Microscopy

Description

Mind Map on Microscopy, created by Hazelcatrina on 01/06/2014.
Hazelcatrina
Mind Map by Hazelcatrina , updated more than 1 year ago
Hazelcatrina
Created by Hazelcatrina about 11 years ago
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Resource summary

Microscopy
  1. using a microscope
    1. make things look bigger and clearer to the naked eye
      1. microscope
        1. objective lens
          1. lens closest to the object or specimen. the information gathering lens of an optical system
          2. eye piece
            1. projection lens system projecting an image onto the retina of the eye
            2. substage condenser
              1. focuses light onto the specimen
                1. controls resolution versus contrust
                2. iris diaphragm
                  1. resolution versus contrast
                    1. controls the ratio between resolution and contras
                    2. light intensity control is the sole means to adjust the brightness.
                  2. Light Microscopy
                    1. uses electromagnetic radiation in the ultraviolet or visible wavelength range to obtain a magnified imagine of an object
                      1. the resolution of the imaging is limited by the minimum focus of the radiation due to difraction
                        1. the difraction limit is approximately 1um
                        2. magnification and imagine formation
                          1. magnification is the enlargement of an image
                            1. magnification restricts clear image formation the following are affected
                              1. resolution
                                1. the ability to see objects as being separate
                                  1. if you have two objects and and put them closer and closer together at some point you will not be able to see them as separate objects. and that is the limit of resolution.
                                  2. the ABBE equation
                                      1. D relates the distance between two objects
                                        1. as alpha increases D gets smaller and resolution increases
                                        2. as d decreases, resolution increases
                                          1. as wavelength becomes smaller, D becomes smaller, increasing resolution
                                            1. you can improve the resolution by changing the wavelength. i.e by decreasing the wavelength
                                            2. relates refractive index and wavelenght
                                              1. defines resolution
                                                1. as wavelength decreases, d decreases and resolution increases
                                              2. depth of field
                                                1. affects the image you are actually seing.
                                                2. contrast
                                                  1. relates to the colour light absorption of one material in comparison to another e.g black on white would have high black on black will have no contrastcontrast whereas
                                                    1. staining helps to eliminate the issue with no contrast
                                                      1. used when looking at proteins, lipids or antybody
                                                        1. stains can damage living cell samples, limiting problem as it is inappropriate for delicate or living material
                                                  2. optical aberrations
                                                    1. spherical abberation
                                                      1. for monochromatic light the focus point varies with lens position
                                                        1. may result in fuzzy images that are not clear
                                                          1. can be solved by reducing the amount of light. or reducing the amount of lens that is being exposed to light, can use apertures, this narrows the light source, ligtht more likely to hit the centre of the lends and focus in one point
                                                          2. chromatic aberation
                                                            1. for polychromatic light the focus point varies with wavelegth
                                                              1. rainbow effect but the red diffracts less than the blue because its a shorter wavelength. the imaging will be the product of two different points. the light focuses over a range therefore you get a fuzy image
                                                                1. can be resolved by using a combination of lens with different refractive indices allowing you to recombine the colour, put it back together to white light using a second lens, however this method is expensive
                                                      2. wavelength of light restricts image resolution
                                                      3. visible light: phase contrast microscopy
                                                        1. light paths varies therefore phase varies.
                                                          1. converts invisible phase difference into intensity differences
                                                            1. manipulates phase differences to improve contrast using interference. manipulating the waves to get them in the right place and size
                                                            2. two unique components in phase contrast system
                                                              1. annular diaphragm which directs a hallow cone of light through specimen
                                                                1. where the light comes from, (shines through a ring and forms a ring of light)
                                                                2. a diffraction or phase plate in the objective lens
                                                                3. cone of light converges on he sample, both directed light and diffracted light passes up into the objective lends and then to the phase plate, phase plate separate the diffracted and the direct light, and it alters their relative intensity (so they are roughly the same) and phase so they combine in the eye piece to form a visible image resulting in interference between the two beams. where you se no light in the microscope you have destructive interference and where you have constructive interference you get bright light. (contrasting)
                                                                4. UV light: fluoresence microscopy
                                                                  1. UV light is directed onto the sample (BUT allows only visible light to return to the eye)
