Bio 2415- Module 2

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Bio 2415- Module 2
2015-09-30 14:11:16
methods cell biology

Methods in Cell biology
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  1. what are the factors that determine science? (what is science?) (6)
    • focuses on the natural world
    • aims to explain the natural world
    • uses testable ideas
    • relies on evidence
    • involves the scientific community
    • leads to ongoing research
    • benefits from scientific behavior
  2. what are the 6 qualities that allow science to be used as a tool for exploration?
    • adaptable, not rigid
    • only falsifies, does not prove
    • cumulative, builds upon previous results
    • self-critical, because science is social activity
    • devoid of moral content
    • is limited in its application
  3. describe the 2 main approaches to scientific inquiry.
    • observational science: scientists use some aspect of the world and use inductive reasoning to draw general conclusions
    • Hypothesis-driven science: scientists use "scientific method" and deductive reasoning to explain nature
  4. inductive reasoning
    • an initial observation leads to the discovery of a certain pattern
    • allows a tentative prediction to be made which leads to a general theory about how things work
    • general principles from a large number of specific observations
  5. what is an example of observational science and inductive reasoning?
    • the cell theory
    • after 2 centuries of scientists discovering cells in every biological specimen they observed, they could conclude "all organisms are made of cells"
  6. what are the problems with observational approach?
    • cannot establish cause and effect (can form correlation)
    • groups are not random
  7. deductive reasoning
    • refers to something known as the "if...then...logic"
    • flows in the opposite direction from inductive reasoning: from general to specific
  8. what are the 7 steps in the scientific method?
    • observations
    • questions
    • hypotheses
    • methods and predications
    • data collection
    • interpretations
    • conclusions
  9. what are the 3 important properties to a good hypothesis?
    • must be:
    • predictive
    • testable
    • falsifiable
  10. hypothesis
    statement consistent with most of the data, may take the form of a model (an explanation that appears to account for the data); must be testable
  11. theory
    a hypothesis that has been extensively tested by many investigators, using different approaches, widely accepted
  12. law
    a theory that has been tested and confirmed over a long period of time with virtually no doubt of it's validity
  13. what are the 3 main points to remember during the experimenting stage of the scientific method?
    • tests should be reproducible
    • measurements that are quantitative are better than those that are qualitative
    • experiments designed to test hypothesis must be "controlled" experiments
  14. describe the 2 types of variable.
    • Independent Variable: values that can be changed in a given model or equation
    • Dependent variable: values that result from the independent variable
  15. 3 reasons controlled groups are important.
    • 1. clarify experimental results by providing a standard for comparison
    • 2. without control, it's no possible to say if experimental outcome is due to variable or some other variable
    • 3. used to minimize the unintended influence of other variables on the same system
  16. cofounding factors
    an extraneous variable in an experiment that correlates with the variable and can lead to erroneous "false positive" conclusions
  17. artifact
    • type of cofounding variable
    • is some aspect of the study process itself that produces results independent of what we are testing
  18. statistics
    a mathematical body of science pertaining to the collection, analysis, interpretation, or explanation, and presentation of data
  19. N-values
    • number of the same samples in the experiment
    • greater the N-value, greater the value of interpretations of scientific method
  20. repetitions
    • important because it tells you if you're consistent
    • have to have at least 3 repetitions in bio to perform statistics
  21. what are the 5 sections in a research paper?
    • abstract
    • introduction
    • materials and methods
    • results
    • discussion and conclusion
  22. peer review and it's important
    • is the evaluation of work b one or more people of similar competence to the producers of the work
    • important because it's a form of self-regulation by qualified members of a profession within the relevant field
  23. 3 main approaches of experimenting in cell biology
    • In vitro: using purified chemicals and cellular components or live cells
    • cells in petri dish of multicellular organism
    • In vivo: using live cells (from unicellular organism such as yeasts) organisms
    • In silico: using computer analysis of large amounts of data
  24. what are the 3 common laboratory models using in cell biology?
    • E.coli (prokaryote): easy to grow in agar medium
    • S. cerevisiae (eukaryotic unicellular-yeast): easy to grow in simple growth medium
    • A. thaliana (photosynthetic eukaryotic cell): clonal growth
  25. who was Robert Hooke?
