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3.1D: Magnification and Resolution - Biology

3.1D: Magnification and Resolution - Biology


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Magnification is the enlargement of an image; resolution is the ability to tell two objects apart.

Learning Objectives

  • Define magnification and resolution

Key Points

  • Magnification is the ability to make small objects seem larger, such as making a microscopic organism visible.
  • Resolution is the ability to distinguish two objects from each other.
  • Light microscopy has limits to both its resolution and its magnification.

Key Terms

  • airy disks: In optics, the Airy disk (or Airy disc) and Airy pattern are descriptions of the best-focused spot of light that a perfect lens with a circular aperture can make, limited by the diffraction of light.
  • diffraction: the breaking up of an electromagnetic wave as it passes a geometric structure (e.g., a slit), followed by reconstruction of the wave by interference

Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called “magnification. ” The term magnification is often confused with the term “resolution,” which describes the ability of an imaging system to show detail in the object that is being imaged. While high magnification without high resolution may make very small microbes visible, it will not allow the observer to distinguishbetween microbes or sub-cellular parts of a microbe. In reality, therefore, microbiologists depend more on resolution, as they want to be able to determine differences between microbes or parts of microbes. However, to be able to distinguish between two objects under a microscope, a viewer must first magnify to a point at which resolution becomes relevant.

Resolution depends on the distance between two distinguishable radiating points. A microscopic imaging system may have many individual components, including a lens and recording and display components. Each of these contributes to the optical resolution of the system, as will the environment in which the imaging is performed. Real optical systems are complex, and practical difficulties often increase the distance between distinguishable point sources.

At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks (or, in other words, the ability of the microscope to distinctly reveal adjacent structural detail). It is this effect of diffraction that limits a microscope’s ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by the wavelength of light (λ), the refractive materials used to manufacture the objective lens, and the numerical aperture (NA) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field. This is known as the diffraction limit.


Difference between Resolution and Magnification in tabular form

Resolution and magnification are two terms used in Optics which are related to each other. The Basic difference between resolution and magnification is that Resolution is the ability to separate two closely placed objects while magnification is the mean of increasing the size of the object.


Talk Overview

The resolution of a microscope can be defined as the smallest distance at which two small objects can still be seen as separate objects. This lecture discusses various criteria for resolution, the factors that influence resolution in the lateral and axial planes, and how to sample an image adequately using a camera or confocal microscope, such that the full optical resolution is retained.

Questions

  1. You have a 100ࡧ.4na objective, no additional magnification in your microscope and can choose between 4 different cameras, varying in pixel size. Which camera should you choose to fulfill the Nyquist sampling criterium, assuming that you resolution limit is described by the Rayleigh criterium and you work with light of 500nm?
    1. 16 micron pixels
    2. 10 micron pixels
    3. 6.5 micron pixels
    4. 4.0 micron pixels
    1. 64吼 mm
    2. 128𴡘 microns
    3. 64吼 microns
    4. 40吤 microns
    1. Higher wavelength
    2. Higher refractive index of immersion medium
    3. Lens with higher numerical aperture
    4. Lens with lower numerical aperture
    5. Lens with better chromatic correction

    Answers

    1. B (or C): Rayleigh criterium: 0.61* lambda/NA -> 0.61 * 500 / 1.4 = 218nm. 100x magnification leads to 21.8 micron at the camera plane. Nyquest sampling: 2 pixels per resolvable element -> 21.8 /2 = 10.9 micron pixels. Camera B (10 micron pixels) will satisfy Nyquist, camera C will give 3 pixels per resolvable element.
    2. C: Rayleigh criterion: 0.61 * 575 / 1.4 = 251 nm. Nyquist: 2 pixels per resolvable element > 125nm per pixel. 512𴧘 pixels > 64吼 microns
    3. B and C

    Magnification & resolution in light & electron microscopy (Edexcel A level Biology B)

    A Science teacher by trade, I've also been known to be found teaching Maths and PE! However, strange as it may seem, my real love is designing resources that can be used by other teachers to maximise the experience of the students. I am constantly thinking of new ways to engage a student with a topic and try to implement that in the design of the lessons.

