechanism for adjusting the distance between the lens system and object being observed. There are a number of important components and it is essential that you be able to identify them and understand their function before you can proceed. By going through the different modules in Biolabo and using the microscope you will develop a competency for bright field microscopy. Identify the following components using Biolabo (Parts and functions figure) and your microscope:
REVOLVING NOSEPIECE: Supports the various objectives ? You will only use the 4x, 10x and 40x objectives in the BIO1140 labs (not the 100x). STAGE: Supports the specimen being observed. A system of knobs on the side of the stage allows you to move the specimen under the objective on the X and Y axes. Try and move the stage. COARSE FOCUS KNOB: Permits rapid change in distance between the specimen and the objective thereby allowing for rough focussing – Do not use when focusing with the 40x objective
FINE FOCUS KNOB: Permits small changes in distance between the specimen and the objective and thereby allows for final focussing of the image. 10 Lab1 ? Microscopy OCULAR OR EYEPIECE: A magnifying element in the microscope, usually 10X. It is through the ocular, or eyepiece that one looks at the specimen. All our microscopes are parfocal, so that when an object is in focus with one objective, the focus will not be completely lost when changing to the next objective. OBJECTIVES: The magnifying element which is closest to the specimen.
See figure 1 to find out about the engravings on the side of each objective. CONDENSER: System of lenses that concentrates the light furnished by the illuminator. It does not magnify the object. CONDENSER HEIGHT ADJUSTMENT KNOB: Allows one to focus the concentrated light onto the specimen. APERTURE IRIS DIAPHRAGM: Used to reduce glare from unwanted light by adjusting the angle of the cone of light that comes from the condenser; Production of Image by a Compound Microscope The most important part of a microscope is the objective.
All the other parts of the instrument are designed to help the objective produce the best possible image. The best image is not the largest; it is the clearest. There is no value to a high magnification. If the resolution is poor you will have no better understanding of the specimen. light beam ocular lens Magnification Numerical aperture (NA) Determines the resolving power of the objective* Optical tube length / max. coverslip thickness in mm prism objective lens specimen condenser lens Figure 1: Objectives engravings light source
Figure 2: Image production in a compound microscope. 11 Lab1 ? Microscopy *Resolving power is the ability to see two objects that are very close as two separate objects. The human eye will resolving power is about 100µm. Using the compound microscope Always handle the microscope GENTLY! It is an expensive, delicate and heavy instrument. Carry it with two hands, one hand on the arm, and the other hand under the base. If the ocular or objective is dirty, wipe it clean using ONLY Kimwipes or special lens tissue and cleaning fluid supplied.
If you use anything else you may scratch the lens. Wipe up any cleaning fluid immediately; otherwise it will dissolve the glue which holds the lens in place. REMEMBER, your demonstrator is here to help, so... ASK! 1. Make sure that the power cord is plugged into the back of your microscope and into a power outlet. 2. Using the letter “e” microscope slide provided, follow steps 2 through 13 in the Setup and Bright field alignment procedure of Biolabo. Remember, observation can be done on screen or through the oculars. Orientation and working distance . Starting your examination with the 4X objective, position the letter "e" slide on the stage. 2. Draw what you see in the microscope:_________________ 3. What would a slide with the letter “t” look like under the microscope? _________________ 4. Using the knobs located on the side of the stage and looking through the microscope, move the slide slowly to the right, then to the left. Record your observations. ___________________________________ 5. Now, move the slide slowly away from you, then towards you while observing through the microscope.
Record your observations ____________________________________ 6. Focus on the slide at 10X. Check the distance between the objective lens and your slide (= the working distance, see also the reference at the end of this chapter). Now switch to the 40X objective and check the working distance. What happens to the working distance as your magnification increases? 12 Lab1 ? Microscopy Depth of field (depth of focus) Lenses have a depth of focus. It is the number of planes in which an object appears to be in focus.
