cells

• Microscopy


  The use of microscopes to analyse cell components and observe organelles
• Magnification


  How many times bigger the image produced by the microscope is than the real-life object you are viewing
• Resolution


  The ability to distinguish between objects that are close together (i.e. the ability to see two structures that are very close together as two separate structures)
• Types of microscopes


  • Optical (light) microscopes
  • Electron microscopes
  • Laser scanning confocal microscopes

  
• Optical (light) microscopes


  • Use light to form an image
  • Have a maximum resolution of around 0.2 micrometres (µm) or 200 nm
  • Can be used to observe eukaryotic cells, their nuclei and possibly mitochondria and chloroplasts
  • Cannot be used to observe smaller organelles such as ribosomes, the endoplasmic reticulum or lysosomes
  • Have a maximum useful magnification of about ×1500

  
• Electron microscopes


  • Use electrons to form an image
  • Have a maximum resolution of around 0.0002 µm or 0.2 nm (i.e. around 1000 times greater than that of optical microscopes)
  • Can be used to observe small organelles such as ribosomes, the endoplasmic reticulum or lysosomes
  • Have a maximum useful magnification of about ×1,500,000

  
• Types of electron microscopes


  • Transmission electron microscopes (TEMs)
  • Scanning electron microscopes (SEMs)
• Transmission electron microscopes (TEMs)


  • Use electromagnets to focus a beam of electrons
  • The beam of electrons is transmitted through the specimen
  • Denser parts of the specimen absorb more electrons, making these denser parts appear darker on the final image produced
  • Give high-resolution images (more detail)
  • Can only be used with very thin specimens or thin sections of the object being observed
  • Cannot be used to observe live specimens
  • Do not produce a colour image

  
• Scanning electron microscopes (SEMs)


  • Scan a beam of electrons across the specimen
  • The beam bounces off the surface of the specimen and the electrons are detected, forming an image
  • Can be used on thick or 3-D specimens
  • Allow the external, 3-D structure of specimens to be observed
  • Give lower resolution images (less detail) than TEMs
  • Cannot be used to observe live specimens
  • Do not produce a colour image

  
• Laser scanning confocal microscopes


  • The cells being viewed must be stained with fluorescent dyes
  • A thick section of tissue or small living organisms are scanned with a laser beam
  • The laser beam is reflected by the fluorescent dyes
  • Multiple depths of the tissue section/organisms are scanned to produce an image
  • Can be used on thick or 3-D specimens
  • Allow the external, 3-D structure of specimens to be observed
  • Produce very clear images due to the laser beam being focused at a very specific depth
  • It is a slow process and takes a long time to obtain an image
  • The laser has the potential to cause photodamage to the cells

  
• Optical microscopes have a maximum resolution of around 0.2 micrometres (µm) or 200 nm
• Electron microscopes have a maximum resolution of around 0.0002 µm or 0.2 nm
• The maximum useful magnification of optical microscopes is about ×1500

  
• The maximum useful magnification of electron microscopes is about ×1,500,000
• Preparing a slide using a liquid specimen


  1. Add a few drops of the sample to the slide using a pipette

  2. Cover the liquid/smear with a coverslip and gently press down to remove air bubbles

  3. Wear gloves to ensure there is no cross-contamination of foreign cells
• Preparing a microscope slide using a solid specimen


  1. Take care when using sharp objects and wear gloves to prevent the stain from dying your skin

  2. Use scissors to cut a small sample of the tissue

  3. Peel away or cut a very thin layer of cells from the tissue sample to be placed on the slide (using a scalpel or forceps)

  4. Apply a stain

  5. Gently place a coverslip on top and press down to remove any air bubbles
• Preparing a microscope slide using a solid specimen (alternative method)