    Share this

    pptx, 3.29 MB docx, 13.8 KB docx, 13.03 KB pptx, 537.17 KB

    This fully-resourced lesson describes how magnification and resolution can be achieved using light and electron microscopy. The engaging PowerPoint and accompanying resources have been designed to cover the content of point 2.1 (vi) of the Edexcel A-level Biology B specification and the importance of specimen staining is also briefly introduced so that students are prepared for the next lesson.

    To promote engagement and focus throughout this lesson, the PowerPoint contains a quiz competition with 7 rounds. The quiz rounds found in this lesson will introduce the objective lens powers, the names of the parts of a light microscope and emphasise some of the other key terms such as resolution. The final round checks on their understanding of the different numbers that were mentioned in the lesson, namely the differing maximum magnifications and resolutions. Time is taken to explain the meaning of both of these microscopic terms so that students can recognise their importance when considering the organelles that were met earlier in topic 2. By the end of the lesson, the students will be able to explain how a light microscope uses light to form an image and will understand how electrons transmitted through a specimen or across the surface will form an image with a TEM or a SEM respectively.

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    Magnification and Resolution

    Magnification is the enlargement of an image resolution is the ability to tell two objects apart.

    Learning Objectives

    Define magnification and resolution

    Key Takeaways

    Key Points

    • Magnification is the ability to make small objects seem larger, such as making a microscopic organism visible.
    • Resolution is the ability to distinguish two objects from each other.
    • Light microscopy has limits to both its resolution and its magnification.

    Key Terms

    • airy disks: In optics, the Airy disk (or Airy disc) and Airy pattern are descriptions of the best-focused spot of light that a perfect lens with a circular aperture can make, limited by the diffraction of light.
    • diffraction: the breaking up of an electromagnetic wave as it passes a geometric structure (e.g., a slit), followed by reconstruction of the wave by interference

    Aging Tissue and Vision Loss: These are micrographs of a section of a human eye. Using computer algorithms and other technology the panel on the right has a higher resolution and is therefore clearer. It should be noted that both panels are at the same magnification, yet the panel on the right has a higher resolution and gives more information on the sample. The labels represent various parts of the human eye: Bruch membrane (B) choroid (C) retinal pigment epithelium (RPE) and retinal rod cells (R). The scale bar is 2um.

    Magnification is the process of enlarging something only in appearance, not in physical size. This enlargement is quantified by a calculated number also called “magnification. ” The term magnification is often confused with the term “resolution,” which describes the ability of an imaging system to show detail in the object that is being imaged. While high magnification without high resolution may make very small microbes visible, it will not allow the observer to distinguish between microbes or sub-cellular parts of a microbe. In reality, therefore, microbiologists depend more on resolution, as they want to be able to determine differences between microbes or parts of microbes. However, to be able to distinguish between two objects under a microscope, a viewer must first magnify to a point at which resolution becomes relevant.

    Resolution depends on the distance between two distinguishable radiating points. A microscopic imaging system may have many individual components, including a lens and recording and display components. Each of these contributes to the optical resolution of the system, as will the environment in which the imaging is performed. Real optical systems are complex, and practical difficulties often increase the distance between distinguishable point sources.

    At very high magnifications with transmitted light, point objects are seen as fuzzy discs surrounded by diffraction rings. These are called Airy disks. The resolving power of a microscope is taken as the ability to distinguish between two closely spaced Airy disks (or, in other words, the ability of the microscope to distinctly reveal adjacent structural detail). It is this effect of diffraction that limits a microscope’s ability to resolve fine details. The extent and magnitude of the diffraction patterns are affected by the wavelength of light (λ), the refractive materials used to manufacture the objective lens, and the numerical aperture (NA) of the objective lens. There is therefore a finite limit beyond which it is impossible to resolve separate points in the objective field. This is known as the diffraction limit.


