TIRF Microscopy Advantages and Disadvantages
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Questions and Answers

What is the main advantage of multiphoton microscopy compared to conventional excitation wavelength?

  • Produces more out-of-focus fluorescence
  • Causes less photobleaching
  • Better z-resolution (correct)
  • Requires lower power lasers

    • Which of the following is a disadvantage of multiphoton microscopy?

  • Requires lower power lasers
  • Expensive equipment (correct)
  • Produces less photobleaching
  • Achieves better resolution due to longer wavelength

    • What is the main application of multiphoton microscopy?

  • Deep tissue imaging, such as neuronal activity in deeper brain layers (correct)
  • Performing fluorescence lifetime imaging microscopy (FLIM)
  • Measuring temperature and viscosity
  • Studying neuronal activity in shallow tissue layers

    • Which of the following is a method used to measure fluorescence lifetime in FLIM?

    <p>Both TCSPC and frequency domain measurements</p> Signup and view all the answers

    What is the primary advantage of using FLIM compared to other techniques?

    <p>All of the above</p> Signup and view all the answers

    Which of the following is a disadvantage of multiphoton microscopy?

    <p>Requires high power lasers that can boil samples</p> Signup and view all the answers

    What is the main advantage of spinning disk confocal microscopy compared to traditional confocal microscopy?

    <p>It is much faster and has less bleaching</p> Signup and view all the answers

    What is the main disadvantage of spinning disk confocal microscopy?

    <p>It has cross-talk between pinholes, especially in thick samples</p> Signup and view all the answers

    Which of the following is a common use of spinning disk confocal microscopy?

    <p>Studying cell adhesion and cover slip bound molecules</p> Signup and view all the answers

    What is the main advantage of TIRF (total internal reflection fluorescence) microscopy over spinning disk confocal microscopy?

    <p>TIRF has better z-resolution</p> Signup and view all the answers

    What is the main disadvantage of TIRF (total internal reflection fluorescence) microscopy?

    <p>All of the above</p> Signup and view all the answers

    Which of the following is a common use of TIRF (total internal reflection fluorescence) microscopy?

    <p>Studying membrane-associated processes and cover slip bound molecules</p> Signup and view all the answers

    What is FRET used for?

    <p>Measuring energy transfer between molecules</p> Signup and view all the answers

    Which technique requires specialist training for frequency domain measurements?

    <p>TCSPC</p> Signup and view all the answers

    What is a disadvantage of using FRET for environmental measurements?

    <p>Requires destructive acceptor photobleaching</p> Signup and view all the answers

    How does FRET provide definitive proof of intermolecular interactions?

    <p>By measuring energy transfer between molecules</p> Signup and view all the answers

    In what manner does FRET transfer energy to the donor when other fluorescent proteins are in close proximity?

    <p>Through dipole-dipole interactions</p> Signup and view all the answers

    What is a limitation of sensitized emission on a conventional microscope for quantifying FRET efficiency?

    <p>Cannot be done multiple times per sample</p> Signup and view all the answers

    Study Notes

    Two-Photon Microscopy

    • No pinhole needed as infrared light can penetrate tissue deeper than conventional excitation wavelength
    • Advantages: less photobleaching, better z-resolution
    • Disadvantages: high power lasers needed, bleaching in focal volume happens faster, expensive, lower resolution due to longer wavelength excitation
    • Usage: deep tissue imaging, neuronal activity from deeper layers inside the brain

    FLIM (Fluorescence Lifetime Imaging Microscopy)

    • Measures the time between excitation and emission, which is achieved by measuring differences between the excitation light and the emission light
    • Uses differences in lifetimes of fluorophores to map variations in environmental components such as temperature, viscosity, refractive index, glucose concentration, and pH
    • Methods: Time Correlated Single Photon Counting (TCSPC), Frequency Domain Measurements (phase shift, modulation depth)
    • Advantages: concentration independent, can be combined with FRET/TIRF/others
    • Disadvantages: expensive, difficult to set up, TCSPC takes long, frequency domain measurements require specialist training
    • Usage: environmental measurements, FRET quantification

    FRET (Fluorescence Resonance Energy Transfer)

    • Physical process of energy transfer from one fluorophore (donor) to another (acceptor) by resonance, not via photon emission of the donor
    • Process occurs at distances below 10nm and can be used to measure interaction between molecules
    • Energy transfer can occur through emission of a photon, going into a triplet state, delayed fluorescence, undergoing a photochemical reaction, or transfer of energy to donor
    • Ways to quantify FRET efficiency: sensitized emission, acceptor photobleaching, fluorescence lifetime imaging, spectral imaging, fluorescence polarization imaging
    • Advantages: definitive proof of intermolecular interactions, scale of interactions below the Abbe limit (1-10nm)
    • Disadvantages: many controls needed for sensitized emission, acceptor photobleaching is destructive and can only be done once per sample

    Spinning Disk Microscopy

    • Advantages: faster than confocal microscopy, less bleaching, more sensitive detection with camera
    • Disadvantages: fixed pinhole diameter, different disks needed for different magnifications, cross-talk between pinholes, spinning disk frequency and exposure time need to be aligned otherwise striping artifacts
    • Usage: high speed live cell imaging

    TIRF (Total Internal Reflection Fluorescence)

    • Uses total internal reflection to create an evanescent wave to excite fluorophores in a 50-200 nm space above the coverslip
    • Advantages: better z-resolution, faster imaging due to camera, no out of focus excitation and thus no blur
    • Disadvantages: limited to membrane of cover slip bound samples, samples need to be bound to cover slip, expensive objective needed, difficult optical configuration
    • Usage: membrane studies, cell adhesion, cover slip bound molecule studies

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    Description

    Explore the advantages and disadvantages of Total Internal Reflection Fluorescence (TIRF) microscopy, including faster imaging, less bleaching, and more sensitive detection with a camera. Learn about fixed pinhole diameter, cross-talk between pinholes, and other challenges in TIRF microscopy.

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