Cells - Methods of Studying Cells Flashcards
What are the equations for magnification?
magnification = image size / actual size
magnification = objective lens x eyepiece lens
How do you draw scientific diagrams?
- large
- boundary lines are thin and clear
- use blank paper
- labelling communicates cell features observed
- do not cross label lines
- include magnification or a scale
- most features that are visible are included
- evidence of 3D structure that is visible using controlled focussing
- dotted lines are used for features that are out of the plane of focus
- no sketching, shading, colouring, crossed lines or hanging lines
- use stippling to show darker areas
- if possible, place all labels at the right-hand side of the drawing
What is magnification?
The number of times bigger the image/drawing is compared to the object/real size. It is controlled by the power of the lenses used.
What is resolution?
The minimum distance needed to differentiate between 2 adjacent objects - it is a measure of the clarity of the image. It is controlled by the wavelength of the illumination used.
What is a millimeter?
1x10-3 m
What is a micrometer?
1x10-6 m
What is a nanometer?
1x10-9 m
What is a picometer?
1x10-12 m
What is a light microscope?
A type of microscope which has a condenser, objective lens, and eyepiece lens and light is passed through the thin specimen and up through the objective and eyepiece lenses to the eye. Light microscopes use a pair of convex glass lenses that can resolve images that are 0.2um apart. The reason for this is that this is the wavelength of light and therefore restricts the resolution that a light microscope resolves to.
What is an electron microscope?
Beams of electrons are used to visualize structures in a vacuum. The vacuum environment is needed so that particles in the air do not deflect/scatter the electrons (which are very small) out of the beam alignment. Electrons have a smaller wavelength than light so electron microscopes have a higher resolution than light microscopes. Electron microscopes can distinguish between items 0.1nm apart. Electrons produce an image when focused onto a fluorescent screen.
How do you prepare a temporary mount?
- Add a drop of water at the centre of the microscopic slide.
- Cut a thin slice of the sample and lay it at the centre of the microscopic slide (the drop of water will help flatten it).
- Add a drop of stain to make the sample more visible.
- Some parts of the sample will absorb more stain than others, producing a black and white image on the screen.
- Gently lay a microscopic coverslip on top and press it done gently using a needle to remove air bubbles.
- Touch a blotting paper on one side of the slide to drain excess iodine/water solution.
- Place the slide on the microscopic stage under low power to observe.
What are the differences between a light microscope and an electron microscope?
LIGHT MICROSCOPE
- use a beam of light
- use a light bulb which only requires a low voltage
- use a condenser, objective and eyepiece lens
- image can be seen directly by the eye
- the radiation medium that is used is air
- low magnification (up to x1,500)
- low resolution (200nm)
- cheap
- use glass lenses to focus light rays
- easy to use
- can show the structure and arrangement of tissues, microscopic organisms, living or dead, but it cannot show the cell ultrastructure
ELECTRON MICROSCOPE
- use a beam of electrons which require high voltages for generation
- use an electron gun
- use a condenser, objective and eyepiece lens
- a fluorescent (TV) screen is required to see the image
- the radiation medium that is used is a vacuum, since electrons are easily scattered by the particles in air
- high magnification (up to x500,000)
- high resolution (0.2nm)
- expensive
- use electromagnets arranged around the path of the electron beam, which can deflect and thus focus electron beams
- specialised training required
- can only show dead structures, but will show the cell ultrastructure including the fine structure of cell organelles
What is a scanning electron microscope (SEM)?
All the limitations of the TEM apply to the SEM, except that specimens need not be extremely thin as electrons do not penetrate. In a scanning electron microscope, a beam of electrons is reflected off the prepared surface of the specimen. The sample is covered in gold metal. The beam is then passed back and forth across a portion of the specimen in a regular pattern. The pattern of scattering builds up a 3D image depending on the contours of the specimen. We can build up a 3D image by computer analysis of the pattern of scattered electrons and secondary electrons produced. The basic SEM has a lower resolving power than TEM, but is still better than a light microscope.
