Total Internal Reflection Fluorescence

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Prism-based TIRF Microscopy

Prism-based TIRF microscopy.  Total Internal Reflection Fluorescence (TIRF) has become a method of choice for single molecule detection, super-resolution microscopy and other studies that require the excitation of fluorescence confined in space.  TIRF provides the  best optical confinement, the thinnest optical slicing - it excites only a dozen of nanometers of the specimen. In comparison, the confocal scheme excites ~1,000 nm. Prism-based geometry ensures the best quality of the evanescent wave, the lowest auto-fluorescence and scatter, and the best signal-to-background ratio. See Refs. [1-5], web page TIRF Microscopy, the brochure Compare TIRF Geometries, and the white paper “Selecting the optimal TIRF Geometry....pdf”   

If your application permits, prism-TIRF is the geometry to be considered at the first place.  pTIRF ensures the closest profile to theoretically predicted exponential decay of the evanescent wave, the “cleanest” TIRF effect, the crispest TIRF images, and the highest contrast for reliable detection of single molecules.  pTIRF can be used for a variety of applications, including super resolution microscopy methods, analysis of biomolecular interactions, characterizing of antibody-based and nucleic acid-based assays, real-time microarrays, membrane biophysics, the dynamics of lipid rafts, and many other. Prism-TIRF is so efficient that allows for using even low-cost, moderate sensitivity CCD and CMOS cameras for detecting single molecules [5].

The schemes in Fig. 1 illustrate seven most popular implementations of the prism-TIRF geometry. Each of the schemes is suited for certain applications, but is not suited for others. Contact TIRF Labs  to better determine which geometry is better suited for your specific studies. TIRF Labs performs in-house the entire circle from conception of ideas, to rapid prototyping and manufacturing. In fact, many of our products have been inspired by our customers and collaborators. We will be delighted to customize our existing products or develop an “ab initio” product based on your idea. As alternative to objective- and prism-based TIRF, we are offering exceptionally flexible lightguide-based TIRF. Similar to pTIRF, the excitation path  in lgTIRF is naturally independent from the emission channel.

Record-high Signal-to-Background Ratio. Among TIRF geometries that include objective- and lightguide-TIRF,  prism-based scheme, as mentioned above, ensures the closest profile to theoretically predicted exponential decay of the evanescent wave, the cleanest TIRF effect with the best signal-to-background ratio. In the case of through-objective TIRF, excitation and emission channels share the same optical elements; the intensity of undesirable stray light is large,  and the  quality of TIRF effect is compromised [2, 3]. In prism-TIRF, the excitation is naturally independent from the emission channel. This fact and the absence of additional auto-fluorescence, light-scattering and reflecting surfaces ensures  the best signal-to-background ratio  [1].

 XY translation stages. TIRF Labs offers a broad range of prism-based TIRF systems configured  for inverted and upright microscopes, with fixed and variable angles of incidence (contact TIRF Labs for more information). This web page describes only the most popular prism-TIRF systems. pTIRF systems are designed as an add-on accessories for inverted and upright microscopes. The photo in Fig. 2 shows the puTIRF system installed into a K-frame window of a motorized XY translation stage of an inverted microscope. puTIRF is supplied on a platform of nested design, which also can be used with manual XY translation stages, round 4-inch diameter windows of microscopes or Gibraltar platforms, or rectangular windows with the footprint of 96-well SBS plate.  Optional arm shown on the photo along Y axis provides travel of the excitation spot and EW together with objective. Contact us for details:

pTIRF Systems are compatible with dry, water- and oil-immersion objectives. In pTIRF geometry total internal reflection occurs at the interface between a slide (or a coverslip) and water or aqueous solution, as shown in the scheme below in Figure 2. The TIRF prism and slide are brought in optical contact by a droplet of refractive-index-matching fluid. For excitation light, the prism and the slide represent continuous optical medium. In the case of puTIRF system shown in Fig. 2, a thin layer of aqueous solution and an optical window separate the TIRF surface from the objective.

