Microscopy Flashcards
What are the three principles of cell theory?
1) All living organisms are composed of one or more cells
2) The cell is the basic unit of structure and organisation in organisms
3) Cells arise from pre-existing cells
What did Robert Hooke contribute to cell theory?
- He observed cells in cork
- Was the first man to use the word ‘cell’
- Wrote a journal of his micro graphs called micrographia.
What did Antonie van Leeuwenhoek contribute to cell theory?
- First person to document the microscopic observations on LIVE samples
- Such as bacteria, red blood cells, spermatozoa, muscle fibres
- Called these microbes ‘tiny animals’
- Found that living things were indeed made up of cells.
What did Barthélemy Dumortier contribute to cell theory?
- First person to observe cell division in plants using microscopy
- This was used as evidence for cell theory, that cells were created from old ones, rather than spontaneously created.
What did Theodor Schwann contribute to cell theory?
-Proposed the extension of cell theory from just plants to animals as well.
What did Robert Remak contribute to cell theory?
- Discovered that the origin of cells was by the division of pre existing cells
- Observed cell division occur in frogs eggs immediately after fertilisation.
What did Louis Pasteur contribute to cell theory?
- Had many discoveries in vaccinations, microbial fermentation and pasteurisation.
- FERMENTATION provided firm evidence against the spontaneous generation of life and cells
- Allowed full acceptance of full cell theory.
What was Louis Pasteur’s pasteurisation experiment?
- Heated three swan neck flasks
- One was left open and no bacteria was present
- Bacteria couldn’t reach fermentable liquid, which went against spontaneous creation
- One had the long neck removed
- Bacteria could reach fermentable liquid
- One was tipped to the side
- Bacteria could reach fermentable liquid.
What are the disadvantages of electron microscopy?
- Expensive to buy and operate
- Specimens have to be dead
- Complex sample preparation
How does a light microscope work?
- Light is focused by glass lenses
- Light is passed through specimen
- Light absorbed creates the image
- Image is magnified by objective lens, then again by eyepiece lens.
What are the advantages of a light microscope?
- Inexpensive to buy and operate
- Don’t require a lot of training to use
- Vacuum isn’t required (can use on live samples)
- Simple sample preparation.
What are the disadvantages of a light microscope?
- Relatively low resolution 200nm
- Relatively low magnification 2000x
What is the equation for magnification?
Magnification=Image
_____
Actual
One eyepiece graticule division equation
Number of micrometers on the slide micrometer
______________________________________
Number of eyepiece graticule divisions.
How to prepare a dry mount microscope slide?
- Solid specimen viewed whole or cut into thin slices
- Specimen is place on centre of slide
- Cover slip placed over specimen
- Used for pollen, insects, etc.
How to prepare a wet mount slide?
- Specimen is suspended in liquid such as water or immersion oil
- Cover slip placed over specimen at an angle (avoid air bubbles)
- Aquatic samples can be viewed this way
How to prepare a squash slide?
- Prepare a wet mount slide
- Use lens tissue to gently push down on cover slip to squash sample
- Can also squash sample between to microscope slides as cover slip is delicate
- This can be used for root tip squashes to observe cell division.
How to prepare a smear slide?
- Edge of microscope slide used to smear sample onto another microscope slide
- This creates a thin, even coating
- Cover slip is place over sample
- Used on samples of blood to look at cells.
Why must specimens be thin?
To allow light to pass through
Why must the refractive index of medium used in wet mount be the same as glass?
Because this will keep the rays of light straight so an in-focus image is produced.
How to focus a microscope?
- Move stage to the highest level using coarse focus mechanism
- Start with the lowest magnification objective lens
- Place your microscope slide on stage, held in place by stage clips
- Move the stage down until it comes into view and focus using the fine focus mechanism.
Why do we need to calibrate microscopes?
Because the magnification stated on each lens can vary, so we need to calibrate so we can find the true magnification.
Why do we stain specimens?
- Higher contrast because different organelles take up more stain than others
- More internal structure visible
What are the rules for a good scientific drawing?
- Drawing and label lines done in sharp pencil
- Drawing should take up at least half the page
- Lines need to be continuous
- Ensure proportions are correct
- Add labels of what you see
- Add extra ANNOTATIONS (colour, texture)
- State magnification
- Have a title and name stain used
- Make sure label lines touch what you’re labelling
- NO shading or colour
What are the differences between the eyepiece graticule and stage micrometer?
- Eyepiece graticule remains constant regardless of the magnifications, whereas the stage micrometer increases in size
- This way, you can calibrate the microscope at every magnification.
How does a transmission electron microscope work?
- Beam of electrons, in a vacuum, are transmitted through a sample from an electron gun
- The beam is focused by electromagnetic lenses
- Beam passes through specimen and either scatter or hit a fluorescent screen at the base of the microscope, which magnifies the image.
How are samples prepared for TEM microscopy?
Samples are embedded in resin, stained with heavy metal compounds and cut into into ultra thin sections.
How does a scanning electron microscope work?
- A beam of electrons, in a vacuum are focused by electromagnetic lenses
- The fine beam of electrons scans the surface
- The secondary electrons emitted by atoms which have interacted with the electron beams are detected using secondary electron detector.
- These secondary electrons that are collected, create the image.
What are the advantages of electron microscopy?
- Much higher resolution
- Allow a cell’s ultrastructure to be seen
- Resolving power of TEM 0.5nm
- Resolving power of SEM 3-10nm
- Higher magnification over 50,000X
- TEM produces detailed images of cell ultrastructure
- SEM produces 3D images and great surface detail of an object inside a cell
How are samples prepared for SEM microscopy?
- Have to be small enough to fit on the specimen stage
- Needs special preparation to increase sample’s electrical conductivity
- Non-conduction specimens are coated with an ultra thin layer of gold, platinum, chromium, etc.
What are artifacts?
Anomalies apparent in both microscopy and photography.
What is an example of an artefact?
Mesosomes
- People believe they were part of a cell’s ultrastructure but they actually don’t exist
- They in fact were artefacts
What are the disadvantages of electron microscopes?
- Expensive to buy and operate
- Compex sample preparation
- Vacuum is required
- Specimens have to be dead
How does a fluorescence microscope work?
- Uses fluorescence to create an image of a specimen
- Fluorescent dyes contain fluorophores
- Fluorophores emit longer wavelengths of light
- This creates a high resolution image
- The dichroic mirror reflects only one colour of light which passes through the specimen.
How does a laser scanning confocal microscope work?
- Is a refinement of the fluorescence microscope
- Single spot of focused light focused over specimen and causes fluorophores in sample to fluoresce
- A confocal pinhole blocks any out of focus light, and therefore the image is higher resolution.
How does a super resolved fluorescent microscope work?
It creates a higher detail image by combining many smaller and overlapping images of a specimen
AND
Digitally superimposing many images on top of each other to create a higher resolution image.
RESOLUTION BREAKS THEORETICAL LIMIT OF 0.2nm for optical microscopes
How does an atomic force microscope work?
Microscope generates images by scanning a cantilever over the surface of a sample.
Can generate images of things the size of fractions of a nanometer
-1000x more than optical limit
-Shape traced with cantilever is processed and an image is created.
What is the definition of magnification?
How many times larger the image is than the actual size of the object being viewed
What is the definition of resolution?
Ability to see individual objects as separate entities
How to convert mm to nm?
1x10^6
How to convert mm to um?
1x10^3
