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
Function of membranes
They’re selectively permeable and control the movement of substances into and out of the cell and organelles