Imaging and Studying Cells Flashcards
Define ultrastructure
Level of detail obtainable with the EM
Define magnification
The ration of an objects image size to its real size
Define resolution
The measure of the clarity of the image or the minimum distance of two distinguishable points
Define contrast
Visible differences in brightness or colour between parts of the sample
How does the bright field microscope work?
Light passing through specimen (specimen must be partially transparent)
What is the resolution of light microscope?
0.2 microns
How does a fluorescent light microscope work?
Fluorescent substances absorb short wavelengths and emit longer wavelengths emitting visible light
How does a confocal light microscope work?
Uses lasers for optical sectioning. Regions with a narrow depth are focussed
Outline the main steps for light microscope sample preparation
Small transparent specimens can be used
Tissues must be sectioned. Fixation occurs using chemical fixation. Water removed by dehydration and clearing. Embedding; the specimen is infiltrated with molten wax. Cut into thin sections. Wax is removed and tissue is stained.
How does a advanced light microscope work?
Light phase shifts to generate contrast
What is deconvolution microscopy?
Algorithms remove out of focus light and sharpens the image and improves resolution.
How does super resolution microscopy work?
Gathers light from individual fluorescent molecules and records their position.
Why does the electron microscope have a higher resolution?
Electrons have a very short wavelengths (resolution is 100xs bigger)
Why do electron microscopes require a vacuum?
Electrons have poor penetrating power
Describe how a transmission electron microscope works
Electron beam passes through specimen
Image focussed by magnetic field
Electron image is converted into visible image by a fluorescent screen which is viewed through a glass window
Photographs can be taken
Describe the process of transmission electron microscope sample preparation
Whole mount examined Fixation in Glutaraldehyde then in Osmium tetroxide Dehydration in an ethanol series Specimen embedded in plastic resins Cut Heavy metal stain
Which microscope always gives 3D appearance?
SEM- scanning electron microscope
Describe the process of scanning electron microscope sample preparation
Sample fixed and dried
Fixation in Glutaraldehyde then in Osmium tetroxide
Dehydration by ethanol replacing water
Critical point drying to remove ethanol and minimise shrinking
Coat in thin layer of gold
Why is SEM specimen coated with gold?
To prevent electron beam damage
Why is TEM specimen stained with heavy metals such as lead?
To improve contrast
What is the purpose of cell fractionation?
Allow major organelles to be individually separated out. They’re homogenised to release organelles.
Define differential centrifugation
Isolates cell components on the basis of size and density by using increasing durations and g forces