    Types of Microscopes

    1. Compound Microscope


    By far the most popular kind of microscope, the compound microscope uses two lenses to achieve up to 1000x or 2000x magnification. Specimens are backlit and may be viewed using either a monocular or binocular eyepiece.

    You can find compound microscopes in one form of another in homes, science labs, and even hospitals. Oddly enough, it was the work of Robert Hooke using one of the first compound microscopes that inspired the invention of the simple microscope.

    2. Confocal Microscope

    Providing higher resolution than a compound microscope, a confocal microscope allows for 2D or 3D images of the subject matter. A slide containing a dyed sample is inserted into the microscope. The sample is then scanned using a laser light and, with the aid of a dichromatic mirror, appears on a computer monitor.

    As laser light penetrates deeper than regular light, the user can get either a highly detailed look at opaque objects as far as the laser can penetrate, or the interiors of more translucent objects. This type of microscope is useful in cell biology, as well as various medical applications.

    3. Fluorescence Microscope

    A high-energy, short wavelength light is used for this microscope, exciting the electrons of certain molecules. These electrons shift into a higher orbit briefly. When they settle back, they emit a low energy, low wavelength (visible) light.

    The amount of spatial resolution is limited, but the microscope is powerful enough to detect the presence of a single molecule. While fluorescence was first described in 1852 by Sir George G. Stokes, its almost essential use in biology and biomedical science wasn’t explored until the 1930s.

    4. Scanning Electron Microscope (SEM)

    An electron microscope uses electrons instead of light, allowing for incredible resolution. Scanning electron microscopes are used exclusively to view the surface of an object.

    The object must be dehydrated, then lightly coated in a highly conductive material such as gold or palladium. A beam of focused electrons is bounced off of the specimen in a manner similar to sonar.

    The resulting data is translated into a black and white image on a computer screen at a resolution chosen by the user. These microscopes have a wide range of scientific uses in both physical and medical science.

    5. Scanning Probe Microscope

    This optical microscope uses a physical probe to examine the sample. The scan is done using a raster (line by line) method. As a result, the scans can take some time but produce high-quality computer images.

    These have a more limited magnification than electron microscopes but do not require a vacuum. Another great advantage is that the sample can be stimulated and the reactions or response may be observed, as well as the specimin’s properties.

    In use since 1986, scanning probe microscopes are not only valued in the fields of biology and chemistry, but also physics.

    6. Simple Microscope

    As the name implies, this is the most basic type of microscope. It was created in the 17th century by Antony van Leeuwenhoek and involves a single convex lens and specimen holder.

    Capable of magnifying 200x to 300x. This form of microscope is rarely used today.

    7. Stereo Microscope

    Sometimes referred to as a dissecting microscope, this type overcomes the need for slides, allowing the user to study opaque objects. While the magnification is only 300x, users can view and even manipulate 3D objects.

    Stereo microscopes are used not only for biological and medical science, but can often be found in electronic fields such as circuit making. The tool works by having two optical paths set up at different angles, allowing for a detailed surface view of even living or inanimate objects.

    8. Transmission Electron Microscope (TEM)

    The counterpart to the SEM, a transmission microscope uses ultra-thin samples prepared on a slide. Once coated in a high conductivity material, the sample slide is scanned in a vacuum.

    This allows the electrons to pass through the object with the beam being reflected by the denser parts. As a result, the black and white image allows for a high degree of magnification and resolution.

    These microscopes are useful in a wide range of fields, from physical and biological science to forensics. It is also extremely useful in the development of nanotechnology and metallurgical analysis.

    9. UV Microscope

    Using ultraviolet light produced by a mercury arc or xenon burner, UV microscopes are able to get twice the resolution of visible light microscopes. Images are either photographed or scanned using a digital sensor to avoid harming the observer’s eyes.

    10. X-Ray Microscope

    Used in the observation of living cells, X-ray microscopes use electromagnetic radiation to create highly detailed images. This type of microscope is popular in both biological research and metallurgy.


