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Total internal reflection fluorescence microscopy (TIRFM) is a cutting-edge optical technique that excites the fluorophores in a fragile axial region to visualize the cellular events occurring close to the cell surface. The evanescent field, a near-field wave which decays in intensity over a sub-wavelength distance, is used as the basis for total internal reflection fluorescence microscopy (TIRFM). The evanescent field occurs when the incident light is totally reflected at the interface of two transparent media having different refractive indices. The TIRFM has a wide range of applications in visualizing the biological events and quantifying their kinetic rates. The total internal reflection fluorescence microscopy is used to study the protein-protein and protein-nucleic acid biochemical interactions. The total internal reflection fluorescence microscopy has made it easier to understand the mechanism and function of cellular components including signaling cascades, membrane proteins, and molecular motors. The relative ease of use of TIRF and the high sensitivity in single-molecule detection makes it an indispensable technique in biomedical science to tackle a wide array of cellular questions.


The total internal reflection fluorescence microscopy is based on the evanescent field, which exclusively illuminates a thin plane just below the glass coverslip. The evanescent field occurs as a result of the total internal reflection of the light rays at the interface of the imaging surface and an aqueous medium. The refractive index of the optical medium tells about the propagation of the electromagnetic waves through it relative to the propagation of the wave through the vacuum. When light rays traveling through one medium strike at the interface of another medium with a different refractive index, the subsequent direction of the light rays is changed depending on the angle at which the light meets the interface. The energy of the evanescent field decreases as it travels to the interface, only the fluorophores close to the coverslip are excited. This creates the images with an outstanding signal-to-noise ratio, as the rest of the fluorophores in the cell are hardly excited. Therefore, the membrane-associated processes like cell adhesion, molecule transport, hormone binding, and exocytotic and endocytotic processes are observed (Yildiz. & Vale., 2015).


The total internal reflection fluorescence microscopy consists of an objective lens, an excitation beam path which passes the light through the objective lens to the sample, and a coupling element arranged in the back focal plane of the objective lens. The coupling element consists of two areas; one for relaying light to the objective lens for total internal reflection illumination and the second for separating the light emitted by the sample and passing it through the excitation beam path in reverse direction. The laser beams are joined with a dichroic mirror and expanded via Gaussian beam expander. These laser beams are focused on the back focal plane of the objective with an achromatic doublet lens. A set of multiband dichroic and emission filters reflect the laser beams on the objective and transmit the fluorescence simultaneously. The fluorescence is then separated by a Dual View instrument which is equipped with a dichroic mirror to split the fluorescence, and band-pass emission filters to reduce the cross talk between the two fluorescence channels (Fish, 2009).

Procedure for observing exocytosis in the endocrine cells (Trexler. & Taraska., 2017)

Coverslip preparation

  1. Place the coverslips in a ceramic or Teflon coverslip holder.
  2. Place the coverslip holder in the bottom of a 2-liter glass beaker and add 300 mL water in it.
  3. Add 60 mL of the 30 % hydrogen peroxide (H2O2) to the beaker and place it in a fume hood.
  4. Add 60 mL of 27 % ammonium hydroxide to the beaker. And place the beaker on a hotplate and turn it to high. Wait for 5 minutes and check for gentle bubbling in the beaker. After the gentle bubbling begins, incubate the coverslips for 15 minutes. After the incubation, make sure that the solution is vigorously bubbling at 80–90 °C.
  5. Remove the beaker from the hotplate. With the help of wire tongs, transfer the coverslip holder to a 2-liter beaker filled with 1 liter of water. Then transfer the coverslip holders to smaller containers of 100 % ethanol for long-term storage.
  6. Coat the coverslips according to the cell type. Remove the coverslips from ethanol inside the safety cabinet. Air-dry the coverslips and transfer one coverslip to each well of a six-well plate. Add 100–200 μL of poly-l-lysine (PLL) solution to each coverslip.
  7. Incubate them for 10 minutes at room temperature and then aspirate the PLL solution from the coverslip. Wash the coverslips twice with 2 mL media and then cover it with 2 mL media for cell addition. Rinse away the unbound PLL.

Cell culture and transfection

  1. Rinse the cells with Dulbecco’s phosphate buffered saline (DPBS). After rinsing the cells in DPBS, perform trypsinizing and pelleting, and resuspend the cells in media and add dropwise to coverslips in six-well plates.
  2. After plating, allow the cells to rest overnight in an incubator.
  3. Transfect the cells after incubation. Cells should be transfected with a vesicle cargo marker.
  4. Label the vesicles with suitable probes. If needed co-tag the vesicles with a second fluorescent probe.
  5. After transfection, allow the cells to rest overnight. Visualize the cells 1–2 days post-transfection.

Microscope and sample preparation

  1. Place a 25 mm coverslip in the coverslip chamber. Add the buffer on the coverslip and rub the coverslip with the forefinger to stick the beads to the glass. Add 500 μL of the imaging buffer to the coverslip followed by the addition of 5 μL diluted fluorescent beads. Place the bead-coated coverslip on the microscope.
  2. Focus the beads stuck to the coverslip by adjusting the microscope.
  3. Adjust the TIRF angle as needed for the shallow evanescent illumination. 

