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Flow Cytometry - Coggle Diagram

- - - - Glass envelope containing gas or vapour at high pressure
      - Initial high voltage spark between 2 electrodes creates a plasma arc
      - Plasma arc maintained by application of high current at low voltage
      - Prone to flicker and average life of arc lamps is short
      - Cheap
    - - Coherent, plane-polarised, intense, narrow beam of light
      - Light is monochromatic
      - Expensive
      - Plasma tube contains gas under pressure
        
        Fluoresces under application of current
      - Light emitted is reflected along tube
      - Photons strike an atom in excited state they release another photon of the same \(\lambda\)
  - - - Cells can be stained with a fluorescent dye
      - Flurochrome conjugated by an Ab
        
        In amount proportional to the quantity of binding constituent (DNA,RNA, surface Ag)
      - Intensity of emitted fluorescence is proportional to the fluorescing cellular constituent
      - Examples
        
        FITC
        
        Bright
        
        Absorption max. close to emission lines from argon laser + mercury arc lamp
        
        Yellow-green spectrum
        
        APC
        
        R-Phycoerythrin
        
        Can be excited at 488nm so only one laser required
        
        Orange-red spectrum
        
        Cascade blue
        
        Texas red
      - Useful for fluorochromes to emit light in different spectrums so they can be distinguished
  - - - Doesn't interfere with light sources
  - - - Dichroic mirrors (beam splitters)
        
        Allow certain \(\lambda\) to be reflected whilst rest passes through
      - Shortpass filters
        
        Allow light below a certain \(\lambda\) through
      - Bandpass filters
        
        Only allows a specified band of \(\lambda\) through
      - Longpass filters
        
        Allow light above a certain \(\lambda\) through
    - - Photodiodes
        
        Newer
        
        Good in VS
        
        No adjustable gain/amplification
        
        Must be kept cool
        
        Pick up forward scatter
        
        Bigger cell = more scatter
      - Photo multiplier tubes (PMT)
        
        Fluorescence
        
        Signal can be amplified
        
        Photoemissive cathodes + dynodes
        
        High sensitivity
        
        Most common
        
        Inexpensive
    - - Provides data on internal structures
      - Dead cells have high side scatter
        
        Rough membrane
    - - Mirrors and filters set up in certain way so that light beams can be split and sent to the specific detectors
    - - This cannot be analysed by a computer
- - - - Can see cells with 1 or another Ag, both or neither
  - - - No reagents needed
      - Cell size
      - Granularity
      - pigment content e.g. Hb
    - - Reagents required
      - DNA, RNA content + base ratios
      - Functional
        
        Surface + intracellular receptors
        
        DNa synthesis
        
        Apoptosis (DNA degradation)
        
        Gene expression
- - - - Single cell suspension
      - Into centre because it aligns the cells singularly
    - - Filtered isotonic saline
      - Continuously flows through machine
  - - - Lines cells up in single file
  - - - Stream velocity of 10ms\(^{-1}\)
        
        10\(\mu\)m particles traverse their own diameter in 1\(\mu\)s
        
        Interrogation must be rapid
        
        High speed electronics
        
        Lasers
  - - - No sample?
        
        Check sample tube has no cracks
- - - - Allows coupling of Abs, Ags, peptides, oligonucleotides or receptors
- - - - Droplet with charge on it
        
        Charge added depends on the colour of the cell that passes
      - Droplets fall between 2 oppositely charged plates
        
        Separates charges
  - - - High wastage
- - - - Older flow cytometers
    - - Common
    - - Due to emission spectrums, some of the detection from one detector is from another fluorochrome
      - If not corrected, results will be falsely high
      - Known beads run through the system
        
        Subtract voltage until corrected
- - - - Size
      - Granularity
      - Fluorescence
    - - Rate
        
        Order of 500-4000 cells per second
        
        Cells moving in a fluid stream
  - - - Delivers particles individually to a specific point
    - - Excitation source and data collection optics
    - - Allow conversion of signals from optical into electronic for data analysis