Exam 1 Book Notes Chapter 18 Methods in Cell Biology 18 1 The Light Microscope o resolution o o light rays focused on specimen by condenser lens collected by microscope s objective lens o now 2 types of rays to consider those that specimen has altered and those that are unaltered unaltered forms background altered shows image of specimen d 0 61 lambda NA d is the minimum distance that two points in the specimen must be separated to be resolved o magnification without resolution is empty o NA numerical aperture which is a constant for each lens and is a measure of light gathering qualities oil can be used to increase the resolution by increasing NA limit of resolution visibility o o can be found by substituting the minimum possible wavelength of illumination and greatest possible NA in equation concerned with factors that allow an object actually to be observed can also be termed contrast which is the difference in appearance between adjacent parts of an object or between an object and its background can deal with this by creating a dye to stain a specimen a dye absorbs certain wavelengths and ones that are not absorbed are transmitted to the eye causing stained object to appear colored Feulgen stain specific for DNA causing chromosomes to appear colored most stains cannot be used with living cells preparation of specimens for bright field light microscopy 2 main categories whole mount and section o o whole mount is an intact object either living or dead and may be entire microscopic organism or a small part of a larger organism sections are prepared by first killing cells in immersion of a fixative which immobilizes structures to keep cell intact I e formaldehyde alcohol acetic acid tissue is then dehydrated and embedded usually in paraffin which is readily dissolved by organic solvents phase contrast microscopy o o small unstained specimens can be difficult to see which a bright field microscope and the phase contrast microscope makes highly transparent objects more visible distinguishes parts of cell by their refractive indices converting differences into that of brightness intensity useful for viewing intracellular components of living cells at high resolution negatives produces halos and shades o o o Nomarski interference differential interference contrast is another type that delivers a 3D quality image fluorescence microscopy and related techniques allows viewers to observe location of certain molecules called fluorochromes or fluorophores these absorb invisible ultraviolet radiation and release portion of energy in longer visible wavelengths called fluorescence light source produces beam of ultraviolet light that travels through filter which blocks all wavelengths except those capable of exiting the fluorophore excites the specimen causing it to emit light of visible wavelength that is focused by objective lens into image seen by viewer o o o o o o o o 18 2 Transmission Electron Microscopy o o o o o o o o o o high contrast common application would be that of small fluorochrome like rhodamine or fluorescein that is covalently linked to conjugated an antibody to produce fluorescent antibody used to determine location of a specific protein within a cell this is called immunofluorescence video microscopy and image processing can be very sensitive to light allows them to image specimens at low illumination preventing damage from light can detect and amplify small differences in contrast laser scanning confocal microscopy confocal microscope produces an image of a thin plane situated within a much thicker specimen specimen is illuminated by a finely focused laser beam that rapidly scans across it at a single depth used with fluorescence optics aperture and illuminated plane are confocal out of focus points in the specimen become invisible super resolution fluorescence microscopy limit of resolution of light microscope 200nm o o mutation of GFP polypeptide converts protein into photoactivatable molecule PA GFP nonfluorescent until hit with violet light development of photoswitchable proteins on and off with pulses of light o i e process called STORM allows localization of single fluorescent molecule within resolution of less than 200nm electrons transmitted through very thin sections of specimen greater resolution than light microscope o derives from wave properties of electrons shorter wavelength better resolution o wavelength of electron lambda sqrt 150 V V accelerating voltage in volts lambda wavelength in Angstroms electrons drawn from hot tungsten filament accelerated by fine beam by high voltage applied between cathode and anode air is pumped out of column prior to avoid collisions with gas molecules beam of negatively charged electrons is focused by electromagnetic lenses condenser lenses placed between electron source and specimen focus electron beam on specimen electrons passing through specimen brought to focus on phosphorescent screen at bottom of column electrons strike screen excite coating of fluorescent crystals which emit own visible light image formation depends on differential scattering of electrons by parts of the specimen o o metals become selectively complexed with different parts of organelles o increased by fixing and staining with heavy metal solutions the more metal atoms the darker the image image can be recorded onto film best or video not as good specimen preparation for electron microscopy o fixation more critical than for light microscopy fixative must stop cell life without significantly altering the cell structure requires extremely small pieces of tissue to be fixed and embedded o o o o o o fixatives are chemicals that denature and precipitate cellular macromolecules may cause coagulation or precipitation of materials into an artifact had no structure in living cell most common fixatives are glutaraldehyde and osmium tetroxide after fixation water removed by alcohol tissue space filled with material that supports tissue sectioning tissues to be sectioned usually embedded in epoxy resins studies with antibodies use acrylic resins because they are more permeable to large molecules cryofixation stops metabolic processes and preserves structure without fixatives embedding less likely to lead to artifacts does not alter cell s macromolecules difficulty is preventing formation of ice crystals use ultrarapid freezing techniques plunging into extremely low temp or high pressure freezing frozen sections can be viewed under light or electron microscope
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