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PSU BMB 251 - Methods in Cellular Visualization

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BMB 251 1st Edition Lecture 9 Outline of Last Lecture I. Review Lecturea. Cellular Respirationb. Noncovalent Interactionsc. Blottingd. Protein PurificationOutline of Current Lecture II. Optical diffraction effectsIII. ResolutionIV. Light Microscopya. Dark Fieldb. Bright FieldV. Fluorescent MicroscopyVI. AntibodiesCurrent Lecture- Optical diffraction effects: light waves travel through optical system by several slightly different routes and interfere with one anothero If trains of waves are in phase, they will increase the brightness when coming into contact o If trains are out of phase, they will cancel each other out partly or entirely- Resolution: the minimum distinguishable distance, depends on wavelengtho **”The resolving power of the microscope depends on the width of the cone of illumination and therefore on both the condenser and the objective lens.”o If you have two things which are closer together than the wavelength of the light, you cannot use light to measure it o Violet light is preferred because of its short wavelength Put in higher level photons and get out a lower energy photono Resolution is proportional to wavelength- Limit of resolution: limiting separation at which two objects appear distinct o Depends on wavelength of light and numerical aperture of lens system used (measure ofwidth of entry pupil of microscope; scaled according to distance from object  wider the microscope opens its eye, more sharply it can see - Light Microscopy:o Uses thin slices of substance to be studied under microscopeo The sample is fixed to the preserve its structureo The sample is thinly sliced so that it transmits lightThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o The sample is stained so that it can be seen by bright field microscopyo If the sample wasn’t stained, you wouldn’t be able to see it; this is why it is “fixed”- to make it easier for stain to get in/be see and preserves the cell- Dark-field microscope: illuminating rays of light are directed form the side so that only scattered light enters microscope  but, cell appears as a bright object against a dark background- Bright- field microscope: light passing through cell in culture form image directly - Imperfections in a light microscope can be overcome by a image processing (electronic/digital imaging) system- Tissues are to thick to see individual cells, so they must be cut into very small slices, or sectionso Must be treated with fixative to immobilize, kill and freeze the cells- Fluorescent molecules absorb light at one wavelength and emit it at a different, longer oneo Shows up against a dark backgrounds and is visible through a fluorescent microscopeo Advantages: Highly selective tags help determine specific location of molecules in cell Combining tags might show you two proteins located in the same place  MIGHT work together Fluorescence intensely reflects the concentration of tagged molecules Fluorescently tagged molecules can be introduced into cells and their behaviors can be viewed in a live cell- Antibodies: proteins produced by the vertebrate immune system as a


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