Chapter 2 Microscopy Flashcards
Why is staining used?
Increases contrast of the cells/organelles/image
Allows internal structures e.g organelles to be more easily seen, also helping distinguish between them
Can help distinguish between different types of cells
Effectively increases clarity of the image
What is staining?
Add a chemical which is absorbed by certain cells/organelles of a sample, increasing contrast
How should samples be generally prepared?
Using a sharp blade, cut the sample very thinly to allow the light to pass through the specimen, increasing resolution
Place on a slide with forceps
Use some sort of mount ideally- wet mount or smear or squash, to allow more cells to be seen
How should a stain be added?
Place the very thin sample on the slide with forceps
Add one or two drops of the stain to the edge of the sample
Use blotting paper to remove excess stain
Lower the cover slip at an angle with an angled needle
What is the purpose of a wet mount? What is the purpose of squashing?
Wet- prevents dehydration/distortion of the tissue
Squash- allows light to penetrate more easily
What is the resolution, magnification, image and use of TEMs?
Around 0.2nm x500000
2D, black and white image with a very high resolution
Used to find details of cell ultrastructure
What is the resolution, magnification, image and use of SEMs?
Around 3-10nm x100000
3D, black white images
Used to determine information of cell surface, as 3D
What is the resolution, magnification, image and use of light microscopes?
Around 200nm x2000
2D, coloured images
Info about live samples, cells
How do TEMs work? What are some disadvantages of them?
Beam of electron through sample
Can produce artefacts, require complex sample preparation, including very, very thin samples
What is an artefact?
Distortions in the image caused by the sample preparation, mostly with electron microscopes, can be air bubbles with light
How does SEMs work? What are some advantages of them?
Beam of electrons send across the surface of a specimen, electrons detected to form a 3D image
Very expensive, lower resolution than TEM
What are the general advantages/disadvantages of light microscopes?
Cheap, Portable, Easy to prepare samples, Alive or dead sample, natural colour, no vacuum needed
But lower resolution
What is the process of electron sample preperation?
Chemical Fixation
Cryofixation
Dehydration, ethanol to preserve
Embedding in resin
Sectioning
Staining with heavy metals
Mounting
What the types of slide preperation of light microscopes?
Dry Mount- cover slip parallel
Wet Mount- in liquid e.g oil, cover slip angled
Squash- Wet then squash
Smear- with cover slip
What is fluorescence?
The absorption and re-emittance of of light at lower energy and higher wavelength, so magnified
How does a LSCM work?
Laser Scanning Confocal Microscope
Dye in sample
Point illumination- moving a laser across the specimen
Light emitted as fluorescence, only light closest to focal plane allowed through pinhole aperture to increase resolution, remove background noise
What are advantages of LSCM?
Very high resolution
Depth selectivity
Define magnification
How many times larger an image is compared to the real size
Define resolution
The ability to see two points which are close together as two separate entities
What must be increased to see greater detail?
Magnification and Resolution
What happens with light that limits resolution?
Tendency for light to spread when passing through a specimen, diffraction
If distances between points closer than 0.5 wavelength of light, cannot be seen individually, as waves overlap
Wavelength of electrons lower, so small distances between points able to be seen
What is the equation with microscopes?
Image Size= Real Size x Magnification
What is an eyepiece graticule?
Glass disc on microscope with arbitrary units, used with a stage micrometer to measure the length of samples
What is a stage micrometer?
A microscope slide containing a total distance of 1mm, with 100 divisions. Each division is 10 micrometres
Used to calibrate at each magnification
How can a microscope be calibrated?
Align stage micrometer with eyepiece graticule
Divide to get the value of one EPU
Multiple any measurement of sample at this magnification by the value of one EPU
How can you tell an image is from an TEM?
High resolution and magnification
As very small organelles such as ribosomes can be seen, and more detail of organelles such as nuclear pores
