Microscopes Flashcards
LM
Bright field
used for looking at stained samples
LM
Phase contrast
Enhance the natural contrast of a specimen
LM
fluorescence
Tag/label specific components
Light shined onto a specimen and fluorescent molecules are excited
LM
Deconvolution
Remove out of focus information
LM
Confocal
Remove out of focus information
LM
what does focal length depend on?
curvature of a lens
A long focal length lens is less curved and a short focal length lens is highly curved
LM
Convex lens
Converges parallel beams to a focal point.
A single convex lens produces an enlarged virtual image if the object is closer than the focal point.
If the object is further than the focal length a real image is formed
LM
Concave lens
Diverges parallel beams
The focal point is the point they would have converged from
LM
Objective lens
Lens to produce a magnified real image. A real image can only be seen if you place a screen.
LM
Eyepiece lens
Produces a magnified virtual image of the real image
Bright field microscopy
Light is passed through a specimen. Scattering of light and interference produces an image. This image is magnified and focused on the retina or detector
FM
Fluorescence microscopy
How does FM work?
Different parts of a cell are stained using specific dyes or antibodies which are attached to fluorescent molecules.
specimens are illuminated with light of a specific wavelength to excite a fluorophore. A fluorophore will emit light with a longer wavelength then the image is magnified and focused on a retina/detector.
How are fluorescent tags attached to antigens?
A primary antibody will bind to an antigen. A secondary antibody with a fluorescent tag will then recognise and bind to the primary antibody.
Filters in fluorescent microscopy
A light source shines onto a filter which filters out wavelengths of light that are not wanted. The specimen is illuminated with light of a specific wavelength,
Explain fluorescence
A specimen is illuminated with a wavelength to excite a fluorophore/electron moving the electron into a higher energy level. As the electron moves back down an energy level the light is emitted.
Green fluorescent protein
A chromophore is held within the green fluorescent protein- this is the molecule that is excited by a light wavelength.
This protein can be genetically tagged with other proteins.
GFP and tubulin
Tublin can be tagged with GFP as a tubulin gene is added into GFP. Stick plasmid containing these genes into a mammalian cell, transcription and translation occurs. There is a shorter linker peptide between the tubulin and GFP genes as otherwise the tubulin would not be incorporated into microtubules.
Confocal or deconvolution microscopy
Improve the clarity and resolution of fluorescence images.
Confocal microscopy pinhole prevents out of focus light reaching the detector so filters it out.
TEM
Transmission electron microscopy
2D projection image of a thin specimen
Electrons scattered and shadow image created of the specimen
SEM
Scanning electron microscopy
Electrons scattered across a sample surface and electrons are emitted off the surface
TEM components compared to a light microscope
TEM occurs in a vacuum, no glass lenses only electromagnetic lenses
Describe how SEM works
Primary beam from an electron beam is focused to a fine point
Beam scanned across a sample surface
Secondary electrons emitted from the specimen as the beam passes over it
Secondary electrons are collected by a detector and a picture is built up
EM
condenser lens
Focuses electrons onto specimens
Controls diameters of beams- wide at a low magnitude