Extend your fist at arm’s length in front of you and hold your thumb up. Concentrate on your thumb and notice that the objects past your thumb on the other side of the room are not clearly seen. Similarly with a microscope, when it is focussed on one surface, the surfaces lower or higher will be out of focus. 1. Position a prepared slide with coloured threads upon the stage. At low power, 4X, focus on the area where the threads cross. 2. Using the fine focus adjustment, focus up and down slowly. 3. Repeat using different objectives.
What can you say about the depth of field at different magnifications? Has it increased or decreased? (i. e. , can you see more threads in one focal plane at 4X or 40X? ) ____________________________________________________________ Magnification The magnification given by objectives and oculars is engraved on them. The total magnification for any combination of objective and ocular is the product of the magnification of each lens. Objective magnification Ocular magnification Total Magnification Light intensity Working distance 4x 10x 40x High 22mm 10x 10x 100x
Medium 10. 5mm 40x 10x 400x Low 0. 56mm Table1 . Comparison magnification, working distance and brightness at three different objective magnifications. You also can calculate the magnification of your picture using the following formula: Magnification factor= measured size of object = ( X) Actual size of object 13 Lab1 ? Microscopy Specimen size and Magnification of the picture Before you start this exercise, make sure you have carefully read the website section relevant to the software you will use to take digital pictures (ImageJ/Qcapture).
The goal of this section is to teach you different techniques that will allow you to determine the size of objects you’re observing under the microscope. The general principle is fairly simple: 2 objects have the same relative size (expressed as a ratio) in the real world and under the microscope. actual size of object A = on? screen size of object A ? A1 = A2 actual size of object B on? screen size of object B B1 B2 The following exercises are applications of this formula. Place a slide under the microscope.
Choose the right objective and adjust the focus and light level. Then, choose a structure you want to measure and take a picture. A? First method: Measuring an object using the field of view (FOV): The simplest way to determine the size of an object is to use the known size of the whole field of view (FOV, the whole picture from left to right). 1? On the computer screen (using a ruler and without writing anything of the screen), measure the object of which you want to determine the size (= A2) 2? Then, measure the width of the whole picture on the screen (=B2). ? Refer to table 2 on page 20 to know the actual size of the field of view for the objective you’re using (=B1) 4? Use the following formula: Actual size of the object (A1) = Actual size of the FOV (B1) x on? screen size of the object (A2) on? screen size of the FOV (B2) Example: On a snapshot using the 4x objective, an insect has an on? screen length of 10cm. The whole picture is 20cm wide. What is the actual size of the insect? ______________________________ 14 Lab1 ? Microscopy B? Second method: Measuring an object using a scale bar file:
From Image J (using the file / open command), open the file that contains the relevant scale bar in the (T:/BIO/BIO1140): new10X. jpg for the 10x objective, and new40X. jpg (for the 4x and 40x objectives). Then, using a ruler measure the following distances directly on the computer screen: 1? The on? screen length (or width) of the object whose size you wish to determine (=A2) 2? The width of the scale bar on the screen (=B2) You now can calculate the actual size of the object using the formula: actual size of object = on? creen length of object x actual size of scale bar* on? screen length of scale bar ? A1 = A2 x B1 B2 *The actual size of the scale bar is indicated on the scale bar file (ex: on the new10x. jpg file, the bar represents 0. 2mm at 10x or 0. 02mm at 100x) = B1 Example: I took a picture of a small insect larva, using the 4x objective. The larva length is 60mm on the screen. The scale bar on the new40x. jpg is 30mm and represents 0. 2mm. What is the actual size of the larva? _________________________
Do not put the compound microscope back in the cupboard you will need it later this afternoon. Points to remember concerning microscopes 1. Always work with a clean microscope. Use only the lens paper provided. Don't forget to clean the slide too! 2. Always locate the specimen under low power and work your way up to the high power objective. 3. Never use the coarse focusing knob when the high power lens is in position. Use only the fine focus knob. 4. Never use the 100x in 1st year labs (we didn’t teach you how) 5.