  1. Treat the tissue sample with chemicals to kill/make the tissue rigid

  2. Fix the specimen using formaldehyde (preservative)

  3. Dehydrate it using a series of ethanol solutions

  4. Impregnate it in paraffin/resin for support

  5. Cut thin slices from the specimen using a microtome

  6. Remove the paraffin from the slices/specimen

  7. Apply a stain

  8. Mount the specimen using a resin and apply a coverslip
• Preparing a microscope slide using a solid specimen (alternative method)


  1. Freeze the specimen in carbon dioxide or liquid nitrogen

  2. Cut the specimen into thin slices using a cryostat

  3. Place the specimen on the slide and add a stain

  4. Gently place a coverslip on top and press down to remove any air bubbles
• When using an optical microscope always start with the low power objective lens

  
• Adding a drop of water to the specimen (beneath the coverslip) can prevent the cells from being damaged by dehydration
• Using a graticule to take measurements of cells


  1. Place a graticule (a small disc with an engraved ruler) into the eyepiece of the microscope

  2. Calibrate the graticule by using a stage micrometer (a scale engraved on a microscope slide)

  3. This allows the number of micrometers each graticule unit is worth to be worked out

  4. The graticule can then be used as a ruler in the field of view

  
• The size of cells or structures of tissues may appear inconsistent in different specimen slides
• Cell structures are 3D and the different tissue samples will have been cut at different planes resulting in inconsistencies when viewed on a 2D slide
• Optical microscopes do not have the same magnification power as other types of microscopes and so there are some structures that cannot be seen

  
• The treatment of specimens when preparing slides could alter the structure of cells
• Staining in light microscopy


  • Coloured dyes are used to make naturally transparent tissues visible
  • The dyes absorb specific colours of light while reflecting others, making the structures within the specimen that have absorbed the dye visible
• Common stains used in light microscopy


  • Toluidine blue
  • Phloroglucinol
• Staining for electron microscopy


  • Heavy-metal compounds are commonly used as dyes because they absorb electrons well
  • Any colour present in electron micrographs is not natural and is added using image-processing software
• Chloroplasts don't need stains as they show up green, which is their natural colour
• The internal structure of the mitochondrion can be seen using a TEM and staining
• No colours have been added to the image of the spiracle found on the exoskeleton of an insect using image-processing software
• Biological drawings


  Line pictures which show specific features that have been observed when the specimen was viewed
• Mitochondrion


  The site of aerobic respiration within all eukaryotic cells
• Mitochondrion


  • Surrounded by double-membrane with the inner membrane folded to form cristae
  • The matrix formed by the cristae contains enzymes needed for aerobic respiration, producing ATP
  • Small circular pieces of DNA (mitochondrial DNA) and ribosomes are also found in the matrix (needed for replication)
• Chloroplast


  Found in the green parts of a plant, the green colour a result of the photosynthetic pigment chlorophyll
• Chloroplast


  • Larger than mitochondria, also surrounded by a double-membrane
  • Membrane-bound compartments called thylakoids containing chlorophyll stack to form structures called grana
  • Grana are joined together by lamellae (thin and flat thylakoid membranes)
  • The light-dependent stage of photosynthesis takes place in the thylakoids
  • The light-independent stage (Calvin Cycle) takes place in the stroma
  • Also contain small circular pieces of DNA and ribosomes used to synthesise proteins needed in chloroplast replication and photosynthesis
• Ribosome


  Formed in the nucleolus and are composed of almost equal amounts of RNA and protein
• Ribosome


  • Found freely in the cytoplasm of all cells or as part of the rough endoplasmic reticulum in eukaryotic cells
  • Each ribosome is a complex of ribosomal RNA (rRNA) and proteins
  • 80S ribosomes (composed of 60S and 40S subunits) are found in eukaryotic cells
  • 70S ribosomes (composed of 50S and 30S subunits) in prokaryotes, mitochondria and chloroplasts
  • Site of translation (protein synthesis)
• Rough Endoplasmic Reticulum (RER)


  Surface covered in ribosomes, processes proteins made by the ribosomes
• Smooth Endoplasmic Reticulum (ER)


  Involved in the production, processing and storage of lipids, carbohydrates and steroids