2.1,2,3 Microscopy Flashcards
What is the cell theory?
Both plants and animals are made of cells
Cells are the basic unit of life
Cells only develop from existing cells
How does a light microscope work?
There are 2 lenses - objective lens and eyepiece lens
Objective lens produces a magnified image, which is magnified again by the eyepiece lens
Illumination from a light underneath the sample
Types of sample preparation
Dry mount
Wet mount
Squash slides
Smear slides
Dry mount
Specimens are viewed whole or cut into thin slices
Specimen is placed on a slide and a cover slip on top
Wet mount
Specimens are suspended in a liquid such as water or immersion oil
Coverslip is placed on from an angle
Squash slides
Wet mount is prepared
Lens tissue is used to press down on the coverslip
Squash slides are used to look at soft samples
Smear slides
Edge of a slide is used to smear the sample
Creates a thin, even coating on another side
Cover slip is placed over the sample
What is bright field microscopy?
Sample being illuminated from below with white light and observed from above
What is wide field microscopy?
Whole sample is illuminated at once
Why is staining used?
Interior of cells is usually transparent
Increases contrast between organelles
To differentiate between 2 types of organisms
What charge is crystal violet and methylene blue?
Positively charged - attracted to negatively charged materials in the cytoplasm, so cell components are stained
What charge is Nigrosin and Congo red?
Negatively charged, so they are repelled by negatively charged cytosol. These dyes stay outside the cell, so cells stand out against stained background
What is negative staining?
Using negative/acidic stains that do not bind to the specimen
Clear specimen is observed against a dark coloured background
The negative stain does not penetrate the cell surface membrane
What is differential staining?
Distinguishing between 2 types of organisms that would be hard to identify otherwise. It can also differentiate between different organelles of an organism
What is the gram stain technique?
Used to separate bacteria into 2 groups - gram positive and gram negative
Crystal violent is added, then iodine (fixes the dye)
Slide is washed with alcohol
Gram positive bacteria will retain the crystal violet stain and appear blue/purple
Gram negative bacteria have thinner walls, so will lose the stain
These are stained with safranin dye to turn red
Gram positive/negative bacteria and penicillin
Gram positive bacteria are susceptible to antibiotic penicillin - prevents formation of cell walls
Gram negative bacteria have thinner cell walls and are not susceptible to penicillin
What is the acid fast technique?
Used to differentiate mycobacterium from other species
Lipid solvent is used to carry cabolfuchsin dye into the cells
Cells are washed with a dilute acid-alcohol solution
Mycobacterium aren’t affected by the acid-alcohol solution and retain the stain (bright red)
Other bacteria lose the stain and are exposed to methylene blue stain
Preparing slides
Fixing - chemicals like formaldehyde are used to preserve specimens
Sectioning - specimens are dehydrated with alcohol, placed in a mould with wax/resin to form a hard block, can be slices with a knife called microtome
Staining - sample are treated with multiple stains to show different structures
Mounting - specimens are secured to a microscope slide and a cover slip on top
How does electron microscopy work?
Beam of electrons with wavelength less than 1nm illuminates the specimen
What are artefacts?
Structures produced due to the preparation process
2 types of electron microscope
Transmission electron microscope
Scanning electron microscope
TEM
Beam of electrons transmitted through a specimen
Best resolution - 0.5nm
SEM
Beam of electrons sent across the surface of the sample and reflected electrons are collected
Resolving power of 3-10nm
Produces 3D images
Why is the inside of an electron microscope a vacuum?
Ensures the electron beams travel in straight lines
Advantages of light microscope
Inexpensive to buy and operate Small and portable Simple sample preparation Natural colour of sample is seen or stains are used Specimens can be living or dead
Disadvantages of light microscopes
Lower magnification up to x2000
Lower resolution around 200nm
Advantages of electron microscopes
Large magnification of over x500 000
High resolutions
Disadvantages of electron microscopes
Expensive to buy and operate Large and needs to be installed Complex sample preparation that could damage sample Vacuum is required Specimens have to be dead
How does a laser scanning confocal microscope work?
A single spot of focused light is moved across a specimen
This causes fluorescence from the components labelled with a dye
The light emitted from the specimen is filtered through a pinhole aperture
Why is a laser used instead of light?
This results in higher intensities and improves the illumination
How is a 3D image produced in LSCM?
By creating images at different focal planes
What is magnification?
How many times larger the image is than the actual object being viewed
What is resolution?
The ability to see objects as separate entities
What is resolution limited by?
The diffraction of light as it passes through samples and lenses
Diffraction is the spreading out of light waves
Magnification formula
Size of image/actual size of object
What is an eyepiece graticule?
Glass disc marked with fine scale of 1-100
Remains unchanged for all objective lens
What is a stage micrometer?
Microscope slide with a very accurate scale in micrometres engraved on it