    Magnification & resolution (Edexcel Int. A-level Biology)

    A Science teacher by trade, I've also been known to be found teaching Maths and PE! However, strange as it may seem, my real love is designing resources that can be used by other teachers to maximise the experience of the students. I am constantly thinking of new ways to engage a student with a topic and try to implement that in the design of the lessons.

    Share this

    pptx, 3.29 MB docx, 14.67 KB docx, 13.8 KB pptx, 537.17 KB docx, 13.03 KB

    This fully-resourced lesson describes how magnification and resolution can be achieved using light and electron microscopy. The engaging PowerPoint and accompanying resources have been designed to cover the content of points 3.7 (i) & (ii) of the Edexcel International A-level Biology specification and also considers how specimens are stained.

    To promote engagement and focus throughout this lesson, the PowerPoint contains a quiz competition with 7 rounds. The quiz rounds found in this lesson will introduce the objective lens powers, the names of the parts of a light microscope and emphasise some of the other key terms such as resolution. The final round checks on their understanding of the different numbers that were mentioned in the lesson, namely the differing maximum magnifications and resolutions. Time is taken to explain the meaning of both of these microscopic terms so that students can recognise their importance when considering the organelles that were met earlier in topic 3. By the end of the lesson, the students will be able to explain how a light microscope uses light to form an image and will understand how electrons transmitted through a specimen or across the surface will form an image with a TEM or a SEM respectively.

    Get this resource as part of a bundle and save up to 36%

    A bundle is a package of resources grouped together to teach a particular topic, or a series of lessons, in one place.

    Topic 3: Cell structure, Reproduction & Development (Edexcel International A-level Biology)

    The locus and linkage, meiosis, differential gene expression and protein transport within cells lessons have been uploaded for free and by downloading these, you will be able to observe the detail of planning that has gone into all of the lessons that are included in this bundle. This intricate planning ensures that the students are engaged and motivated whilst the detailed content of topic 3 (Cell structure, Reproduction and Development) of the Edexcel International A-level Biology specification is covered. The 12 lesson PowerPoints and accompanying resources contain a wide range of activities which cover the following topic 3 specification points: * All living organisms are made of cells * Cells of multicellular organisms are organised into tissues, organs and organ systems * The ultrastructure of eukaryotic cells * The function of the organelles in eukaryotic animal cells * The role of the RER and Golgi apparatus in protein transport within cells * The ultrastructure of prokaryotic cells * Magnification and resolution in light and electron microscopes * The gene locus is the location of a gene on a chromosome * The linkage of genes on a chromosome * The role of meiosis in ensuring genetic variation * Understand how the mammalian gametes are specialised for their functions * The role of mitosis and the cell cycle in growth and asexual reproduction * The meaning of the terms stem cell, pluripotent, totipotent, morula and blastocyst * The decisions that have to be made about the use of stem cells in medical therapies * Cells become specialised through differential gene expression * One gene can give rise to more than one protein through post-transcriptional changes to mRNA * Phenotype is the interaction between genotype and the environment * Some phenotypes are affected by multiple alleles or by polygenic inheritance Due to the detail included in all of these lessons, it is estimated that it will take in excess of 6 weeks of allocated A-level teaching time to complete the teaching of the bundle


    Focusing On Microscopic Objects

    Start with Clean Lenses:

    It is important that microscope lenses be very clean. Before viewing through a microscope, use lens paper to gently clean the lenses.

    Begin at Low Power Magnification:

    Always begin by viewing the object through a low power lens. Depending on how small the object is, start with the scanning or low-power objective.

    Using low-power objective lens, get the target object centered in the field-of-view and focus as much as possible, first by using the coarse focus and then fine-tuning the clarity of the image with the fine focus.

    Once the object is in focus, switch to the next higher objective power. Do not change the focus or manipulate the focus knobs in any way while changing objectives.

    Adjustments for oil immersion objective:

    Without changing the adjustment of high power, turn to oil immersion objective. One drop of oil is added into on the slide. The nose piece is turned such that the oil immersion objective touches on the drop of oil. Open the iris diaphragm completely. Use only fine adjustments for focusing.

