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Intro to cell bio, best --> worst resolution - Coggle Diagram

- - - - vitamins
      - salts
      - minerals
      - glucose
      - amino acids
      - serum
        
        insulin
        
        growth factors
      - 37 degrees
      - CO2 incubator
    - - done with mechanical fragmentation
        
        trypsin
        
        an enzyme that chews up the glycoproteins around the cell
        
        EDTA
        
        soaks up ions on cell that cause stickiness between cells
  - - - cell cultures stick on bottom
    - - cell cultures don't stick on bottom
        
        agitation
        
        roller bottles
  - - - cells that come directly from an organism
        
        also undergo contact inhibition
        
        after cells become packed, they stop dividing
      - can only be divided until cell reaches Hayflick limit
        
        must create new culture afterwards
    - - cells that are transformed and can keep on growing/dividing with no limit
        
        possible due to dysregulation in their cell cycle
      - less likely to have contact inhibition
      - first cell line came from Henrietta Lacks
- - - - remove inner cell mass from blastocyst (embryo)
        
        culture them off fibroblast feeder cells
        
        dissociate survived embryonic stem cells
        
        plate these dissociated cells on new feeder cells
        
        embryonic cells are now established and form colonies
    - - testing for pluripotency
        
        put cells on suspension cultures
        
        cells will try to revert to embryo stage
        
        forms embryoid bodies
        
        embryoid bodies have pluripotent cells inside that can give rise to the three germ layers
        
        pluripotent
        
        1 more item...
  - - - the adjacent cells give signals to stem cells either to renew or to differentiate
        
        intestine tract cells
  - - - mature cells can be reprogrammed to pluripotent with Yamanaka factors (found often in embryonic stem cells)
        
        Sox2
        
        Klf4
        
        Oct4
        
        c-Myc
        
        often also in cancer cells
      - could have potential for transplantation medicine
        
        have safety and ethical issues
        
        application to medicine
        
        repair iPS cells and differentiate to reintegrate to damaged tissue
        
        differentiate the damaged iPS cells to understand what went wrong and study the diseased state
        
        can then determine what therapies/drugs may be effective to fix the problem
- - - - light shines onto condenser lens
        
        condenser lens shines light on specimen
        
        light then goes to ocular lens
        
        ocular lens focuses image on eye
      - magnification
        
        determined by the obj. and ocular lens
        
        total magnification multiplies them together
      - cannot have resolution that allows objects to be less less than 0.2 micrometers apart
      - exploiting changes in light phase
        
        as light goes through some parts of specimen, the phase changes
        
        causes light going through specimen to be out of phase
        
        creates dark spots
        
        light spots seen have higher amplitude
    - - similar to modern compound microscope
        
        differences
        
        phase plate
        
        puts wave length 1/4 out of phase additional to 1/4 from specimen
        
        make dark spots more apparent
        
        annular diaphram
        
        allows a larger angle/radius of light to hit the specimen
      - good to see intracellular details
      - a downside is it make a phase halo on round spots of specimen
    - - good to see cell surface details
        
        can see the edge and provide a 3D view of cell
      - uses plane polarized light (light in 1 direction) to contrast cell details on the edge
        
        microscope is equipped with a polarizer
    - - uses the fact some molecules can fluoresce (emit a wavelength after they absorb light at a specific wavelength)
        
        parts that are labeled with fluorochromes will fluoresce and can be imaged
        
        can see many protein and cell structures at once
        
        fluorochromes emit light because when they are excited, they are unstable
        
        e- fall to normal orbital releasing energy as light
        
        there are diff. fluorochromes that when excited, will emit different light which allows to view more proteins/organelles at once
        
        can overlay to see interactions between labeled parts
        
        to localize molecules of interest in cell, antibodies can be used
        
        process
        
        inject mouse with antigen to produce spleen cells with antibodies (are primary cells)
        
        cells are fused with mouse myeloma cells to allow them to grow nonstop
        
        1 more item...
      - how it works
        
        uses light that is more intense with broader wavelength than normal microscopes
        
        light goes through excitation filter
        
        reflects off dichroic mirror
        
        objective lens
        
        excitation light excites the specimen
        
        1 more item...
        
