2 2.1.4 Comparison of relative sizes of molecules, cell membrane thickness, viruses, bacteria, organelles and cells, using the appropriate SI unit.Refer Figure 1.18, Clegg p20Size relationshipsof biological and chemicallevels of organization arecompared-Notice the diversity andhow the power of 10 is used-Although sizes are expressedin length and diameter, cellsand organisms are 3-D
3 Size of various cells and structures: Molecules: 1 nmMembranes (on organelles): 10 nmViruses: 100 nmBacteria: 1 umOrganelles: up to 10 umMost cells: up to 100 umMeasurements above in 2D, remember all structures have 3D shape.
4 The Metric System Know how to convert from one unit to another. BasicUnitKilo-1000UnitsHecto-100unitsDek-10Deci-0.1Centi-0.01Milli-0.001MultiplyDivide
5 What units are used to measure cells? 1 mm = 1000 micrometers (um)1 mm = 1,000,000 nanometers (nm)Or…A micrometer is 1 x 10-3 mm (0. 001)A nanometer is 1 x 10-6 mm ( mm)
7 Magnification and Scale bars Specimen size how large the specimen actually isImage size how large the specimen appears in a drawing or photographMagnification how much larger the image is than the actual sizeFormula used for these calculations:Magnification = size of imagesize of specimenMicroscopy Calculations Youtube video
8 Calculating Linear Magnification What is the actual size of this specimen in um?60mm/5 = 12mm12mm x 1000 um =12,000 umMagnification x560 mmMeasuring picture
9 Refer to image on p16 and problem 9 on p17 2.1.5 Calculate the linear magnification of drawings and the actual size of specimens in images of known magnificationMagnification could be stated (for example, ×250) or indicated by means of a scale barMagnification = Image size = 58mm = 2.5mm x 1000 = x Size of specimen mm(size of specimen meas with scale bar)Scale bar1umRefer to image on p16 and problem 9 on p17
10 Calculating image size using Scale Bar Magnification = Image size = 129mm = 460umSize of specimen 28mmX100um (scale bar)Refer to problem 5, p12Scale bar0.1mm
11 If we want to see a cell… We have to magnify it Magnification: making something that is small appear largerA cell from the inside of your cheek
12 Antony vanLeeuwenhoek Father of microbiologyEyeglass maker who invented first microscope in 1600’s
13 Eyepiece Body tube Arm Stage Base Stage clips Diaphragm Light source Revolving nosepieceArmObjective lensStageStage clipsCoarse adjustment knobFine adjustmentDiaphragmLight sourceBase
14 Light Microscopy Advantages Can view living specimensInexpensive and easy to use
15 Light Microscopy Disadvantages Resolution is limited.Resolution: the ability to form separate images of objects that are close togetherResolving power: the minimum distance two points can be separated and still be individually distinguished as two separate points.The smaller the resolving power, the better the resolution.
16 Light Microscopy Disadvantages Can only magnify a limited number of times (ours go up to 1000x; best light microscopes magnify up to 4000x)Limited by focal length of lens
17 Electron MicroscopesTo magnify an image a large number of times, you must use an electron microscope.Specimen has a beam of electrons passed through it
18 Electron Microscopes There are different types of electron microscopes In a transmission electron microscope (TEM), an electron beam passes through a very thin section of materialAn image is formed because the electrons pass through some parts of the section but not othersIn a scanning electron microscope (SEM), a narrow beam of electrons is scanned in a series of lines across the surface of the specimenThe electrons that are reflected or emitted from the surface are collected by a detector and converted into an electrical signal, which is used to build a 3-D image, line by line, on a TV screen
19 Electron Microscope Advantages Images can be magnified thousands of times (up to 250,000x)A lot of detail can be seen
20 Electron Microscopy Advantages Can magnify 1000’s of timesDetails are easily visibleHIV, magnified 24,000x
21 Electron Microscopy Disadvantages Expensive ($$$$$)Must use heavy metal dyes, which kill organisms
22 Transmission vs. Scanning EM Transmission EM’s view cross-sectionsSEM’s view surfaces only
23 Comparison of Light and Electron Microscopes Light MicroscopesElectron MicroscopesMaterial can be prepared easily for examination. Often, a sample can simply by placed on a slide with a few drops of water and a cover slip. An image can be obtained within secondsPreparation of material for examination always involves a long series of procedures. These take several days to complete and often involve the use of toxic chemicalsLiving material can be examined, so specimens do not always have to be killed. There is less danger of artificial structures appearing and causing confusion if the specimen is still aliveLiving material cannot survive in the vacuum inside electron microscopes. Tissues therefore have to be killed as the first stage in the preparation of them for examinationMovement can be observed if living material is examined, including the flow of blood, streaming of cytoplasm inside cells and the locomotion of microscopic organismsNo movement can be observed as the material is always dead. Movement can only be deduced indirectly by complex experiments, often involving radioactive tracersColors can be seen – both natural colors and artificial colors caused by stainingOnly monochrome images are produced, with black, white, and shades of greyThe field of view (the area which can be observed at once) is relatively large ~2mm across at low power with typical microscopesOnly a small field if view can be examined at once – in a TEM the max uninterrupted view is about 100mm acrossThe resolution of light microscopes is relatively poor – about .25mm so the max useful magnification is only about x600. Many structures within cells cannot be seen clearlyThe short wavelength of electrons gives very good resolution – about .25nm. This allows magnification of up to x500,000. Very small objects therefore become visible including many of the details of cell structure
24 To calculate total magnification: Multiply the magnification on the objective by the magnification found on the eyepieceYou will need this for every specimen you draw under the scope!
25 Field of View (FOV)Field of View: Sometimes abbreviated "FOV", it is the diameter of the circle of light that you see when looking into a microscope.As the power gets greater, the field of view gets smaller. You can measure this by placing a clear metric ruler on the stage and counting the millimeters from one side to the other. Typically you will see about 4.5mm at 40X, 1.8mm at 100X, 0.45mm at 400X and 0.18mm at 1000X.
26 Calculating FOV Measuring the microscope field of view on lowest power Place a clear plastic ruler with mm markings on top of the stage of your microscope. Looking through the lowest power objective, focus your image. Count how many divisions of the ruler fit across the diameter of the field of view. Multiply the number of divisions by 1000 to obtain the field of view in micrometers (µm).Record this in µm (1mm = 1000 µm ).Magnified at 40X, the lines of the ruler are clearly visible.
27 FOV Mathematical Calculation Total Magnification Low Power = FOV at Other PowerTotal Magnification at Other Power FOV at Low Power
28 Practice calculating FOV Example:If a 5x FOV is 3 mm, what is the 40x FOV of that microscope?Total Magnification Low Power = FOV at Other PowerTotal Magnification at Other Power FOV at Low Power5 = FOV at Other Power40 3mm(3)(5) = (FOV of higher power)(40)=0.375 mm FOV of higher power
29 Website for microscope calculations Calculating with microscopes