Microscopy Flashcards
What is magnification?
How many times larger the size of an image is compared to the size of the object.
What is resolution?
The degree to which it is possible to distinguish between two objects that are close to each other (higher resolution means a sharper image)
What are the two types of microscope?
- Light
- Electron
What are advantages/ disadvantages of light microscopes?
Advantages:
- Prep of specimen is relatively easy
- Cheaper than electron microscopes
- Natural colour of specimen
- Can view living organisms
- Small/portable
Disadvantages:
- Lower magnification
- Lower resolution
What are advantages/ disadvnatges of electron microscopes?
Advantages:
- Can produce artifacts
- High magnification
- High resolution
Disadvantages:
- Only B/W images
- ONly dead material (in a vacuum)
- Prep of specimen is complex an requires expertise
- Large and operates in a special room
What are the two types of electron micoscope?
- Transmission electron microscope (TEM)
- Scanning electron microscope (SEM)
What are features of a TEM?
- Electron beam passed through interior of specimen
- Produces a 2D image (usually with a white background)
- Higher magnification and resolution than SEM
What are features of a SEM?
- Electron beam is scattered along specimen’s surface and transmitted to detector
- Produces a 3D image (usually with a black background)
- Can view interior and exterior
How do you prepare a temporary slide (onion) for light microscopes?
- Cut small piece of onion
- Use forceps to remove a thin layer of tissue
- Place in centre of slide
- Add a drop of stain to edge of specimen.
- Use mounting needle to gently lower coverslip at an angle (prevents air bubbles)
How do you use the irrigation method to prepare a temporary slide for light microscopes?
- Coverslip is already mounted
- Place stain on edge of coverslip
- Use filter paper to draw stain to other side
How do you prepare a permanent slide for a light microscope?
- Fixation: using chemical reagents to preserve the specimen with minimum distortion
- Dehydration: Using alcohols to remove water and prevent decomposition
- Clearing: Remove excess alcohol, leaving a transparent specimen
- Embedding: Fixing the specimen in resin/wax block
- Sectioning: Cutting block into thin slices using a microtome
- Staining: Using chemical reagents to stain different structures different colours (differential staining)
- Mounting: Securing specimen on slide with coverslip.
Why do we use stains?
- Provides contrast between different cells/structures and between cells and their backgrounds to enable them to be indentified
- Different molecules making up cell structure take up the stain to different degrees.
What is differential staining?
Using multiple stains to identify differences between two cells or two organelles (would be difficult to differntiate between them otherwise)
What is the equation for magnification?
magnification= image size / actual size
How do you calibrate an eyepiece graticule and then find the size of a specimen?
- Align eyepiece graticule with stage micrometer
- Count number of eyepiece units (epu) in each division of stage micrometer
- Use length of 1 epu =
no of micrometres/ no of epu - Then repeat with medium/ high power obective lenses
To find size of specimen: Count number of epu across spec and multiply by length of each, according to lens being used.
How many micrometres are there in one mm?
1000
