BIS104 Note3 - Lecture 3 Bio Sci 104 How are cells and cell...

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Lecture 3 1 Bio Sci 104 Winter 2010 How are cells and cell function studied? (part II) A. Microscopes - 1. Light microscopes- quite remarkable. Simple solid-state lens that can magnify up to 1000x . Resolution limit of unaided eye = 100-200 µ m (0.1 - 0.2 mm). Typical animal cells are 10 to 20 µ m in diameter, typical bacteria are 0.5 to 1 µ m. a. Resolution of light microscopes is limited by wavelength of light , to about 0.2 µ m (note a hydrogen atom is about a thousand times smaller). Thus bacteria and mitochondria are the smallest objects whose shapes can be directly visualized by light microscopes . b. Other limitations- most biological material thick and colorless. Must be fixed, thin sectioned to single cell thicknesses and/or stained to use in microscope. c. Visible stains extremely powerful approach for differentiating different types of cells and different subcellular components (e.g. hematoxylin stains RNA, DNA, and negatively charged proteins, FYI only). d. Fluorescent stains - Can be excited by light with a specific wavelength then emit light at a specific wavelength (ie. only fluoresce at a specific wavelength). Very sensitive, with correct filters on microscope, can detect very small quantities. e. Joining of these dyes to antibodies permits specific types of molecules to be visualized. e.g. To visualize microtubule: first, add anti-tubulin antibody, which binds only to tubulin (major component of the microtubule) to permealized cells (first antibody treatment). Then, add anti- XXX antibody that is coupled to a fluorescent dye (second antibody treatment). XXX is determined by the source of the first antibody. For example, if the first antibody is raised in rabbit, the secondary anti-XXX Ab=anti-rabbit Ab; if the first Ab is raised in mouse, the secondary anti-XXX Ab=anti-mouse Ab…etc. f. Many (but not all dyes) require cell to be fixed (i.e. killed). Thus, cannot always follow a process in real time. g. Phase contrast or Nomarski -optics microscopes rely on changes in refractive index of different cell components to create an image in absence of dye . Useful for following living processes . Can be further enhanced using electronic image processing. h. Above methods still unable to visualize thick samples, resulting in multiple images stacked above one another. One solution is to slice samples very thinly. Yet, even at this level the resolution is not good enough. Recent approach- Confocal fluorescence microscope, which can avoid this problem by restricting excitation of a fluorescent image to a single plane within the cell, using special apertures and lenses to exclude out-of-focus lights.
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Lecture 3 2 2. Electron microscopes. How to avoid inherent limit of light microscope resolution (0.2 µ m)? Might use a form of radiation with shorter wavelength. X-rays and gamma rays: not yet useful, no efficient method of focusing. (The greater the energy, the shorter the wavelength.) Accelerated electrons can be diffracted analogous to
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BIS104 Note3 - Lecture 3 Bio Sci 104 How are cells and cell...

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