- has a lower magnification than TEM
- specimen doesn’t have to be thin
- higher resolving power than light microscope
- can produce 3D image
What is a transmission electron microscope (TEM)?
A type of electron microscope that passes a beam of electrons through or past a very thin section of the specimen (which often has been stained with heavy metals to show up the fine internal structures) on their way to the fluorescent screen or photographic film. The beam of electrons is focused onto the specimen by a condenser electromagnet. Areas that absorb the electrons appear darker on the electron micrograph that is produced. Other parts of the specimen allow the electrons to pass through and so appear bright. The image that is produced on the screen can be photographed to give a photomicrograph.
- specimen has to be thin
- higher magnification than SEM
- higher resolving power than light microscope
- produces detailed images
What is cell fractionation?
The process where cells are broken up and the different organelles they contain are separated out so that they can be studied in detail.
The most common method of cell fractionation is differential centrifugation.
What is homogenation?
The first stage of cell fractionation when cells are broken up by a homogeniser (blender) and organelles are released from the cell. The resultant fluid, known as homogenate, is then filtered to remove any complete cells and large pieces of debris.
What is ultracentrifugation?
The second stage of cell fractionation when the fragments in filtered homogenate are separated in a machine called a centrifuge. This spins tubes of homogenate at very high speed in order to create a centrifugal force.
What are the limitations of using electron microscopes?
- The whole system must be in a vacuum so living specimens cannot be observed.
- A complex staining process is required and even then the image is not in colour and may introduce artefacts into the image.
- Artefacts are things that result from the way the specimen is prepared. Artefacts may appear on the finished photomicrograph but are not part of the natural specimen. It is therefore not always easy to be sure that what we see on a photomicrograph really exists in that form.
- Specimens have to be very thin, particularly for TEM so that the electrons can pass through. The result is therefore a flat, 2D image. You can partly get over this by taking a series of sections through a specimen. We can then build up a 3D image of the specimen by looking at the series of photomicrographs produced. However, this is a slow and complicated process.
- SEM has a lower resolving power than TEM, but both have greater resolving power than a light microscope.
What is the process of cell fractionation?
- The cells are first blended in an homogeniser forming the resultant fluid called the homogenate. This tube of homogenate is then placed in a centrifuge and spun at a slow speed.
- The heaviest organelles, the nuclei, are forced to the bottom of the tube where a thin sediment or pellet forms.
- The fluid at the top, called the supernatant, is removed which leaves just the sediment of the nuclei. The supernatant is then transferred to another tube and spun at a slightly faster speed. This time the pellet that forms contains the next heaviest organelle, the mitochondria, which are forced to the bottom of the tube.
- This process continues so that each time the speed is increased the next heaviest organelle is sedimented and separated out.
What is the limit of resolution?
The limit of resolution is defined as the smallest distance at which it is possible to distinguish two separate items. Up to this point, increasing the magnification will reveal more detail but beyond this point, increasing the magnification will not do this. The object, while appearing larger, will just be more blurred.
Under what conditions does cell fractionation occur? Why?
Buffer solution - keeps pH constant so enzymes in the cell’s organelles don’t denature.
Isotonic water potential - prevents water from moving in and out the organelles by osmosis and bursting them (cell lysis) or shrinking them under osmotic pressure.
Cold - so the enzymes work more slowly, preventing destructive enzymes from destroying organelles.
What are the relative sizes of the organelles, largest to smallest?
- nucleus
- chloroplasts, mitochondria
- lysosomes, endoplasmic reticulum
- ribosomes
Explain how to calibrate and use an eyepiece graticule.
To calibrate a graticule, you observe a special slide called a stage micrometer. When the stage micrometer and graticule are lined up, you can calculate the length that each division on the graticule is equivalent to.
How would you prepare cheek cell slides?
- Using a cotton bud, remove some cells from the inner part of your cheek.
- Smear them onto a microscopic slide.
- Add a drop of methylene blue solution to the cells.
- Carefully place a coverslip over the cells.