        filter only lets specfic wavelengths that will excite fluorescence to max
      - types
        
        immunofluorescence
        
        process
        
        kill cell and freeze structures
        
        antibodies from before (see above) are added by using detergents (allows them to cross membrane)
        
        add secondary antibodies with fluorochromes to bind to primary antibodies
        
        1 more item...
        
        requires you to kill the cells to work with them
        
        Green fluorescent protein (GFP)
        
        uses a natural protein in jelly fish that has a short sequence of aa that can fluoresce when excited with blue light
        
        gene can be isolated and modified so can encode protein that can be used to fluoresce live cells
        
        modifying GFP can create different colours
        
        takes gene and GFP gene to create hybrid DNA in plasmid form
        
        transfect a euk. with plasmid
        
        protein is made from cell naturally (transcrip + transl)
        
        cell lights up with right light due to the presence of the GFP protein
        
        confocal microscopy
        
        is expensive
        
        uses a laser to excite a point of focus on the cell
        
        emitted light is focused through pinhole and detected
        
        pinhole prevents the light emitted that is out of focal plane from entering (above/below)
        
        the cell is slowed scanned bit by bit to create an image of the whole cell
        
        deconvolution microscopy
        
        can take images quicker
        
        cheaper alternative
        
        disadvantage is the image must be analyzed after which may take a long time
        
        uses the point spread function to remove fluorescence that contributes to out of focus parts
        
        takes image at each focal plane that is tightly controlled to give details with no blur
        
        Two-photon excitation microscopy
        
        uses 2 photons opposed to 1 which can penetrate deeper in tissues and give more accurate imaging
        
        fluorescence resonance energy transfer (FRET)
        
        detects protein interaction
        
        FRET is programmed to only detect a certain absorption wavelength and certain emission wavelength
        
        when they are detected, it means that there is a protein interaction
        
        means one wavelength is exciting one protein, causing the other protein to emit other light
    - - Offers better resolution than fluorescent miscopy (FM)
      - Can only work with dead cells
      - to see image, one needs fixed/sectioned samples or metal-coated samples
      - process
        
        has a wire filament as an e- source where when heated, e- go towards anode
        
        a magnet concentrates the e- on the specimen
        
        the specimen is stained with electron-dense heavy metals
        
        organelles/lipids that take up the stain better appear darker under the microscope
        
        e- that pass through certain areas of the cell are light spots (these areas did not take up the stain well)
      - types
        
        scanning electron microscopy(SEM)
        
        takes section of cells to analyze them intracellularly
        
        can see intracell components
        
        Transmission electron microscopy(TEM)
        
        has scanning coils
        
        uses preserved dead cells and outside is coated with e- dense materials (good to see external structure)
      - immunoelectron microscopy
        
        can detect a protein of interest
        
        adds an antibody to detect the protein
        
        add a gold particle which has a high affinity for Fc domain
        
        gold particle acts like secondary antigen with fluorochrome
        
        gold particle can bud, creating dark spots
  - - - smaller the resolution= better
    - - for low resolution
        
        short wavelength
        
        higher refractive index (N)
        
        wide light angle entering objective lens
        
        brings more light into obj lens
    - - FM
        
        EM
- - - - aligned
        
        organized neatly and grows in parallel arrays
        
        has contact inhibition
        
        good to study cells in their physiological state
  - - - hair like processes
        
        disorganized growth by growing on top of each other
        
        loss of contact inhibition
        
        studies disease cells that are easy to grow, have more predictable results but may not reflect cells in their physiological state
- - - - turns into differentiated cell if require regeneration of a specific kind of cell
      - turns into progenitor cell if required to regenerate more stem cells