Stimulation and imaging

  1. Rinse the coverslip in imaging buffer for three times before placing them in the coverslip chamber.
  2. Place the coverslip in a coverslip chamber and cover the cells with 500 μL of imaging buffer and put on the microscope.
  3. Position the perfusion tip close to the focal plane of the cells.
  4. After positioning the perfusion tip, perfuse the cells with imaging buffer briefly.
  5. Move the stage in the x-y plane in all directions to ensure that the tip is not touching the coverslip or the cells.
  6. If required, add an aspirating pipette to the microscope stage insert, and position the tip just over the desired buffer level in the coverslip
  7. Turn on the fluorescent illumination and scan the coverslip for a cell suitable for imaging.
  8. Adjust imaging parameters to get the maximum intensity from the fluorescent channels.
  9. Perfuse the cells for 5 s.
  10. Acquire the images and rinse the coverslips with 3-5 ml of the imaging buffer to wash away the residual stimulation solution.
Protocol for live-cell imaging of vesicle trafficking (Loder., Tsuboi., & Rutter., 2013)

Cleaning and coating of dishes

  1. Place the glass-bottom dishes into a 500 mL beaker containing 300 mL of Milli-Q water and put the beaker in an ultrasound bath for 1 hour.
  2. Dip the dishes into another 500 mL beaker containing 300 mL of 70% ethanol for 1 hour.
  3. Take out the dishes from the beaker inside a cell culture clean bench, and sterilize them with the help of a UV lamp for 30 minutes.
  4. Apply 100 mL of poly-l-lysine (PLL) solution on each dish.
  5. After 30 minutes, wash the dishes with 1 ml sterile phosphate buffer solution for three times.

Cell transfection

  1. Culture the cells in a 10-cm Petri dish and incubate them in 5% CO 2 at 37°C.
  2. For TIRF microscopy, plate the cells in PLL-coated glass-bottom dishes (35 mm).
  3. Transfect the cells with 3 mg of NPY–Venus vector using the Lipofectamine.

Preparation of mouse β cells

  1. Isolate the pancreatic islets by collagenase digestion of the pancreas of female CD1 mice and select the cells by hand-picking.
  2. Culture the islets RPMI medium (10 mM glucose and 2 mM glutamine supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin) for 1 day.
  3. Incubate the cells and dissociate the islets in Ca2+ -free buffer or Trypsin ethylenediamine triacetic acid EDTA for 5 minutes.
  4. Replace the solution with RPMI medium, and disrupt the islets by pipetting through a glass pipette.
  5. Culture the cells for 1 day on PLL-coated glass-bottom dishes or coverslips.


Infect the cells with adenoviruses at the rate of 30–100 infectious particles per cells for 4 hours, and then change the culture medium.

Imaging of exocytosis

  1. Culture the cells in Krebs Ringer buffer (KRB) for 30 minutes at 37°C.
  2. Place the glass-bottom dishes to the thermostat-controlled heating stage (37°C) of the TIRF microscope.
  3. Locate the fluorescent NPY–Venus-expressing cells or infected dispersed primary β-cells.
  4. Adjust the incident angle of the laser to obtain total internal reflection.
  5. Focus the beam on the cell surface.
  6. Obtain the images at 30- to 300-ms intervals.
  7. Change the buffer from KRB to either high glucose containing KRB and continue imaging for 20 minutes.


Assessment of GLUT4 trafficking across the membrane (Wasserstrom., Morén., & Stenkula., 2018)

Insulin-responsive GLUT4 storage vesicles (GSV) helps in the glucose uptake by translocating to the cell surface. In the study, the total internal reflection fluorescence microscopy was used to understand the events involved in glucose uptake and insulin-regulated GLUT4 translocation in both cultured 3T3-L1 adipocytes and primary adipocytes isolated from the rodents and humans. The cells were prepared, transfected, and imaged under the TIRF microscope. It was found that the GSV traffic is decreased as the vesicles lead to the plasma membrane followed by fusion. It was observed that the insulin-induced GSV fusion is followed by the release of GLUT4 monomers into the plasma membrane. The total internal reflection fluorescence microscopy has been found as a powerful tool to observe the vesicle translocation across the plasma membrane.

Quantification of receptor pharmacology (Fang, 2015)

The total internal reflection fluorescence (TIRF) microscopy has been widely used to visualize single molecules in the cells to unveil fundamental aspects of cell biology as it selectively excites a very thin fluorescent volume close to the substrate on which the cells are grown. TIRFM has been used to track single receptors having a SNAP-tag, and to compare their arrangement, mobility, and supramolecular organization. The studies presented that the G-protein coupled receptors (GPCRs) possess varying degrees of di-/oligomerization. Whereas β1- or β2-Andregenic receptors are freely diffusive on the cell surface. These results suggest that GPCRs are located on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be stimulated or targeted, in a ligand-regulated manner, to different cell-surface micro-domains. The total internal reflection fluorescence microscopy has become a promising technique in profiling the receptor pharmacology in clinical applications. 