    The Importance of Par focal:

    A set of objectives on a microscope are said to be par focal if the viewer can change from one to another and still have the specimen nearly in focus. This is a very convenient feature, because as the magnification increases, even small manipulations of the focus knob can take a specimen far out of focus.

    After changing to a higher objective (such as high-dry or oil-immersion) the viewer needs only manipulate the fine focus knob. Never manipulate the coarse focus at oil immersion. Manipulating the coarse focus at high power can smash the lens into the slide, potentially damaging the scope and the specimen.


    AP Lab 1 Osmosis Sample 3

    Atoms and molecules are constantly in motion. This kinetic energy causes the molecules to bump into each other and move in different directions. This motion is the fuel for diffusion. Diffusion is the random movement of molecules from an area of higher concentration to an area of lower concentration. This will occur until the two areas reach a dynamic equilibrium. When this dynamic equilibrium is reached the concentration of molecules will be approximately equal and there will be no net movement of molecules after this point. The molecules will still be in motion but the concentrations will remain the same.

    Osmosis is a special kind of diffusion in which water moves through a selectively permeable membrane. A selectively permeable membrane allows diffusion for only certain solutes (the substance being dissolved) and water, the most common solvent (a dissolving substance). The most common selectively permeable membrane is the cell membrane. Water moves from an area of high water potential to an area of low water potential. Water potential is the measure of free energy of water in a solution and is represented by the symbol ψ (psi). Water potential is affected by two physical factors: the addition of a solute (ψs) and pressure potential (ψp). The addition of solutes to a concentration will lower the water potential of that solute, causing water to move into the area. Water movement is directly proportional to the pressure potential. Water potential can be determined by the equation:

    Pure water has a water potential of zero. The addition of solutes will cause the water potential value to be negative, while an increase in pressure potential will cause a more positive water potential value.

    There are three relationships that can occur between two solutions. When two solutions have equal solute concentrations, they are isotonic and no net movement of solute occurs. There is also no net movement of water. If the two solutions differ in solute concentrations, they will either be hypertonic or hypotonic. The hypertonic solution has a lower concentration of solute. Water will move out of a hypertonic solution, while solute will move in, (moving up the concentration gradient-similar to water potential). This depends on the selective qualities of the membrane. Pertaining to cells, this will cause the cell to shrivel or become flaccid. The hypotonic solution has a higher concentration of solute, and therefore has less water. This solution will gain water, while losing solute. This movement between the hypotonic and hypertonic solutions will continue until the point of dynamic equilibrium is reached. A hypertonic cell may also undergo plasmolysis. Plasmolysis is the shrinking of the cytoplasm in a plant cell in response to the diffusion of water out of the cell. When a cell is hypotonic it may lyse. In plant cells, it creates turgor pressure against the cell walls keeping the plant from becoming wilted.

    Besides osmosis and diffusion, molecules and ions can be moved by active transport. This process includes the use of ATP to drive molecules in or out of a cell. Active transport is generally used to move molecules against a concentration gradient, from an area of low concentration to an area of higher concentration of molecules.

    In this experiment, diffusion and osmosis will occur until dynamic equilibrium is reached. This experiment is done in a theoretical condition with no other variables affecting the movement of the solute except water potential.

    This exercise requires a 30 cm of 2.5 cm dialysis tubing, 250 ml beaker, distilled water, 2 dialysis tubing clamps, 15 ml of 15% glucose/1% starch solution, 4 pieces of glucose tape, 4 ml of Lugol’s solution (Iodine Potassium-Iodide or IKI), and clock or timer.

    This experiment requires six strips of 30 cm dialysis tubing, 250 ml beaker, 12 dialysis tubing clamps, distilled water six cups, scale, timer or clock, paper towels, and about 25 ml of each of these solutions: distilled water, .2 M glucose, .4 M glucose, .6 M glucose, .8 glucose, and 1.0 M glucose.