                                                                    1. Fluorescent materials re-emits visible light
                                                                      1. Fluorescent microscopy gives specificity of detection since only fluorescent material is observed
                                                                        1. can only see things that are fluorescent, objects can be marked with flourescent markers or stain with fluorescent
                                                                        2. Tissue specific, cell specific and molecule specific fluorescent probes are commercially available
                                                                          1. useful with antibodies
                                                                          2. excite the fluorescent material with UV light, electrons go from normal state to excited state because they take in a photon of energy from the UV light, and they fall back to a lower state, the energy diference is given out as light in the visible spectrum. the longer the wavelength the lower the energy
                                                                          3. electron microscopy
                                                                            1. wavelength of an electron beam is much smaller than light hence theoretically vastly improved resolution
                                                                              1. Light microscopy can achieve up to 97% of theoretical resolution Electron microscopy achieves only 5% of theoretical resolution due to technical problems
                                                                                1. the electrons travel in waves. the waves are much smaller wavelengths. the only reason EM is better is because the wavelengths are shorter
                                                                                  1. Electron beam is easily attenuated, so sample sections need to be very then (<1m)
                                                                                    1. due to having very short wavelength
                                                                                    2. EM Sample preparation
                                                                                      1. 1 Fixation: chemical stabilisation 2. Dehydration: baths of increaseing alcohol content 3. Infiltration: infusion of plastic monomer into tissue 4. Polymerisation: catalysis 5. Sectioning: ultra-microtome (< 1) 6. Mounting: on small copper or nickel grid
                                                                                        1. fixing means you locking it in place.
                                                                                          1. if you have water in your sample it will boil due to the conditions and the sample may start to smell. the way to do this is to wash the sample with alcohol and then you wash the alcohol out with the infusion of plastic monomer
                                                                                            1. by the time you finish the sample preparation the sample looks nothing like what you started with. so when you view it you have to try to come up with reasons why that is the live image of what you started with earlier. ARTIFACTS. big problems especially with biological materials. causes change or damage to the sample which causes artifacts which can be very misleading.
                                                                                          2. method dependent on electron density of the sample. most unstained samples look the same, need to bring about some contrast in the electron density in the sample. tend to use metal salts to increase electron density.
                                                                                            1. EM goes on under high vacuum condition
                                                                                            2. scanning electron microscopy
                                                                                              1. Primary electrons hit sample surface and cause emission of secondary (back scattered) electrons Electrostatic grid collects secondary electrons Intensity mapped as a function of scan position Reveals surface structures of 3-D objects
                                                                                                1. For low density surfaces it is necessary to coat the surface with a very thin dense gold layer. This is done by evaporation.
                                                                                                  1. SEM typical resolving power ~10nm (T EM ~0.2nm) This is largely due to reliance on electron scatter to produce an image TEM is limited only by the ability of the sample to transmit electrons SE micrographs therefore often relatively low magnification but excellent resolution because of the short wavelengths. low vacuum ddoesnt harm live animals.
                                                                                                    1. used generally to look at surfaces. electrons are fired at the surface,depending on what they hit and what angle they hit, eletrons are remmitted at the surface, the electrons have higher energy, the electrons are gathered by an electron grid which then maps out the electrons in term of current. what you measure is the attenuation of the flow of electrons.
                                                                                                    2. atomic force microscopy
                                                                                                      1. The tip (very fine) is dragged across the surface Deflections of the cantilever are monitored by a laser The cantilever magnifies the movement of the tip by <1000X An image of the surface is built up Reads like a record player
                                                                                                        1. AFM is often used on soft materials e.g. polymer films, biological surfaces The dragging tip was found to dig into the surface Artifacts resulted, image not representative ‘Tapping’ approach was developed the tip is set close to but not touching the surface it is then vibrated the effect of the surface on the frequency of the vibration is then used to build up an image damage to the surface is greatly reduced
                                                                                                          1. resolution is dependent on tip sharpness, cantilever length and the laser optics.
                                                                                                            1. AFM or SEM surfaces: both are good high resolution techniques but give you different information about the surfaces. AFM can measure in all 3 dimensions in one scan. SEM has a larger depth of field. SEM has low magnification

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