--- module: 102-01 language: en chapter: 102 title: "Experimental Biomedical Methods, Measurement, Omics, and Reproducibility" module_title: "Microscopy, culture, separation, and quantitative assays" source_sha256: 05c02b4f6b6873cb9603943095d179690208bbc5fba415091ae7609d6669fac6 --- # Microscopy, culture, separation, and assays ## Methods as transformations ### Sampling, preparation, measurement, interpretation ### Each method sees only what its principle preserves #### Images and numbers are instrument and model outputs ### What was measured, from what, at which resolution #### Detection limit, controls, alternative signal sources ## Light microscopy and contrast ### Resolution depends on wavelength and aperture #### Empty magnification only enlarges blur ### Contrast from absorption, phase, polarisation, fluorescence ### Fixation and staining can shrink, distort, relocate ### Phase contrast and DIC show live unstained cells ### Fluorescence: excite, detect longer emission #### Filters separate excitation from emission #### Autofluorescence and overlap create background #### Bleaching and cell damage; keep exposure comparable ## Immunofluorescence and specificity ### Direct: fluorophore on primary antibody ### Indirect: labelled secondary amplifies signal ### Manufacturer label is not proof of specificity #### Positive control: staining system works #### Negative control: nonspecific background #### Orthogonal validation by another reagent or method #### Genetic removal of target is most informative ## Confocal, super-resolution, electron imaging ### Confocal pinhole rejects out-of-focus light ### Multiphoton: lower-energy photons image deeper ### Super-resolution surpasses diffraction limit #### Patterned light, fluorophore switching, localisation #### Depends on labelling, motion, photons, algorithm #### Nanoscale structure needs method-specific controls ### Electron beams have short wavelengths #### Transmission EM: ultrastructure in thin sections #### Scanning EM: surface topography #### Vacuum and processing prevent live imaging #### Cryogenic methods keep hydrated state ## Cell and microbial culture ### Primary cells keep tissue features, limited lifespan ### Immortalised lines drift, adapt, get misidentified #### Authentication, passage records, mycoplasma tests ### Medium, oxygen, stiffness, architecture shape phenotype #### Atmospheric oxygen can be hyperoxic #### Monolayers alter polarity and mechanical signals #### Organoids and chips are not whole organisms ### Microbial culture needs viable organisms, right conditions #### Selective, differential, enrichment media #### Positive: infection, colonisation, or contamination #### Negative: low burden, wrong site, antibiotics, delay ## Physical separation ### Centrifugation separates by sedimentation #### A pellet is enriched, not pure ### Chromatography: partition between two phases #### Size, charge, selective binding, hydrophobicity #### Retention time identifies only under calibration #### Co-elution and matrix effects create false peaks ### Electrophoresis moves charged molecules in a field #### Native gels mix charge, size, shape #### A band is not necessarily one species ## Western blotting and optical assays ### Approximate molecular size and abundance #### Transfer, epitope access, saturation affect it #### Housekeeping control fails if treatment alters it #### Total-protein normalisation; keep crop context ### Beer-Lambert holds within a linear range #### Turbidity and scattering break the assumption ### Fluorimetry sensitive but quenched and bleached ## Immunoassays and flow cytometry ### Sandwich assays capture between two antibodies ### Competitive assays infer from reduced binding ### Heterophile antibodies, hook, cross-reactivity, lots #### Dilution checks and recovery expose failures ### Flow records scatter and fluorescence per event #### Scatter only roughly reflects size and complexity #### Compensation corrects spillover between detectors #### Gating is subjective; controls support gates #### Rare events need enough events, debris excluded ## Mass spectrometry ### Ionise and separate by mass-to-charge ratio ### Internal standards correct ion suppression ### Database matching with false-discovery control ### Undetected: absent, below limit, poorly ionised, excluded ## Performance, replicates, triangulation ### Technical replicates: measurement variability ### Biological replicates: independent units #### Averaging wells does not enlarge the sample ### Controls test the whole process ### Triangulate morphology, biochemistry, perturbation #### Independent principles rarely share artefacts #### Disagreement reveals compartments or flawed assumptions