    This experiment requires a large potato, potato corer (about 3 cm long), 250 ml beaker, paper towel, scale, six cups, knife, and about 100 ml of each of these solutions: distilled water, .2 M glucose, .4 M glucose, .6 M glucose, .8 glucose, and 1.0 M glucose.

    This experiment requires a calculator, paper, pencil, and graphing paper.

    This experiment requires onion skin, dye, microscope, slide, cover slip, salt water (15%), and tap water.

    First, soak the dialysis tubing in distilled water for 24 hours. Remove the tubing and tie off one end using the clamp (twist tubing end about 7 times and fold end on self, slide into the clamp). Next, open the other end of the tubing (rubbing end between fingers) and fill it with the glucose/starch solution. Use the glucose tape and record the color change of the tape and the color of the bag. Tie of the end with the tubing clamp (leave empty space, but no air). Fill the beaker with distilled water and add the 4-ml’s of Lugol’s solution, record the color change. Use glucose tap to test for any glucose in the water (record). Set the dialysis tubing in the beaker and let it sit for about 30 minutes. Remove the bag and record the change in water and bag color. Use the last two pieces of glucose tape to measure the glucose in the water and bag and record results.

    First, soak the dialysis tubing for about 24 hours. Tie off one end of each tube with the clamps. Next, fill each tube with a different solution (distilled water, .2 M glucose, .4 M glucose, .6 M glucose, .8 glucose, and 1.0 M glucose) and tie off the end (leave empty space, but no air). Weigh each tube separately and record the masses. Soak the tubes in separate cups filled with distilled water for about 30 minutes. Remove the tubing, blot dry, reweigh, and record the mass.

    First, slice the potato into to 3-cm discs. Use the potato corer and core out 24 cores (don’t get any). Weigh 4 cores together and record the mass. Fill each cup with a different solution (distilled water, .2 M glucose, .4 M glucose, .6 M glucose, .8 glucose, and 1.0 M glucose). In each cup put 4 potato cores and let it sit over night. Take out the cores and blot them dry. Record the change in mass. Calculate the information and compare.

    First, determine the solute potential of the glucose solution, the pressure potential, and the water potential. Then, graph the information given about the zucchini cores.

    First, prepare a wet mount slide of dyed onion skin. Observe under a light microscope and sketch what the cells. Add a few drops of the salt solution, observe, and sketch the change.


    Different kinds of Microscopes:

    Light Microscopy:

    This is the oldest, simplest and most widely-used form of microscopy. Specimens are illuminated with light, which is focussed using glass lenses and viewed using the eye or photographic film. Specimens can be living or dead, but often need to be stained with a coloured dye to make them visible. Many different stains are available that stain specific parts of the cell such as DNA, lipids, cytoskeleton, etc. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification. Light microscopy has a resolution of about 200 nm, which is good enough to see cells, but not the details of cell organelles. There has been a recent resurgence in the use of light microscopy, partly due to technical improvements, which have dramatically improved the resolution far beyond the theoretical limit. For example fluorescence microscopy has a resolution of about 10 nm, while interference microscopy has a resolution of about 1 nm.  

    Components of a light microscope


    All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path, listed here in the order the light travels through them:

    • Light source, a light or a mirror (7)
    • Diaphragm and Condenser lens (8)
    • Objective (3)
    • Ocular lens (eyepiece) (1)


    In addition the vast majority of microscopes have the same 'structural' components:

    • Objective turret (to hold multiple objective lenses) (2)
    • Stage (to hold the sample) (9)
    • Focus wheel to move the stage (4 - coarse adjustment, 5 - fine adjustment)

    These entries are numbered according to the image on the right.