Always readjust illumination whenever you change the objective. Too much light will give you a blurry image that you cannot focus on. 15 Lab1 ? Microscopy The stereoscopic microscope (dissecting microscope) The stereoscopic microscope, also called stereoscope or dissecting microscope, is used to view objects that are too large or too thick to observe under the compound microscope. Stereo microscopes are always equipped with two oculars producing a stereoscopic or three? dimensional image. Unlike the compound microscope, the image is not inverted.
Our stereo microscopes provide magnification in the range of 6. 7X ? 45X using a zoom? type lens system. By rotating a dial located on the right side of the stereo microscope head, the viewer obtains a continuous change of magnification. Our stereo microscopes can be used with reflected or transmitted light. Reflected light is directed unto opaque specimens from above and is reflected to the viewer. Transmitted light is used with translucent specimens and passes through the specimen from beneath the stage and into the viewer's eyes.
Use of the stereoscopic microscope 1. On the Biolabo home page left click on Stereoscope (Dissecting microscope) and then on Stereoscope setup. 2. Click on Step 1 and read it carefully. Obtain a stereo microscope from the same cupboard as your compound microscope if you haven’t yet. 3. Click on and read steps 2 through 7. 4. Place a coin on the stage. 5. Using the focussing knob on either side of the arm, lower or raise the objective until the coin is in focus. Examine it in both reflected and transmitted light.
Which is best for an opaque specimen? Try the various magnifications by turning the zoom knob. The reflected light source is similar to a spotlight and its orientation can be adjusted manually. Try rotating the light upwards and downwards. 6. Examine other materials such as brine shrimp larvae (Artemia) in a watch glass using both reflected and transmitted light. Add 1? 2 drops of “proto? slow” solution to slow down the larvae. Estimate the actual size of one larva: __________ 16 Lab1 ? Microscopy Prokaryotic and Eukaryotic cells
It has long been recognized that living organisms are composed of basic structural and functional units called cells. Cells can be divided into two general types: prokaryotic and eukaryotic, based on the presence of a nucleus and other membrane bound organelles in the latter. Prokaryotic cells belong to 2 big groups: archaea and eubacteria. They are usually smaller than eukaryotic cells (typically 1? 5µm). These unicellular organisms may be small, but they are the most abundant organisms on the planet, representing about half the biomass (Biology, Brooker et al. 010, McGraw? Hill&Ryerson). They are devoid of membrane bound organelle such as the nucleus, mitochondria or chloroplasts. Their genetic material is usually composed of one circular chromosome plus other extra chromosomal elements called plasmids. Eukaryotic cells are usually much larger. They possess a membrane bound nucleus, their organelles are more complex and numerous, and their genome is larger than prokaryotes. Eukaryotic organisms can be uni? or multicellular. You will have a chance to observe many eukaryotic cells during this semester: Amoeba, Lilly, Whitefish….
In today's exercise you will take a first look at the similarities and differences between prokaryotic and eukaryotic cells as well as the diversity within these groups. You should familiarize yourselves with a whole array of cellular structures and organelles you will probably encounter during the course of this exercise. Before your scheduled lab session, write down the definition and function for each of the following terms: plasma (cell) membrane, cell wall, protoplast, cytoplasm, vacuoles, nucleus, nucleolus and chloroplasts.
Eukaryotic Cells: Elodea (plant) 1? Get a young green Elodea leaf from the jar. Mount it in a drop of water on a clean microscope slide with the convex side of the leaf uppermost. Cover the preparation with a coverslip. 2? Observe the preparation at 4X, then at 10X. If you see brownish oval structures on the leaf surface, ignore then. These are probably epiphytic diatoms. Concentrate your attention on the cells near the central rib at the base of the leaf and on the marginal cells at the edge of the leaf. Can you distinguish several layers making up the leaf? ____ ? What is the average length ______ and width ______ of the cells in micrometres? 17 Lab1 ? Microscopy 3? Focussing at 40X locate the cell wall, the vacuole, the cytoplasm and the numerous green chloroplasts. ? What important biological process takes place in the chloroplasts? _____________________________________ ? What pigment is responsible for their green colouration? ________________________________________________ ? What is the shape of chloroplasts? ____________________________________________ ? Are the chloroplasts moving? What sort of movement? _________________________________________________ ? The phenomenon you are observing is called cytoplasmic streaming or cyclosis. What do you think the function of such a process could be? ___________________________________________________ 4? You have probably realised that the plasma membrane cannot be seen in plant cells. It is too thin to be resolved with the compound microscope.
