Basic components of living systems (microscopy, cell structure) Flashcards
How does a compound light microscope work?
A light microscope has two lenses:
• The objective lens - this produces a magnified image
• The eyepiece lens - the magnified image created by the objective lens is magnified by the lens
‣ This configuration allows for much higher magnification and reduced chromatic aberration (blurriness) than an average light microscope
‣ Illumination is usually provided by a light underneath the sample
What ways can you prepare a sample slide?
- Dry mount = specimen viewed whole or cut into thin slices (sectioning), e.g. hair, pollen, dust, insect parts
- Wet mount = specimen suspended in a liquid (e.g. water, immersion oil), and the cover slip is placed on from an angle, e.g. aquatic samples
- Squash slides = wet mount prepared first, then a lens tissue is used to press down the cover slip, e.g. good for soft samples like root tips
- Smear slides = edge of the slide is used to smear the sample and covered with a cover slip, e.g. blood samples
What is the cytosol of a cell and other cell structures?
Cytosol is the aqueous interior and is often transparent
What is gram stain technique?
- This is used to separate bacteria into two different groups: gram-positive and gram-negative. Crystal violet is applied to a bacteria specimen, then iodine, which fixes the dye. The slide the specimen is on is washed with alcohol. If the bacteria is gram-positive, it will retain the dye, appearing blue/purple
‣ A counterstain (safranin dye) is used, making the gram-negative bacteria appear red
‣ Gram-positive bacteria are susceptible to penicillin, inhibiting the formation of cell walls. Gram-negative bacteria have thinner cell walls which aren’t susceptible to penicillin
What is acid-fast technique?
- This is used to differentiate species of mycobacterium from other bacteria
- A lipid solvent carries carbolfuchsin dye onto the cells being studied
- The cells are then washed with a dilute acid-alcohol solution
- Mycobacterium are not affected by the acid-alcohol solution and retain the carbolfuchsin dye, which is bright red
- Other bacteria lose the stain and are exposed to a methylene blue stain
Which stages do pre-prepared slides go through in production?
- Fixing - chemicals like formaldehyde are used to preserve specimens in a near-natural state as possible
- Sectioning - specimens are dehydrated with alcohols and then placed in a mould with wax or resin to form a hard block. This can be sliced thinly with a knife called a microtome
- Staining - specimens are often treated with multiple stains to show different structures
- Mounting - the specimens are then secured to a microscope slide and a cover slip placed on top
What rules do you have to follow to produce a good scientific drawing?
- Include a title
- State magnification
- Use a sharp pencil for drawings and labels
- Use white, unlined paper
- Use as much of the paper as possible for the drawing
- Draw smooth, continuous lines
- Do not shade
- Draw clearly defined structures
- Ensure proportions are correct
- Label lines should not cross and should not have arrow heads
- Label lines should be parallel to the top of the page and drawn with a ruler
What is magnification?
Magnification is how many times larger an image is than the actual size of the object being viewed
What is resolution?
- Resolution is the ability to see individual objects as separate entities
- The resolution of a microscope determines the amount of detail that can be seen - the higher the resolution the more details are visible
- Its limited by the diffraction on light as it passes through samples (and lenses)
- Resolution can be increased by suing beams of electrons which have a wavelength thousands times shorter than light - reduces diffraction, meaning structures can be seen separately with minimal blurriness
How would you calculate magnification?
Magnification = size of image/actual size of object
‣ In a formula triangle, size of image goes at the top
What is a graticule eyepiece?
This piece of equipment (a glass disc) is used to calibrate microscopes. Its marked with a fine scale of 1-100
What is a stage micrometer?
- A microscope slide with a very fine accurate scale in micrometers (μm) engraved on it
- The scale marked on the micrometer slide is usually 100 divisions = 1mm, so 1 division = 10μm
How would you calibrate a lens?
e.g. x4 objective lens
• Put the stage micrometer in place and the eyepiece graticule in the eyepiece
• Get the scale on the micrometer slide in clear focus
• Align the micrometer scale with the scale in the eyepiece. Take a reading from the two scales, then use this information to calculate the calibration factor for the x4 objective lens
What is electron microscopy? What kind of image is produced?
- In electron microscopy, a beam of electrons with a wavelength of less than 1nm is used to illuminate the specimen
- More detail of cell ultrastructure can be seen because electrons have a much smaller wavelength than light waves
- They can produce magnifications of up to x500,000 and still have clear resolution
Advantages and disadvantages to electron microscopy?
- Expensive to buy and operate
- Large and needs to be installed
- Complex sample preparation
- Sample preparation often distorts material
- Vacuum is required
- Black and white images produced (but can be coloured digitally)
- Over x500,000 magnification
- Low resolving powers
- Specimens are dead
What is an artefact?
Structures that are produced due to the preparation process, e.g. air bubbles
What are the two different types of electron microscopes?
- Transmission electron microscope (TEM) - a beam of electrons is transmitted through a specimen and focused to produce an image. This has a resolving power of 0.5nm
- Scanning electron microscope (SEM) - a beam of electrons is sent across the surface of a specimen and the reflected electrons are collected. This has a resolving power of 3-10nm and can produce 3D images
Advantages and disadvantages to light microscopy?
- Inexpensive to buy and operate
- Small and portable
- Simple sample preparation
- Sample preparation does not usually lead to distortion
- Vacuum is not required
- Natural colour pf sample is seen (or stains are used)
- Up to 2000x magnification
- Resolving power is 200nm - this is much higher compared to electron microscopes
- Specimens can be viewed living or dead
What is laser scanning confocal microscopy?
- A laser scanning confocal microscope moves a single spot of focuses light across a specimen (point illumination). It uses a laser to get higher intensities, improving illumination
- This causes fluorescence from the components labelled with a ‘dye’
- The emitted light from the specimen is filtered through a pinhole aperture
- Only light radiated from very close to the focal plane (the distance that gives the sharpest focus) is detected
- Produces a 2D image, however a 3D image could also be produced by creating images at different focal planes
- Currently used in science in the diagnosis of diseases of the eye and is being developed for use in endoscopic procedures
How can fluorescent tags be used?
- By using antibodies with fluorescent tags, specific features can be targeted and therefore studied by confocal microscopy with much more precision than when using staining and light microscopy
- The dye is sourced from jellyfish (𝘈𝘦𝘲𝘶𝘰𝘳𝘦𝘢 𝘷𝘪𝘤𝘵𝘰𝘳𝘪𝘢): green fluorescent protein (GTP) - this emits a bright green light when illuminated by ultraviolet light
What are prokaryotes?
Prokaryotes are single-celled organisms with a simple structure of just a single undivided internal area called the cytoplasm (composed of cytosol, which is made up of water, salts and organic molecules)
What are eukaryotes?
- Eukaryotic cells make up multicellular organisms like animals, plants and fungi
- They have a much more complicated internal structure, containing a membrane-bound nucleus (nucleoplasm) and cytoplasm, which contains many membrane-bound cellular components
What is metabolism?
Metabolism is a term that is used to describe all chemical reactions involved in maintaining the living state of the cells and the organism. Metabolism can be conveniently divided into two categories:
• Catabolism - the breakdown of molecules to obtain energy
• Anabolism - the synthesis (building up) of all compounds needed by the cells