Visualizing the cell-substrate contact regions (Thompson., Pearce., & Hsieh., 1993)

Cell-substrate contact regions demonstration is one of the applications of total internal reflection fluorescence microscopy. The evanescent excitation has been used to image the arrangement of fluorescent probes for different membrane components in cell-substrate contact regions. The fluorescent reporters attached to cytoskeletal elements are visualized in rat myotube membranes adjacent to glass substrates. TIR-FPPR was used to probe the lateral mobility of fluorescent antibodies which linked the rat basophil leukemia cells to supported planar membranes. It was found that the fluorescence is intense if the cell-to-substrate distance is large and is weak if the distance is small. The TIRFM has also been used to measure the spatial distribution of fluorescence intensities that provides a two-dimensional map of cell-to-substrate contact distances and the binding kinetics of the cell-substrate contact.

Live-cell imaging of the estrogen receptor (Kisler. & Dominguez., 2016)

The total internal reflection fluorescence microscopy has been used to visualize the trafficking of plasma membrane-localized intracellular estrogen receptors following estradiol stimulation in living cells. To visualize estrogen receptor trafficking N-38 neurons were used as a model for membrane-initiated estradiol signaling. The TIRFM permits observation of live, intact cells while allowing visualization of the receptor activation cascade following estradiol activation. The TIRFM yielded high-contrast real-time images of fluorescently labeled E6BSA molecules on and just below the cell surface and was found a powerful tool for studying estrogen receptor trafficking in living cells.

Evaluation of intracellular signaling (Mattheyses., Simon., & Rappoport., 2010)

The total internal reflection fluorescence microscopy has also been used to demonstrate different steps of intracellular signaling. The TIRF has been instrumental in delineating plasma membrane recruitment and spatial distributions of signaling molecules. The plasma-membrane-targeted biosensor enabled the imaging of temporal oscillations of cAMP (cyclic Adenosine monophosphate) signaling, instigating the research in the regulation of upstream targets. The single plasma membrane Ca2+ channels have also been imaged with spatial and temporal resolution revealing uneven molecular kinetics. The TIRF and patch-clamp methods have successfully demonstrated the localization and signaling of open calcium channels and calcium-sensing molecules, explaining the spatial dynamics of intracellular calcium signaling.

  • The cells used for TIRF microscopy must be adherent because TIRF illuminates only the region close to the coverslip and cannot be used to image non-adherent cells.
  • It is essential to coat the coverslips with extracellular matrix molecules to ensure cell adherence.
  • If fixed cells are used, then they must be mounted in a low refractive index media.
  • Maintain the live cells at 37°C as the temperature gradients can lead to focal drift.
  • Keep the tagged samples in the dark.
  • Carefully adjust the incident angle to produce the desired effects.


Strengths and limitations
  • The total internal reflection fluorescence microscopy provides the imaging of even 100-nanometer sections.
  • The TIRFM allows restricted illumination which is best suited for the visualization of membrane receptors and events.
  • The TIRFM is much more economical because the technique does not require complex scanning galvanometer systems.
  • The total internal reflection fluorescence microscopy is a powerful technique used for the imaging of cellular events occurring close to the plasma membrane.
  • The total internal reflection fluorescence microscopy is limited to analyze small samples of 10-300 nm sections only.
  • The TIRFM is limited to the specimen regions having an appropriate refractive index.


  1. Trexler., & Taraska., J. W. (2017). Two-Color Total Internal Reflection Fluorescence Microscopy of Exocytosis in Endocrine Cells. Methods Mol Biol, 1563, 151-165.
  2. Mattheyses., S. M. Simon., & Rappoport., J. Z. (2010). Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci, 3621-8.
  3. , & Vale., R. D. (2015). Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc, 9.
  4. Fang, Y. (2015). Total Internal Reflection Fluorescence Quantification of Receptor Pharmacology. Biosensors (Basel)., 5(2), 223-40.
  5. Fish, K. N. (2009). Total Internal Reflection Fluorescence (TIRF) Microscopy. Curr Protoc Cytom.
  6. Kisler., & Dominguez., R. (2016). Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy. Methods Mol Biol, 1366, 175-187.
  7. K. Loder., T. Tsuboi., & Rutter., G. A. (2013). Live-cell imaging of vesicle trafficking and divalent metal ions by total internal reflection fluorescence (TIRF) microscopy. Methods Mol Biol, 950, 13-26.
  8. L. Thompson., K. H. Pearce., & Hsieh., H. V. (1993). Total internal reflection fluorescence microscopy: application to substrate-supported planar membranes. Eur Biophys J, 22(5), 367-78.
  9. Wasserstrom., B. Morén., & Stenkula., K. G. (2018). Total Internal Reflection Fluorescence Microscopy to Study GLUT4 Trafficking. Methods Mol Biol, 1713, 151-159.