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 microscopies 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 ~100 nm of the specimen. In comparison, the confocal scheme excites ~1,000 nm. Prism-based geometry ensures the lowest scatter, and the best signal-to-background ratio. See Refs. [1-5], web page TIRF Microscopy and the brochure Compare TIRF Geometries.  

If your application permits, prism-TIRF is the geometry to be considered at the first place.  pTIRF ensures the “cleanest” TIRF effect, the crispest TIRF images, the highest contrast for reliable detection of single molecules.  pTIRF can be used for a variety of applications, including super resolution 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 prism-TIRF geometries. Each of the geometries is suited for certain applications, but are not suited for others. Contact TIRF Labs  to better determine which geometry is better suited for your 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 ensures 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  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 system. pTIRF systems are designed as an add-on accessories for inverted microscopes. The photo in Fig. 2 shows the puTIRF system installed into a K-frame window of a motorized XY translation stage. 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 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. A thin layer of aqueous solution and an  optical window separate the TIRF surface from the objective.

 Microfluidic Channels Create Planar Low-volumeTIRF Flow Cell.  An advanced microfluidic system embedded into  pTIRF  provides high share rates at small volumetric flow rates, which allows for measuring 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.  An external pump, or gravity flow, which is always by hand,  can be used with a  pTIRF 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:

pTIRF add-on accessory is the state-of-the-art, but robust system, which combines optical, mechanical, and fluidic 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. 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. If decreased depth of penetration is necessary, TIRF Labs offers 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.  Four versions of TIRF slides and fluidic chamber for puTIRF. 1- Use drilled 1”x 3”  slide with pre-installed flow chamber.  2-  Use fluidic block with optical window and  elastic gasket. The flow cell is formed by the gasket between slide and optical window. 3-  Arrangement similar to version 2, but using 1” x 1.5” slide. 4-  Use 1” x 1.5”  slide with flow cell formed by fluidic block.