Part 2: L1, Principles and Basis Flashcards

1
Q

Advantages of fl. imaging

A
  • Resolution and sensitivity
  • Subcellular level
  • More biochemical technique -> detailed
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2
Q

Fl. spectroscopy setup

A
  • Monochromated using a diffraction grating
  • Excitation monochromator perpendicular to emission monochromator where sample re-emits
  • Sweep through wavelengths both going in and out -> see which are most effective together -> characterise sample
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3
Q

Advantages of confocal microscopy (industry standard):

A
  • High sensitivity (nm)
  • Low cost
  • Specific staining and multi-channel detection
  • Responsive; informs on environment
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4
Q

Essential properties of lumophores for fluorescence imaging applications:

A
  • Stability and solubility
  • Toxicity
  • Uptake -> Water solubility
  • Localisation
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5
Q

Structure of phospholipid membrane

A
  • Charged head groups on outside
  • Nonpolar tails (lipidic)
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6
Q

Rules of thumb for lumophore design

A
  • Cationic
  • Lipophilic
  • Mass <500 Da
  • Biocompatible, soluble, non-toxic (heavy metals; release of metals)
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7
Q

Issues in Fl. imaging:

A
  • Background emission (autofl.)
  • Self-quenching
  • Photobleaching (biochemical loss of agent, typical example is formation of ROS)
  • Biocompatability, solubility, toxicity
  • Transport-membrane permeability/active uptake
  • Tissue damage/penetration
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8
Q

Self quenching

A
  • Close in space to another dye molecule in case of low stokes shift
  • Transfer between molecules loses efficiency (re-absorption by neighbouring molecules loses intensity)
  • No overlap if large stokes shift
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9
Q

Abbe’s diffraction limit:

A
  • As light interacts with matter, it is not just absorbed or reflected, it is also diffracted as it passes through gaps (e.g. confocal pinhole, past organelles)
  • Diffraction = scattering, loss of resolution
  • Smallest distance can be resolved, based on wavelength, lens angle and numerical aperture
  • …Simplifies to d = wavelength/3
  • Beyond this, result is a diffuse excitation spot and a blurred image
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10
Q

How can Abbe’s limit be avoided?

A
  • Super-resolution techniques
  • STED (i.e. principle of lasers)
  • Can deplete excited state by irradiation at that energy leading to stimulated emission at different wavelength to normal fluorescence as not dropping to low vibrational level of ground electronic state
  • Requires large stokes shift and broad emission bands
  • Donut shaped depletion spot
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