Basic Components of Living Systems

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2.1. Microscopy

Cell Theory:

(1) All living things are made up of cells

(2) Cells are basic units of structure

(3) Cells only develop from existing cells

  • Cells contain and pass on hereditary information during cell division
  • All cells are relatively the same in relation to chemical composition and metabolic activity

How light travels through a microscope:

Light from bulb/mirror -> Condenser lens focuses the light onto the specimen -> Light passes through specimen and objective lens -> Objective lens contributes the most to enhancing detail -> Lights travels through the objective lens, through the body piece to the eyepiece lens -> Eyepiece lens magnifies the image made by the objective lens -> Light from specimen enters eye.

    

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2.1. Microscopy (part 2)

Sample Preparation:

  • Dry mount - used to view solid specimens. They are sectioned and placed in the centre of the slide, with a cover slip placed over the sample. E.g. hair, pollen and muscle tissue.
  • Wet mount - the specimen is suspended in a liquid such as water or an immersion oil. A cover slip is placed on top at an angle. E.g. aquatic samples and living organisms. 
  • Squash slides - a wet mount is prepared, then a lens tissue is used to gently press down the cover slip. To avoid damage, the sample can be squashed between two slides. E.g. root tips.
  • Smear slides - the edge of a slide is used to smear the sample, creating a thin, even coating on another slide. A cover slip is then placed on the sample. E.g. blood. 

Using staining:

Stains increase contrast as different components within a cell take up stains to different degrees. This increase, in contrast, allows components to become visible so they can be identified. 

  • To prepare a sample for staining it is first placed on a slide and allowed to air dry.
  • This is then heat-fixed by passing through a flame.
  • The specimen will adhere to the microscope slide and will then take up stains. 

Differential staining - can distinguish between two types of organisms that would otherwise be hard to identify. Can also differentiate between different organelles of a single organism within a tissue sample. E.g. gram stain technique ad acid-fast technique. 

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2.1. Microscopy (part 3)

Stages in the production of slides:

1.   Fixing – chemicals are used to preserve the specimen to keep it as close to a natural state as possible

2.   Sectioning – specimens dehydrated with alcohols and then placed in a mould with wax. This can be sliced thinly with a knife called microtome.

3.   Staining – specimens are stained with multiple chemicals to sow different structures 

4.   Mounting – the specimen is then put on a microscope slide and a cover slip is put on top.

Many of the chemicals used to stain are toxic/irritants

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2.2. Magnification and calibration

Magnification = how many times larger the image is than the actual size of the object being viewed. 

Resolution = the ability to see individual objects as separate entities. The resolution of a microscope determines the amount of detail that can be seen - higher the resolution, the more visible details. 

Unit conversions: 

100 nanometres (nm) = 1 micrometre (μm)

1000 micrometres (μm) = 1 milimetre (mm)

100 milimetres (mm) = 1 centimetre (cm)

Calculation for magnification:

magnification = (size of image) / (actual size of object)

Using a graticule to calibrate a light microscope:

  • Eyepiece graticule = a glass disc marked with a fine scale of 1 to 100.
  • Stage micrometer = a microscope slide with a very accurate scale in micrometres (μm) engraved on it. Usually 100 divisions = 1mm, so 1 division = 10 μm.
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2.3. More microscopy

Electron Microscopy:

  • In light microscopy, increased magnification can be achieved easily but if the image is blurred no more detail will be seen. Resolution is the limiting factor.
  • in electron microscopy, a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen, making it easier to identify different organelles in a cell.

There are two types of electron microscope:

  • Transmission electron microscope (TEM) - a beam of electrons is transmitted through a specimen and focused to produce an image.
  • Scanning electron microscope (SEM) - a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected. Can produce 3D images. 

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2.3. More microscopy (part 2)

In fluorescent microscopes:

  • Uses a high light intensity to illuminate a specimen that has been treated with a fluorescent dye
  • This emits light with a longer wavelength and lower energy and this is used to produce a magnified image 

Laser scanning confocal microscope:

  • Moves a single focused spot of light across a specimen, this causes fluorescence from the components that were treated with a fluorescent dye
  • The light that is emitted from the specimen is filtered through a pinhole aperture
  • The laser scans the specimen point-by-point and this is then reconstructed by a computer to show the full image
  • Produces images with high resolution and high contrast 
  • Eliminates out of focus light (blur) to produce sharp images
  • 3-D images can be generated, and also rotate these 3-D images
  • Can scan multiple layers of the sample and create a ‘stack’ of images, using thin optical sectioning so no physical sectioning is needed
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