 Embedded Microfluidic Channels and Fluidics Cartridge Create Planar Low-volume TIRF Flow Cell.  An advanced microfluidic system embedded into  pTIRF  creates closed flow cell encompassing the TIRF surface and provides high share rates at small volumetric flow rates, which allows one to measure k-on and k-off rate constants with minimal amount of bioanalyte solution. Typically, 20-40 uL of bioanalyte is sufficient for measuring a kinetic sensogram.  Alternatively to the embedded fluidics, one can use reusable fluidics cartridges as schematically shown in  Fig. 3 below, panels 2 and 2a. Similar to the embedded fluidics, the cartridge creates 20-40 microliter flow cell around the TIRF surface. Virtually any shape of slide or cover slip with sizes larger than 20 mm can be used with the cartridge, including 1-inch x 3-inches slides (25 mm x 75 mm), half-slides ~25 mm x 38 mm, or round slides or cover slips with diameter larger than 20 mm. An external pump, or gravity flow, which is always by hand,  can be used with TIRF fluidics system for kinetic experiments.

Precision Optical-Mechanical Design of pTIRF provides high reproducibility of TIRF measurements within one experiment and between different TIRF sessions. Figure 2 below, as mentioned above, shows optional optical-mechanical arm installed at the microscope frame, which holds the collimator. The arm allows for relating the excitation spot  with the center of the objective, so that the evanescent wave stays with the objective, if the specimen is moved along Y axis.  

Silica Optics includes an adjustable collimator, TIRF prism, TIRF slides, and an optical window. The range of excitation wavelengths encompasses UV-Vis-Near IR 190-1000 nm. The size of the excitation spot can be adjusted in the range 0.1mm - 12 mm. For more information contact TIRF Labs at:

pTIRF add-on accessory is the state-of-the-art, but robust system, which combines optical, mechanical, and fluidics modules.  Typically, a researcher is capable of TIRFing with pTIRF after reading the Quick Start Guide.

TIRF Labs offers pTIRF systems equipped with closed flow cells or open perfusion chambers, designed for upright or inverted microscopes. For more information contact TIRF Labs at:

The pTIRF systems are compatible with dry, water-, and oil immersion objectives and can be used with 1-mm thick slides or 0.12-0.24 mm glass or silica coverslips as TIRF chips. They feature advanced microfluidics, which allows for operating with sub-microliter amounts of solutions. We also offer pTIRF systems for TIRFing specimens in Petri dishes, as shown in the Fig.1. Most of our pTIRF accessories are factory aligned systems: the angles of incidence are fixed to provide  reproducible intensity of the evanescent wave. We also offer variable-angle pTIRF systems. However, even with fixed-angle pTIRF, one can decrease the depth of penetration using special optical traps that extinguish low angles of incidence, which results in a decreased penetration depth. For more information contact TIRF Labs  

Literature cited:

1. Ambrose W, Goodwin P, Nolan J. Single-molecule detection with TIRF: comparing signal-to-background  in different geometries. Cytometry 1999, 36(3), 224.

2. Brunstein M, Teremetz M, Hérault K, Tourain C, Oheim M. Eliminating unwanted far-field excitation in objective-type TIRF. Part I. Biophys J. 2014; 106(5): 1020.  

3. Brunstein M, Hérault K, Oheim M. Eliminating unwanted far-field excitation in objective-type TIRF. Part II. Biophys J. 2014; 106(5): 1044.   

4. Simon S. Partial  internal  reflections  on  total  internal reflection fluorescent microscopy. Trends Cell Biol, 2009, 19: 661.

5.  Protasenko V, Hull KL, Kuno M. Demonstration of a Low-Cost, Single-Molecule Capable, Multimode Optical Microscope. Chem. Educator 2005, 10, 269282.

Fig. 1.

Fig. 2.


Fig. 3.  Three options for creating a closed flow cell encompassing the TIRF surface on slides and cover slips that can be used with prism-TIRF systems. 1- Embedded fluidics channels, optical window, and diamond-shape gasket to be used with 1”x 1.5”  (25 mm x  38 mm) slide.  2 and 2a -  Use reusable fluidics cartridge with 1” X 3” (25 mm X 75 mm) slide - (2), or 1”x1.5” slide (25mmX38mm) -(2a). 3 -  Use 1”x3”  (25mmx75mm)  slide with pre-installed flow chamber created by 24mmx50mm cover slip attached by a double-sided adhesive tape. Click the figure above to download high resolution version.