    • observed compartments in cork, under a microscope, and first named cells (basic unit of bio)
    • used dead cells with 30X enlargement
  26. who was Anton van Leeuwenhoek?
    • few years later produced better lenses in microscope that magnified up to 300X
    • first to observe living cells including single celled organism in pond water and sperm
  27. Who as Robert Brown?
    • coined the term nucleus
    • discovered in leaves of orchids
  28. who was Felice Fontana?
    • glimpsed the nucleus in epidermal cell so snakes (animal material)
    • described the nucleolus
  29. what were the 2 factors that restricted progress in early cell biology?
    • microscopes had limited resolution (ability to see fine detail)
    • focus was observation, with little emphasis on explanation
  30. define chromatic aberration and how to fix it
    • it's a type of distortion in which there is a failure of a lens to focus all colors to the same convergence point
    • blue and red most impacted when moving through lens; they separate from each other and it depicts a blurred image
    • double lens system used to correct chromatic aberration (with concave and convex lens)
  31. what methods were used to identify intracellular structures?
    • oil immersion and better staining and cutting procedures led to the identification of intracellular structures
    • oil immersion reduces bending & velocity of light
  32. what are the 4 requirements for microscopy?
    • source of illumination
    • system of lenses
    • a method of interpretation
    • a specimen
  33. what is the difference between compound light microscope and electron microscope in terms of lens source of illumination and visualization?
    • compound light microscope: lens is glass
    • directly visualized though eyepiece, or focused on a detector like photographic film or digital camera
    • light is source of illumination

    • electron microscope: lens is electromagnets
    • focused on a florescent screen or film or digitally imaged with a detector
    • source of illumination is beam of electrons from a heated tungsten filament
  34. how does light help us visualize an image under a microscope?
    • photons and electrons behave as waves
    • when wave form is disrupted, object becomes visible under microscope
    • the ability for an object to disturb the wave form is dependent on the size of the object and the wavelength within the waveform
  35. resolution
    how close can two points be in an image before they are no longer seen as two separate points?
  36. what are the 3 things resolution is related to?
    • 1. numerical aperture of the object lens: higher the numerical aperture, the better the resolution (the amount of light you can gather and focus on to the specimen)
    • 2. The wavelength of light passing through the lens: the shorter the wavelength, the better the resolution
    • 3. refractory index: a measure of the change in velocity of light as it passes through one medium to another (oil slows velocity of light)
  37. limit of resolution
    • how far apart adjacent objects must be in order to be distinguished as separate entities
    • smaller the limit of resolution, the greater the resolving power of a microscope
  38. distinguish the difference between magnification and resolution.
    • magnification: expanding the image to get the details; is a characteristic of resolution
    • resolution: clarity
  39. how did microtome improve resolution?
    allowed preparation of very thin slices of samples which improves the limit of resolution
  40. what kind of images do light microscopes create? what can be a problem?
    • bright field image
    • most cells and cells structure are transparent and colourless
  41. how is bright field microscopy improved?
    • phase contrast and differential interference contrast microscopy exploit differences in the phase of light passing through a structure with a refractive index different than the surrounding medium
    • phase contrast uses a phase plate to allow rays of light to differ in wavelength
    • DIC contrast (picks up contrast on membrane)uses a prism to split the light to different wavelengths
  42. what is one way of enhancing contrast? problem with this?
    • dyes: developed by German industrial chemists in second half of 19th century
    • played a key role in staining and identifying subcellular structures
    • problem: have to kill specimen
  43. H&E stain
    • Hematoxylin- blue nuclei
    • Eosin- red to pink proteins
  44. Periodic Acid Schiff Stain
    stains glycoproteins, glycogen, and mucin purple
  45. Trichrome
    • hematoxylin (nuclei) + chromotrope (muscle red) + aniline blue (collagen)
    • ex. staining blood vessels
  46. Methylene blue
    • stains DNA and RNA blue
    • don't have to kill specimen to use
    • can wash away dye with water
  47. Fluorescence microscopy
    • detects fluorescent dyes, or labels, to show locations of substances in the cell
    • can view living cells or subcellular phenomena
    • the excitation filter only allows one wavelength of light from the mercury arc lamp to pass through and that light is the one that will be picked up by specimen (excites the specimen)
  48. what are the 4 types of ways to make cells fluorescent?