    Preparation of Slide Samples

    • Fixation: Chemicals preserve material in a life like condition. Does not distort the specimen.
    • Dehydration: Water removed from the specimen using ethanol. Particularly important for electron microscopy because water molecules deflect the electron beam which blurs the image.
    • Embedding: Supports the tissue in wax or resin so that it can be cut into thin sections.Sectioning Produces very thin slices for mounting. Sections are cut with a microtome or an ulramicrotome to make them either a few micrometres (light microscopy) or nanometres(electron microscopy) thick.
    • Staining: Most biological material is transparent and needs staining to increase the contrast between different structures. Different stains are used for different types of tissues. Methylene blue is often used for animal cells, while iodine in KI solution is used for plant tissues.
    • Mounting: Mounting on a slide protects the material so that it is suitable for viewing over a long period.

    Practical investigation into size and scale of microscopic tissues

    This practical focuses on microscope technique and using graticules and stage micrometers to determine size and scale in biological cells and tissues.

    1. Use a microscope fitted with an eyepiece graticule and stage micrometer
    2. Calibrate the eyepiece graticule using the stage micrometer
    3. Use the calibrated graticule to determine the actual size of microscopic specimens
    4. estimate the accuracy of a measurement
    5. Use the graticule to determine scales
    6. Understand the importance of repeating or validating set of results.

    Safety Information There are no particular hazards in this practical, however you must follow your laboratory rules.

    Background information • The measurement of specimen size with a microscope, is made by using an eyepiece graticule. This is a glass or plastic disc with 8 divisions etched onto its surface, which is inserted into the eyepiece lens. • The size of the eyepiece graticule remains constant, despite the fact that the image viewed will change its size depending upon whether high- or low-power objective lenses are used. For example a cell viewed with the x40 objective will appear much larger than when viewed with the x10 objective. However because the graticule is in the eyepiece it will not change its size. Therefore the value of each of the divisions in the eyepiece graticule varies with the magnification of the objective lens.

    • A stage micrometer is a very accurately etched glass or plastic ruler that is placed on the microscope stage so that the eyepiece graticule scale is superimposed on the stage micrometer scale. The scale is usually 1mm divided into 100 separate divisions so that each division equals 10 micrometres (10μm).

    • It is necessary to calibrate the eyepiece graticule with the stage micrometer placed on the microscope stage for each objective lens used.

    You will observe a TS of plant tissues through a microscope and use an eyepiece graticule and a stage micrometer to determine the size of some of the structures.Plant tissues

    • Read the information above.

    • Ensure that you understand the principles of using an eyepiece graticule and a stage micrometer before you continue with the investigation.

    1. You have been provided with a compound light microscope with both low and high-power objective lenses and an eyepiece lens that has been fitted with a graticule. You have also been provided with a stage micrometer.

    2. You must now calibrate the eyepiece graticule. Place the stage micrometer onto the microscope stage and focus using the low-power objective lens so that the graticule scale becomes superimposed over the stage micrometer scale.

    3. Move the stage micrometer until the start or zero line of each scale is coincident (lined up)

    4. Look along the scale until another coincident point is found.

    5. The relationship between the two scales can now be calculated On the scale shown there are 17 divisions on the stage micrometer scale that line up with 7 divisions on the graticule scale. Thus 17 / 7 = 2.42857 units. Each unit on the stage micrometer scale is 10 micrometres (10μm). Therefore each division on the graticule scale is 24.2857 micrometres rounded to 24.3 μm.

    6. Use the procedure described above to determine the size of each division on the eyepiece graticule using the low-power objective lens of your microscope.

    7. Repeat the procedure to determine the size of each division when using the high-power objective lens.

    1. You are provided with a stained transverse section through part of a dicotyledonous plant root.

    2. Examine the specimen using the low-power of your microscope.

    3. Make a large, plan drawing to show the distribution of tissues, labelling the stele (vascular bundle).

    4. Use the eyepiece graticule to measure the width of the vascular bundle at its widest point in graticule units and then calculate the actual width of the vascular bundle in millimetres and in micrometres.

    5. Draw a straight line on your drawing across the vascular bundle to show where you took your measurement. Write the dimension on your drawing next to the line.

