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  <head>
    <title>102-01 Microscopy, culture, separation, and quantitative assays</title>
    <ownerName>Integrated Medical Foundations</ownerName>
  </head>
  <body>
    <outline text="Microscopy, culture, separation, and assays">
      <outline text="Methods as transformations">
        <outline text="Sampling, preparation, measurement, interpretation"/>
        <outline text="Each method sees only what its principle preserves">
          <outline text="Images and numbers are instrument and model outputs"/>
        </outline>
        <outline text="What was measured, from what, at which resolution">
          <outline text="Detection limit, controls, alternative signal sources"/>
        </outline>
      </outline>
      <outline text="Light microscopy and contrast">
        <outline text="Resolution depends on wavelength and aperture">
          <outline text="Empty magnification only enlarges blur"/>
        </outline>
        <outline text="Contrast from absorption, phase, polarisation, fluorescence"/>
        <outline text="Fixation and staining can shrink, distort, relocate"/>
        <outline text="Phase contrast and DIC show live unstained cells"/>
        <outline text="Fluorescence: excite, detect longer emission">
          <outline text="Filters separate excitation from emission"/>
          <outline text="Autofluorescence and overlap create background"/>
          <outline text="Bleaching and cell damage; keep exposure comparable"/>
        </outline>
      </outline>
      <outline text="Immunofluorescence and specificity">
        <outline text="Direct: fluorophore on primary antibody"/>
        <outline text="Indirect: labelled secondary amplifies signal"/>
        <outline text="Manufacturer label is not proof of specificity">
          <outline text="Positive control: staining system works"/>
          <outline text="Negative control: nonspecific background"/>
          <outline text="Orthogonal validation by another reagent or method"/>
          <outline text="Genetic removal of target is most informative"/>
        </outline>
      </outline>
      <outline text="Confocal, super-resolution, electron imaging">
        <outline text="Confocal pinhole rejects out-of-focus light"/>
        <outline text="Multiphoton: lower-energy photons image deeper"/>
        <outline text="Super-resolution surpasses diffraction limit">
          <outline text="Patterned light, fluorophore switching, localisation"/>
          <outline text="Depends on labelling, motion, photons, algorithm"/>
          <outline text="Nanoscale structure needs method-specific controls"/>
        </outline>
        <outline text="Electron beams have short wavelengths">
          <outline text="Transmission EM: ultrastructure in thin sections"/>
          <outline text="Scanning EM: surface topography"/>
          <outline text="Vacuum and processing prevent live imaging"/>
          <outline text="Cryogenic methods keep hydrated state"/>
        </outline>
      </outline>
      <outline text="Cell and microbial culture">
        <outline text="Primary cells keep tissue features, limited lifespan"/>
        <outline text="Immortalised lines drift, adapt, get misidentified">
          <outline text="Authentication, passage records, mycoplasma tests"/>
        </outline>
        <outline text="Medium, oxygen, stiffness, architecture shape phenotype">
          <outline text="Atmospheric oxygen can be hyperoxic"/>
          <outline text="Monolayers alter polarity and mechanical signals"/>
          <outline text="Organoids and chips are not whole organisms"/>
        </outline>
        <outline text="Microbial culture needs viable organisms, right conditions">
          <outline text="Selective, differential, enrichment media"/>
          <outline text="Positive: infection, colonisation, or contamination"/>
          <outline text="Negative: low burden, wrong site, antibiotics, delay"/>
        </outline>
      </outline>
      <outline text="Physical separation">
        <outline text="Centrifugation separates by sedimentation">
          <outline text="A pellet is enriched, not pure"/>
        </outline>
        <outline text="Chromatography: partition between two phases">
          <outline text="Size, charge, selective binding, hydrophobicity"/>
          <outline text="Retention time identifies only under calibration"/>
          <outline text="Co-elution and matrix effects create false peaks"/>
        </outline>
        <outline text="Electrophoresis moves charged molecules in a field">
          <outline text="Native gels mix charge, size, shape"/>
          <outline text="A band is not necessarily one species"/>
        </outline>
      </outline>
      <outline text="Western blotting and optical assays">
        <outline text="Approximate molecular size and abundance">
          <outline text="Transfer, epitope access, saturation affect it"/>
          <outline text="Housekeeping control fails if treatment alters it"/>
          <outline text="Total-protein normalisation; keep crop context"/>
        </outline>
        <outline text="Beer-Lambert holds within a linear range">
          <outline text="Turbidity and scattering break the assumption"/>
        </outline>
        <outline text="Fluorimetry sensitive but quenched and bleached"/>
      </outline>
      <outline text="Immunoassays and flow cytometry">
        <outline text="Sandwich assays capture between two antibodies"/>
        <outline text="Competitive assays infer from reduced binding"/>
        <outline text="Heterophile antibodies, hook, cross-reactivity, lots">
          <outline text="Dilution checks and recovery expose failures"/>
        </outline>
        <outline text="Flow records scatter and fluorescence per event">
          <outline text="Scatter only roughly reflects size and complexity"/>
          <outline text="Compensation corrects spillover between detectors"/>
          <outline text="Gating is subjective; controls support gates"/>
          <outline text="Rare events need enough events, debris excluded"/>
        </outline>
      </outline>
      <outline text="Mass spectrometry">
        <outline text="Ionise and separate by mass-to-charge ratio"/>
        <outline text="Internal standards correct ion suppression"/>
        <outline text="Database matching with false-discovery control"/>
        <outline text="Undetected: absent, below limit, poorly ionised, excluded"/>
      </outline>
      <outline text="Performance, replicates, triangulation">
        <outline text="Technical replicates: measurement variability"/>
        <outline text="Biological replicates: independent units">
          <outline text="Averaging wells does not enlarge the sample"/>
        </outline>
        <outline text="Controls test the whole process"/>
        <outline text="Triangulate morphology, biochemistry, perturbation">
          <outline text="Independent principles rarely share artefacts"/>
          <outline text="Disagreement reveals compartments or flawed assumptions"/>
        </outline>
      </outline>
    </outline>
  </body>
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