# Microscopes and Cells IB HL Biology.

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Microscopes and Cells IB HL Biology

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 p20 Size relationships of biological and chemical levels of organization are compared -Notice the diversity and how the power of 10 is used -Although sizes are expressed in length and diameter, cells and organisms are 3-D

Size of various cells and structures:
Molecules: 1 nm Membranes (on organelles): 10 nm Viruses: 100 nm Bacteria: 1 um Organelles: up to 10 um Most cells: up to 100 um Measurements above in 2D, remember all structures have 3D shape.

The Metric System Know how to convert from one unit to another.
Basic Unit Kilo- 1000 Units Hecto- 100 units Dek- 10 Deci- 0.1 Centi- 0.01 Milli- 0.001 Multiply Divide

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)

Magnification and Scale bars
Specimen size how large the specimen actually is Image size how large the specimen appears in a drawing or photograph Magnification how much larger the image is than the actual size Formula used for these calculations: Magnification = size of image size of specimen Microscopy Calculations Youtube video

Calculating Linear Magnification
What is the actual size of this specimen in um? 60mm/5 = 12mm 12mm x 1000 um = 12,000 um Magnification x5 60 mm Measuring picture

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 magnification Magnification could be stated (for example, ×250) or indicated by means of a scale bar Magnification = Image size = 58mm = 2.5mm x 1000 = x Size of specimen mm(size of specimen meas with scale bar) Scale bar 1um Refer to image on p16 and problem 9 on p17

Calculating image size using Scale Bar
Magnification = Image size = 129mm = 460um Size of specimen 28mmX100um (scale bar) Refer to problem 5, p12 Scale bar 0.1mm

If we want to see a cell… We have to magnify it
Magnification: making something that is small appear larger A cell from the inside of your cheek

Antony vanLeeuwenhoek
Father of microbiology Eyeglass maker who invented first microscope in 1600’s

Eyepiece Body tube Arm Stage Base Stage clips Diaphragm Light source
Revolving nosepiece Arm Objective lens Stage Stage clips Coarse adjustment knob Fine adjustment Diaphragm Light source Base

Can view living specimens Inexpensive and easy to use

Resolution is limited. Resolution: the ability to form separate images of objects that are close together Resolving 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.

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

Electron Microscopes To magnify an image a large number of times, you must use an electron microscope. Specimen has a beam of electrons passed through it

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 material An image is formed because the electrons pass through some parts of the section but not others In a scanning electron microscope (SEM), a narrow beam of electrons is scanned in a series of lines across the surface of the specimen The 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

Images can be magnified thousands of times (up to 250,000x) A lot of detail can be seen

Can magnify 1000’s of times Details are easily visible HIV, magnified 24,000x

Expensive (\$\$\$\$\$) Must use heavy metal dyes, which kill organisms

Transmission vs. Scanning EM
Transmission EM’s view cross-sections SEM’s view surfaces only

Comparison of Light and Electron Microscopes
Light Microscopes Electron Microscopes Material 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 seconds Preparation of material for examination always involves a long series of procedures. These take several days to complete and often involve the use of toxic chemicals Living 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 alive Living 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 examination Movement can be observed if living material is examined, including the flow of blood, streaming of cytoplasm inside cells and the locomotion of microscopic organisms No movement can be observed as the material is always dead. Movement can only be deduced indirectly by complex experiments, often involving radioactive tracers Colors can be seen – both natural colors and artificial colors caused by staining Only monochrome images are produced, with black, white, and shades of grey The field of view (the area which can be observed at once) is relatively large ~2mm across at low power with typical microscopes Only a small field if view can be examined at once – in a TEM the max uninterrupted view is about 100mm across The 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 clearly The 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

To calculate total magnification:
Multiply the magnification on the objective by the magnification found on the eyepiece You will need this for every specimen you draw under the scope!

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.

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.

FOV Mathematical Calculation
Total Magnification Low Power = FOV at Other Power Total Magnification at Other Power FOV at Low Power

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 Power Total Magnification at Other Power FOV at Low Power 5 = FOV at Other Power 40 3mm (3)(5) = (FOV of higher power)(40) =0.375 mm FOV of higher power

Website for microscope calculations
Calculating with microscopes

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