    6. Make a high-power drawing to show a group of four xylem vessels from inside the vascular bundle.

    7. Use the eyepiece graticule to measure the width of the xylem vessel at its widest point in graticule units and then calculate the actual width of the vessel in micrometres, remembering to use the appropriate calibration of the eyepiece graticule for the high-power objective lens.

    8. Draw a straight line on your drawing across the xylem vessel to show where you took your measurement. Write the dimension on your drawing next to the line.

    9. Look at your two measurements and check on their accuracy. The actual size of the xylem vessel should be smaller than the size of the vascular bundle even though it looked larger using the high power objective lens.

    10. You are now going to determine the magnification of your drawing of the xylem vessels. Use a ruler to measure the length of the line that you drew across the xylem vessel. Use your knowledge of the actual size of the vessel to calculate the magnification of your drawing. Write your answer x at the bottom right hand corner of your drawing.

    • Compare your results with other members of the class and check for consistency of readings.


    • Did any member of the class have anomalous results? What are the potential causes of such an anomalous result in this investigation?


    • Write up your procedure including a discussion of the benefits of comparing your results with other students.

    Electron Microscopy

    This uses a beam of electrons, rather than electromagnetic radiation, to "illuminate" the specimen. This may seem strange, but electrons behave like waves and can easily be produced (using a hot wire), focused (using electromagnets) and detected (using a phosphor screen or photographic film). A beam of electrons has an effective wavelength of less than 1 nm, so can be used to resolve small sub-cellular ultrastructure. The development of the electron microscope in the 1930s revolutionised biology, allowing organelles such as mitochondria, ER and membranes to be seen in detail for the first time.

    [1]Nucleolus [2]Nucleus (3) Ribosomes (little dots) (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth endoplasmic reticulum (ER) (9) mitochondria (10) vacuole (11) cytosol (not cytoplasm as that includes all the organelles) (12) lysosome (13) centrioles within centrosome

    The main problem with the electron microscope is that specimens must be fixed in plastic and viewed in a vacuum, and must therefore be dead. Other problems are that the specimens can be damaged by the electron beam and they must be stained with an electron-dense chemical (usually heavy metals like osmium, lead or gold). Initially there was a problem of artefacts (i.e. observed structures that were due to the preparation process and were not real), but improvements in technique have eliminated most of these.

    There are two kinds of electron microscope. The transmission electron microscope (TEM) works much like a light microscope, transmitting a beam of electrons through a thin specimen and then focusing the electrons to form an image on a screen or on film. This is the most common form of electron microscope and has the best resolution. The scanning electron microscope (SEM) scans a fine beam of electron onto a specimen and collects the electrons scattered by the surface. This has poorer resolution, but gives excellent 3-dimentional images of surfaces.

    • Thin sections of specimen are needed for transmission electron microscopy as the electrons have to pass through the specimen for the image to be produced.  
    •  This is the most common form of electron microscope and has the best resolution 

    • Electrons are reflected off the surface of the specimen as it has been previously coated in heavy metals. 
    • It is these reflected electron beams that are focussed of the fluorescent screen in order to make up the image.  
    • Larger, thicker structures can thus be seen under the SEM as the electrons do not have to pass through the sample in order to form the image. This gives excellent 3-dimensional images of surfaces 
    • However the resolution of the SEM is lower than that of the TEM.

    Comparison of the light and electron microscope

    Light Microscope Electron Microscope
    Cheap to purchase (£100 – 500) Expensive to buy (over £ 1 000 000).
    Cheap to operate. Expensive to produce electron beam.
    Small and portable. Large and requires special rooms.
    Simple and easy sample preparation. Lengthy and complex sample prep.
    Material rarely distorted by preparation. Preparation distorts material.
    Vacuum is not required. Vacuum is required.
    Natural colour of sample maintained. All images in black and white.
    Magnifies objects only up to 2000 times Magnifies over 500 000 times.
    Specimens can be living or dead Specimens are dead, as they must be fixed in plastic and viewed in a vacuum
    Stains are often needed to make the cells visible The electron beam can damage specimens and

    they must be stained with an electron-dense chemical