BIOL123 Lecture Notes - Lecture 7: Confocal Microscopy, Fluorescence Microscope, Optical Microscope
17 Jun 2018
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· Polarisation (polarised might microscopy)
• Used to analyse structures that have two different refractive indices
(cellulose microfibrils)
• Provides information on the orientation of molecular structures in a
specimen
• Polariser positioned in light path somewhere before the specimen
• Analyser (2nd polariser) placed after the objective
· Dark field illumination
• Used with stereo and inverted microscopes
• Like phase, allows transparent specimens to be visible (important in
tissue culture)
• Central light along optical axis is blocked out so light is at oblique
angles to specimen
· Reflection and differential interference
· Fluorescence and Laser Scanning Confocal microscopy
• Fluorescent microscopy:
• Certain dyes are fluorescent under UV light
• Used to observe distribution of biological materials in cells and
tissues (e.g. proteins, lipids, nucleic acids, ions)
• Multiple colours available
• Sensitivity of fluorophores (fluorescent chemical compound that
can re-emit light upon light excitation) means lower
concentrations of biological material can be detected than with
conventional dyes
• Confocal microscopy:
• Several advantages over optical microscopy:
• Controllable depth of field
• Eliminates out of focus information
• Able to collect optical sections from thick specimen
• Used with fluorescence microscopy
• Image captured by photomultiplier
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