In order to see the true limiting boundary of the cytoplasm it is necessary to treat the cells in such a manner that the plasma membrane becomes withdrawn away from the rigid cell wall. This can be done by placing the cell in a strong salt solution. This will cause water to diffuse out of the cell by osmosis, thereby decreasing the cell volume. The unaffected cell wall remains in its original state. What can then be seen is a space between the cell wall and the limiting boundary of the protoplast (the cell minus the cell wall) which thereby becomes visible. Remove your Elodea slide from the microscope stage. Delicately remove the coverslip, add one drop of 5% NaCl solution then put back the coverslip on your preparation ? Refocus at 40x (don't forget: you must first focus at 4X, then 10X and finally at 40x). ? Are the cells plasmolyzed? (If not wait a while longer). How do they look like now? ______________________ ? Has the cell wall been affected? _________________ ? What becomes of the large central vacuole during plasmolysis? ______ _______________________________________________ Take a picture of a plasmolyzed Elodea cell. How does it compare to the previous picture? 18 Lab1 ? Microscopy Prokaryotic Cells: Lyngbya (eubacteria: cyanobacteria) 1. Take a close look at the sample in the jar. Which colour would best describe its appearance? ___________________ 2. Prepare a wet mount of fresh Lyngbya by the following procedure: ? With forceps or an eye dropper, put a very small amount of green matter on a clean slide ? Add a drop of water from the jar. ? Carefully place a coverslip over it. Make sure it lies flat on the preparation.
Don't worry if there are just a few air bubbles. With practice, your skills will improve. However, if too many air bubbles are present, your preparation risks to dry out very quickly during viewing, compromising your observations. 3. Starting with the 4X objective, focus on your preparation. ? Can you see numerous green filaments? _______ ? Are the filaments moving? __________ 4. Switch to the 10X then the 40X objective and focus using the fine focus knob only: ? Do you see the individual cells making up each filament? ________ ? Estimate the width of one filament in micrometres:_______ What’s the filament width in millimetres (mm)? ________ ? REMEMBER: You are working with living cells. Work quickly and keep your specimen wet at all times. Dead, dry or damaged biological preparations are useless. Returning the microscopes after use After completing all observations, turn and click the low power objective (4X) on the compound microscope into position. Remove the slide from the stage and return it to its correct box. Wipe the stages with a clean paper towel. Carefully disconnect the camera from the firewire cable.
Make sure you turned off the light on each microscope, then unplug the power cord and make a loose coil of it around the eyepieces. Return the microscope in the cupboard. 19 Lab1 ? Microscopy TAs will check that you properly returned the microscopes in the cupboard with the cord properly attached and no slide present on the stage. You will lose marks for this lab (and other labs) if you don’t do so. Evaluation A short quiz on microscope components, specimen observations and measurement of objects will take place at the beginning of Lab2.
Be on time, the quiz will start at 2:30. References: 1? Metric system (see also appendix IV at the end of lab manual): 1 centimetre cm = 10? 2 metres (m) 1 millimetre mm = 10? 3 metres 1 micrometre ? m = 10? 6 metres 1 nanometre nm = 10? 9 metres 2? Size of camera field of views (fov): Table 2: Fields of View: Olympus CX41 Compound Microscope Objective 4X 10X 40X 100X Camera field of view (width in mm) 1. 75 0. 70 0. 175 0. 070 Table 3: Fields of View – Olympus SZ61TR Dissecting Microscope Zoom Setting 0. 67X 0. 8X 1X
