Intro to some Optical, Diffraction Methods used in the Biological Sciences Flashcards
Figure showing sizes of cellular and subcellular structures
Whats the magnification and resolution of light microscope
- Up to 1000x
- Resolves details as small as 0.2 micrometres
What are the 3 things required for viewing specimens in a light microscope?
- Bright light focused on the specimen by lenses
- Specimen carefully placed to allow light to pass through it
- Appropriate set of lenses (objective and eyepiece) must be arranged to focus an image of the specimen in the eye
What systems show how you can interchange optical components to view an image differently on a light microscope
- straightforward (brightfield) optics
- Phase contrast optics
- interference contrast optics
Exploit the differences in the way light travels through regions of the cell with differing refractive indexes
Most tissues aren’t small enough to be examined in a microscope. What is done about this?
Chemically fixed and cut into very thin slices or sections that can be mounted on a glass microscope slide and subsequently stained to reveal different components of the cells.
slide on fluorescence microscopy
How does fluorescence microscopy work?
Illuminating light is passed through two sets of filters. The first filters the light before it reaches the specimen, passing only those wavelengths that excite the particular fluorescent dye. The second blocks out this light and only those wavelengths emitted when the dye fluoresces. Dyed objects show up in bright colour on a dark background.
Explain fluorescent probes
Fluorescent dyes absorb light at one wavelength and emit it at a longer wavelength. Some dyes bind specifically to particular molecules and can reveal their location when viewed with a fluorescence microscope. An example is the stain for DNA. Other dyes can be coupled to antibody molecules, which then serve as highly specific and versatile staining reagents that bind selectively to particular macromolecules, allowing us to see their distribution in the cell. In the example shown, a microtubule protein in the mitotic spindle is stained red with a fluorescent antibody
What are the 2 types of electron microscopy?
Transmission and scanning
How does transmission electron microscopy work?
The specimen, which is placed in a vacuum, must be very thin. Contrast is introduced by electron-dense heavy-metal stains that locally absorb or scatter electrons, removing them from the beam as they pass through the specimen.
- Magnification of up to a million-fold
- Resolves detail to 2 nm
How does a scanning electron microscope work?
The specimen, which has been coated with a very thin film of heavy metal, is scanned by a beam of electrons brought to a focus on a specimen by the electromagnetic coils that, in electron microscopes, act as lenses. The quantity of electrons scattered or emitted as the beam bombards each successive point on the surface of the specimen is measured by the detector and is used to control the intensity of successive points in an image built up on a screen.
- Resolve up to 3-20nm
What is X-ray crystallography used for?
A tool for determining the atomic structure of macromolecules is the scattering x-ray radiation by crystals.
Crystals can be considered as a regular array of atoms. What are the 7 basic crystal structures?
How can x-ray crystallography determine atomic structure?
Bragg showed that the scattering of X-rays from a crystal can be described as the scattering from parallel planes of atoms.
Given a monochromatic beam of wavelength lamda = 0.585 Angstroms and a cubic lattice with a particular family of planes with d = 2.86 Angstroms.What is the (n=1 first order) Bragg reflection angle? What would it be for lamda = 6 Angstroms?
nlamda = 2d sin(theta)
sin(theta) = (0.585)/[(2) (2.86)] = 0.491
so theta = 24.1 degrees
If l = 6 Angstroms a reflection will not be observed because lamda > 2d
