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BSC 300, week 4

by: Ashley Bartolomeo

BSC 300, week 4 BSC 300

Ashley Bartolomeo
GPA 3.9

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notes on microscopy
Cell Biology
John yoder
Class Notes
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This 4 page Class Notes was uploaded by Ashley Bartolomeo on Wednesday September 7, 2016. The Class Notes belongs to BSC 300 at University of Alabama - Tuscaloosa taught by John yoder in Fall 2016. Since its upload, it has received 14 views. For similar materials see Cell Biology in Biology at University of Alabama - Tuscaloosa.


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Date Created: 09/07/16
Chapter 18 Techniques in Cell and Molecular Biology: Microscopy Key Concepts  Define magnification and resolution for light microscopy  Describe methods for improving resolution in light microscopy  Outline different light and electron microscopy techniques emphasizing the information they reveal and their advantages and disadvantages The Microscope: Discovery of Cells, Late 1600’s  Robert Hooke first observed and named cells in 1655 using an elegant compound microscope with a magnification power of only about 50x  Anton van Leeuwenhoek refined the lens. A simpler microscope using a single, small spherical lens  Increased magnification to almost 250x and eliminated aberrations and distortions  He described a miniature world of single cells organisms he called animalcules (protists) Compound Light Microscopes  Lenses of a compound scope o Condenser lens: focuses light o Objective lens: magnifies and establishes resolution o Ocular lens: empty magnification  Total magnification is the product of the magnification of the objective and ocular. It is dependent on curvature and composition of the lenses Magnification is Nothing Without Resolution  Objective lens composition, size and shape determine degrees of magnification and resolution  Resolution: the shortest distance between two points on a specimen that can be distinguished as separate entities  Resolution power of any objective is a function of the wave length of light and a value called the numerical aperture (NA) – specific for each lens Resolution is a Function of NA and   Numerical aperture (NA) depends on lens size, curvature and focal length: the distance from the center of the lens to the focal point  A smaller radius of curvature results in a shorter focal length  This is what made van Leewenhoek’s lens so powerful  D= 0.61 x / (NA)  NA = n x sin  D= smallest distance between two points that can be resolved   = the wavelength of visible light (527 nm average)  n = refractive index (the degree to which the media between object and lens bends the light)   = the half angle of scatter between the focused sample and the objective lens  n ranges from 1(air) to about 1.5 (oil)  The shorter the focal distance, the greater the  and therefore the larger the denominator  Thus, increasing resolution  This is why the maximum resolution of compound light microscopes is ~200 nm Contrasting: Enhancing Visibility  Bright-field microscopy: simplest form of optical microscopy  Sample illumination is transmitted white light. Contrast results from absorbance of some transmitted light by dense areas of sample  Works best with fixed, stained samples (the cells have to be killed) and incubated with dyes that preferentially bind subcomponents of the cell Phase Contrast: Live Cell Imaging  Phase-contrast microscopes make highly transparent objects more visible by converting differences in light refraction of some parts of the specimen into differences in light intensity  Can be used in living non-stained cells  A phase ring projects a ring of light onto the condenser which focuses the light onto the specimen  Light travels undeviated through regions of the sample with little density  Light is refracted (deviated) by regions of the sample with high density (like organelles)  The deviated light is out of phase (the wave is shifted backward compared to the undeviated light)  A second ring, the phase plate, increases this shift (up to 180 degrees)  The shifted and unshifted light interfere with one another, effectively cancelling each other out  This reduces the amplitude of the light coming from the denser regions of the cell  And creates a darker image where those regions are located Differential Interference Contrast  Differential interference contrast (DIC) (aka Nomarki) optics gives a 3D quality to the image  Complex system of filters and prisms that polarize and separate light in order to generate contrast  Also works in living cells The Light Microscope Fluorescence Microscopy  Fluorescence microscopy uses fluoroschromes: compounds that absorb one wavelength of light and emit a linger wavelength  Fluorochrome-conjugated molecules and antibodies can bind to specific subcellular structures, revealing location and shape  This also requires killing and fixing the cells Green Fluorescent Protein and Variants  Transgenic systems (introducing foreign DNA to cells) can be used to live (in vivo) imaging of specific structures  Green Fluorescent Protein (aka GFP): a naturally occurring fluorescent protein from certain jellyfish  The gene encoding GFP can be attached to genes encoding other proteins and introduced to cells  When this hybrid protein is produced, it absorbs and emits specific wavelengths of light  Uses include observing where in a cell a protein is localized or in what cells is a specific gene expressed Enhancing Resolution Laser Scanning Confocal Microscopy 1. Laser beam is focused on a thin section of the object (blue) – some above and below fluorescence is unavoidable (green and red) 2. Objective lens focuses emitted light onto a pinhole aperture that allows only emitted light from the focal plan to be collected by the camera 3. Multiple images from different focal planes can be combined (stacked together) to generate a 3D projection of the object Super-Resolution Fluorescence Microscopy Breaking the Resolution Limits of Light Microscopes  New techniques like STORM (Stochastic Optic Reconstruction Microscopy) can break the 200nm barrier  Uses high speed cameras and fluorochromes that only stochastically fluoresce when excited. This pulsed fluorescence is collected and computer algorithms assemble the high-resolution images Super Dooper Resolution Transmission Electron Microscope  Transmission electron microscopes (TEMs) uses electrons instead of light to form images o Extremely small wavelength of electrons allows resolution to about 10-15 A o Samples must be extremely thin, fixed and embedded with heavy metals which absorb electrons o Electron bean passes through the material. Electrons are absorbed by the densest parts of the cell and transmitted through the sample in areas least dense o Electrons then captured by photographic paper or camera CCD Scanning Electron Microscope  Scanning electron microscopes (SEMs): samples must be dehydrated and coated with layers of carbon or gold  Electron beam may be absorbed by the sample  Other electrons are reflected back or secondary electrons emitted and collected by the detector. The more electrons, the stronger the signal  The result is a 3D image allowing even large, thick samples to be images Atomic Force Microscopy  1000x higher resolution than light microscopy  A non-sized tip is attached to a spring like cantilevel  Laser beam strikes the cantilevel causing the tip to interact with and be displaced by the sample  Displacement is detected as strength of reflected laser beam  Utility beyond simple imaging o Samples do not have to be fixed – can generate molecules sized movies o Can be used as a “nanomanipulator” applying push or pull forces to measure mechanical properties of molecules


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