Total Internal Reflection Fluorescence
Total Internal Reflection Fluorescence - Related Videos
02:51
Cell Imaging Using Total Internal Reflection Fluorescence Microscopy
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2025
Source: Daniele, F., et. al TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis. J. Vis. Exp. (2015).This video demonstrates the imaging of human neuroblastoma cells using a total internal reflection fluorescence (TIRF) microscope to visualize fluorophore-tagged synaptic vesicles. The cells are first focused in epifluorescence mode. Adjustments are then made to achieve total internal reflection,...
03:54
Total Internal Reflection Fluorescence Microscopy for Visualization of Exocytic Events
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2025
Source: Winkle, C. C., et al. Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis. J. Vis. Exp. (2016)This video demonstrates total internal reflection fluorescence (TIRF) microscopy for visualizing exocytic events in cortical neurons. A pH-sensitive green fluorescent protein (GFP) marker fluoresces upon vesicle fusion, with selective TIRF excitation capturing membrane-proximal exocytosis in time-lapse imaging.
01:05
Total Internal Reflection Fluorescence Microscopy
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2023
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
09:14
Nanotopology of Cell Adhesion upon Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM)
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Cited by 7 •
2012
Topology of cell adhesion on a substrate is measured with nanometre precision by variable-angle total internal reflection fluorescence microscopy (VA-TIRFM).
14:14
Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
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Cited by 12 •
2012
Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.
FAQs
The decisive optical condition is incidence above the critical angle at the glass–sample boundary. Under that condition, excitation light is reflected within the glass rather than propagating into the sample, while an evanescent field forms at the interface. This field reaches only a shallow region, making excitation preferentially sensitive to fluorophores close to the coverslip.
Excitation depth controls the balance between useful signal and background. Because the evanescent field is shallow, fluorophores farther from the coverslip receive much less excitation than near-surface molecules. Total Internal Reflection Fluorescence therefore separates membrane-proximal signals from fluorescence originating in deeper cellular regions, improving contrast for events occurring at or close to the plasma membrane.
Compared with illumination that excites a larger sample volume, Total Internal Reflection Fluorescence restricts excitation to the interface region instead of lighting fluorophores throughout deeper cellular material. That restriction is important when background would obscure weak or rapidly changing signals. The resulting high contrast supports observation of localized biological activity without making the entire cell equally fluorescent.
Limiting excitation to a shallow region can reduce unnecessary illumination of fluorophores outside the observation zone. In the biological applications described, this contributes to lower phototoxicity while preserving sensitivity near the surface. The combination is especially useful for quantitative measurements of rapid cellular processes and for observing single-molecule behavior over the course of an experiment.
Light is directed through the glass coverslip toward the glass–sample interface, and the incidence angle is set above the critical angle. Researchers then observe fluorophores located within the evanescent field near that surface. This arrangement concentrates the measurement on the coverslip-adjacent region, which is essential when the biological signal of interest occurs at the plasma membrane or nearby.
Use Total Internal Reflection Fluorescence when the question concerns events close to the cell surface rather than fluorescence throughout the cell. In biology, that includes plasma-membrane dynamics, vesicle fusion, cytoskeletal remodeling, and protein interactions near the surface. Each application benefits from restricting excitation to the region where these processes occur, helping distinguish local activity from deeper background.
The technique can reveal both dynamic and highly localized behavior. Its high contrast supports quantitative analysis of rapid cellular processes, while the shallow excitation region allows investigators to examine near-surface protein interactions and single-molecule behavior. These outcomes make Total Internal Reflection Fluorescence valuable when timing, localization, and weak signals must be evaluated together in living cells.