Microscopy 🔬 Flashcards
(21 cards)
How does a light microscope work?
- Light source shines on specimen
- Objective lens magnifies image –> further magnified by eyepiece (10x).
- low contrast images = cells do not absorb light —-> cells are stained.
- Limited resolution: wavelength of light = long –> diffracts through sample.
Advantages of light microscope
- Not expensive
- Portable
- Simple sample preparation
- Specimens can be living or dead
Disadvantages of light microscopy.
Low magnification + resolution (200nm) compared to electron microscopes.
Describe laser scanning confocal microscopy.
- Single spot of light moved across specimen.
- Specimens labelled with antibodies + flourscent dyes.
- GFP = green light under UV
- Filtered through pinhole apeture.
- Only light radiated from focal plane detected.
- 3D image
Advantages of scanning microscopes
- Simple sample preparation
- Specimen can be viewed dead or alive.
Disadvantages of scanning microscopes
Low magnification and resolution (2/500 nm) compared to electron microscopes.
Expensive
Describe scanning electron microscope
- Beam of electrons less than 1nm
- Beam of electrons sent across surface of specimens + refracted electrons are collected.
Advantages of SEM
High resolution: e- wavelength smaller than light
High magnification
Disadvantages of SEM
- Expensive to buy and operate
- Risks of artefacts
- Black and white images
- Specimens are dead
- Large and needs to be installed
Describe transmission light microscope
- Beam of electrons less than 1 nm
- Viewed in a vaccuum so electrons travel in a straight line.
- Preparation includes sample embedded in resin, staining with heavy metals + dehydration.
Advantages of TEM
High resolution (0.1nm)
High magnification
Disadvantages of TEM
- Risks of artefacts —->contamination/airbubbles
- Specimen are dead
- Black and white imaging
- Expensive
- Complex sample preparation
What is resolution?
The minium distance between two objects in which they can still be seen as separate.
- Optical —-> wavelength of light
- Electron —-> wavelenght of electrons
What is magnification?
Refers to how many times larger the image is compared to the object.
What is a dry mount sample slide?
- Thinly sliced/whole specimens viewed.
- Coverslip placed ontop(sectioning)
What is a squash mount sample?
Wet mount but slide pushed down to ensure thin layer of cells so light can pass through
What is a wet mount sample?
Sample suspended in liquid
Coverslip lowered at angle with mounted needle to prevent any air bubbles.
What is a smear sample?
Edge of 1 slide used to smear sample across another slide
Creates thin + evenly coated specimen.
Coverslip placed on top.
Used in blood samples.
What is differential staining?
Uses many chemical stains which stains different parts of the cell different colours.
- Crystal violet/methylene blue: positively charged .: attracted to negatively charged materials
- Nigrosin/Congo red: negatively charged –> cannot enter cell (cytosol repels them)
–> stains background .:unstained cells stand out.
What is gram staining?
Used to separate bacteria into groups.
- Crystal violet added to sample
- Iodine = fix stain.
- Alcohol = remove unbound stains from sample.
Gram positive stains = blue/purple
–> stain retained due to thick peptidoglycan cell walls absorbing dye.
Gram negative stains cannot absorb crytal violet due to thin peptidoglycan.
- Safranin used as a counter stain to turn cells red.
- Helpful to differentiate which antibiotic needed to bacterial infection.
How to calibrate an eye piece graticule?
- Line up stage micrometer + eyepiece graticule while looking through eyepiece.
- Count how many divisions on the eyepiece graticule fit into one division on the micrometer scale.
- Each division on the micrometer is 10um —–> used to calculate what 1 division on eyepiece graticule is at that current magnification.
