TIRF Microscopy Advantages and Disadvantages

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