2.1.1 Microscopy Flashcards
What are the different types of microscopy?
- Light microscopy
- Transmission Electron microscope
- Scanning Electron Microscope
- Laser Confocal microscope
What are the pros and cons of using a light microscope?
Pros : Easy to see large cellular structures, eg cell walls, chloroplasts, nuclei. Samples can be alive or dead, Images in colour
Cons : Low magnification and resolution, ultra-cellular structure cannot be seen
What is the radiation source and radiation used for a light microscope? How is this radiation focused?
The radiation source is light and radiation used is light. This radiation is focused using glass lenses.
How are cell structures seen with light microscope images?
By using staining dyes
What is the highest magnification and resolution in a light microscope
M : x1500
R : 200nm
How does a TEM work
U
How does a TEM work
A beam of electrons is fired through a thin section of a specimen. Denser areas of the cell absorb more electrons making them appear darker on the image
Max Magnification and resolution of TEM?
M : x500,000
R : 0.2 nm
Pros and cons of TEM?
Pros: highest magnification and resolution,
Cons: Always a 2D greyscale image, colour can be added digitally, specimen always needs to be dead due to the vacuum used
How does a SEM work
A beam of electrons is fired at the surface of specimen, knocks electrons off and these reflected electrons are collected t form an image.
Max Magnification and resolution of SEM?
M : x100,000
R : 10nm
Pros and cons of SEM
Pros: Can produce a 3d image of high magnification and resolution
Cons: always a greyscale image although colour can be added digitally, Specimen has to be dead since a vacuum is used
What is the radiation source, radiation used and method of focusing radiation for electron microscopes?
The radiation source is an electron gun, electrons are the radiation used and electromagnets are used to focus the radiation
Why do you need a vacuum for electron microscopes?
Scattering of electrons by air molecules interferes with image production
How does a Laser scanning confocal microscope work?
Cells are stained with fluorescent dye and a thick section of tissue is scanned with a laser beam,
Light is focused through a pinhole on a detector - the pinhole means any out of focus light is blocked so clearer images
Pros and Cons of a Laser Confocal Microscope?
Pros : Clearer images than light microscopes, laser bean can be focused at a specific depth eliminating the blur in optical scopes caused by out of focus lenses so clearer images, 3D images can be reconstructed as images taken can be fed into a computer
Cons:
When were cells first discovered and by who?
1660s by Robert Hooke
What is magnification?
The ability of a lens to enlarge the size of any optical image
Magnification equation
Image / Actual size
What is resolution
The ability to distinguish between two points as separate to see detail
Why are stains used?
Stains are taken up better by different parts of the cell, the contrast is what makes the different parts show up better
Preparing samples for staining - light microscope
Sample left to air dry, heat fixed, can then take up stains
Preparing samples for staining - Electron microscopes
Samples dipped in solution of heavy metals, metal ions scatter creating contrast and some areas show up darker.
Preparing a sample
- fixation - prevent decay - formaldehyde added in a form of phosphate buffered solution
- embedment - infiltrated with paraffin wax
- sectioning - a single layer of cells cut 3-5micrometers with a microtome
- staining
- covered with coverslip