    • Auto-fluorescence: natural emission of light by biological structures when they have absorbed light and used to distinguish light originating from artificially added fluorescent markers
    • Microinjection: use of glass micropipette to inject fluorescence at microscopic level (whole cell fluorescent)
    • Antibody labelling: using antibodies that are directly labeled with a fluorescence dye
    • Genetic Modification: direct manipulation of an organism's genome using biotechnology
  49. confocal scanning/microscopy
    uses a laser beam to illuminate a single plane of a fluorescently labeled specimen and in this way improves resolution
  50. Computer-Enhanced Digital Microscopy
    you can use a computer to subtract the noise (background junk in your image) to get a more focused picture
  51. 3D-Imaging
    • digital reconstruction of 2D images
    • stack the image together using a computer
  52. Time lapse Microscopy
    4D imaging where the 4th dimension is time
  53. Fluorescence recovery after photobleaching (FRAP)
    • used to measure the ability of a molecule to move around over time
    • you can determine how fluid the cells are by seeing how long it takes the fluorescence cells to recover after bleaching
  54. Describe the 2 types of electron microscopy.
    • Transmission Electron Microscopy (TEM): electrons are transmitted though the specimen; 2D imaging  
    • Scanning Electron Microscopy (SEM): the surface of a specimen is scanned, by detecting electrons deflected from the outer surface; 3D imaging
  55. TEM: Negative stain
    • the mount is suspended in an electron dense stain and then the specimen is dripped on top with water so to be able to visualize the light specimen against a darkly stained background
    • darker image shows in electron dense
    • specimen dead
  56. TEM: Shadowing
    • heavy metal vapor is sprayed at an angle across the specimen causing the accumulation of the metal on one side and a shadow region without metal on the other side
    • 3D feel in a 2D image
  57. TEM: Freeze Fracture (7 steps)
    • 1.cells are frozen in liquid nitrogen
    • 2. block of frozen cells is fractures
    • 3. surface ice is removed by a vacuum
    • 4. A thin layer of carbon is evaporated vertically onto the surface to produce a carbon replica
    • 5. surface is shadowed with a platinum vapor
    • 6. organic material is digested away by acid, leaving a replica
    • 7. carbon-metal replica is put on a grid and examined by a transmission electron microscope
  58. tissue contains more than just cells. what other materials might you find in tissue?
    • epithelial cells
    • melanocytes
    • macrophage
  59. how do you isolate cells from acellular material? (separating tissue materials)
    • you need to chemically disrupt the extracellular matrix that holds the cells together
    • treat with proteolytic enzymes (trypsin and collagenase) to digest proteins in the extracellular matrix and with agents (EDTA) that bind Ca2+ on which cell-to-cell adhesion depends
    • tissue can then be teased apart into single living cells by gentle agitation
  60. what are 3 methods of separating different cell types?
    • centrifugation: insert cells in tube, shake tube and dense cells fall to the bottom, less dense float to the top
    • Antibody, bead, or medium based centrifugation: fill tube with antibodies and the antigens will get pulled to the bottom
    • Fluorescence activate cell sorting (FACS): take antibody and flag it with flurochrome and pass it through a machine and one by one it deflects them into diff tubes
  61. Tissue culture dishes and medium
    • culture dishes must be coated with ECM components
    • medium components vary depending on cell type
  62. what is the function of growth factors in tissue culture?
    growth factors are added to ensure that cells can undergo cell division
  63. plant cell culture (media and how cells proliferate)
    • plant cell culture requires growth regulators
    • many types of plant cells proliferate in culture, producing a mass of undifferentiated cells called callus
  64. what is the striking feature that differentiates plant cell culture and animal cell culture?
    phenomenon called totipotency: ability of a single cell to divide and produce all of the differentiated cells in the organism
  65. cell culture assays
    • experiments with cells in culture
    • ex. migration assay
  66. how are organelles and macromolecules separated using ultracentrifugation?
    • disrupt the cell membrane to form a homogenate
    • separate the components of the homogenate by centrifugation at high speeds
  67. Ultracentrifugation
    • repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components
    • smaller the subcellular component, the greater the centrifugal force required to sediment it