Total Internal Reflection Fluorescence

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TIRF Microscopy  

TIRF for Fluorometers


TIRF Biosensors

TIRF Slides, TIRF Prisms

Total Internal Reflection Fluorescence (TIRF)

Single Molecule Biology, Single Molecule Detection, sm-FRET,

Super-resolution Microscopy, Real-time TIRF Microarrays,

Plasma Membrane Studies, Single Cell Analysis, and i-Diagnostics

TIRF Principles. TIRF employs the phenomena of total internal reflection and the evanescent wave to selectively excite fluorophores located at the surface and in close proximity (~100 nm) to the surface of a glass, silica, or plastic slide. Fluorophores that are in the bulk of the solution are not excited and do not fluoresce. Exceptional spatial selectivity  gives TIRF its numerous advantages, including the ultimate limit of detection: single molecule. The TIRF effect allows for the circumventing of the diffraction limit of  resolution and, therefore, is used in many super resolution microscopy methods.  TIRF efficiently minimizes the background and allows you to monitor the molecular dynamics at the level of single molecules in real-time.  More »

TIRF Applications. TIRF spectroscopy and TIRF microscopy are powerful analytical tools for numerous life science areas, including single molecule detection, super resolution microscopies, cell biology,  real-time microarrays for molecular diagnostics, bioassay development, nanoengineering, and drug screening. In particular, TIRF is “a method uniquely suited to image the plasma membrane with its associated organelles and macromolecules in living cells. The method shows even the smallest vesicles made by cells, and can image the dynamics of single protein molecules.” [Steyer JA, Almers W. A real-time view of life within 100 nm of the plasma membrane. Nature Reviews, Molecular Cell Biology, 2001, 2(4), 268-75]. References to the articles published by our customers and TIRF application notes are available at

TIRF Products. TIRF Labs offers a broad range of TIRF products: prism-, lightguide-, and objective-based TIRF microscopy systems,  from simple add-on accessories to sophisticated computer-controlled  turnkey stations, including uTIRF. Our LED and laser fluorescence illuminators include cost-efficient single-color light sources, and two-, three-, four-,  or seven-color computer-controlled  illuminators. For TIRF spectroscopy we supply TIRF accessories with gravity flow and digital fluidics. Our automated TIRF microarray biosensors detect multiple nucleic acid, protein, and metabolite markers in parallel and in a matter of a few minutes. Popular products also include TIRF slides, lightguides, and prisms. More »

Technologies Applications Applications Applications

Digital Fluidics SmartFlow

iDiagnostics - The Cellphone Future of Medicine  visit:

TIRF Geometries.  Prism-, lightguide-, and objective-based optical schemes are three popular geometries that are used for TIRF microscopy. Each geometry has its own set of advantages and limitations. A Prism-based scheme provides the best signal-to-background ratio, however, is difficult to implement with an open perfusion chamber on an inverted microscope. A lightguide-based geometry yields exceptional flexibility and a superior signal-to-noise ratio, can be used with open perfusion chamber on inverted microscopes, and is compatible with dry, water-, and oil-immersion objectives. However, it requires a larger optical power to obtain an equal intensity of the evanescent wave. An objective-based scheme collects the largest portion of the emitted fluorescence, but the quality of TIRF effect is severely compromised, because the excitation light shares the same optics with the emission channel; a large amount (~15%) of the stray light excites the bulk of the specimen and deteriorates the TIRF effect produced by objective-TIRF. TIRF Microscopy webpages will help you choose a geometry suited for your TIRF applications, while TIRF Products webpages - select illuminators, fluidics, optional electrochemical and dielectrophoretic control, and other devices and supplies that are necessary  or useful for TIRFing. TIRF Labs offers the entire range of TIRF instrumentation: from inexpensive add-on TIRF accessories for microscopes and fluorometers -to turnkey TIRF microscopy stations and automated TIRF biosensor instruments.  More »

Prism-TIRF Microscopy  

Lightguide-TIRF Microscopy

Objective-TIRF Microscopy

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TIRF microscopy for single molecule biology, single molecule detection, super resolution microscopy, single cell analysis,   real-time microarrays, analysis of biomolecular interactions, and molecular diagnostics

In addition to traditional objective-based TIRF, TIRF Labs is offering advanced prism- and lightguide-TIRF systems as add-on accessories for fluorescence microscopes. In prism- and lightguide-TIRF optical schemes, the excitation lightpath is separated from the emission channel. Unlike in objective-TIRF, the intensity of stray light in lg- and p-TIRF is minimal; the TIRF effect is clean. Lg-TIRF and p-TIRF are exceptionally flexible geometries that can be used with dry, water-, and oil-immersion objectives, low and high numerical aperture. UV light (>190 nm) can be used for excitation, a feature not available in o-TIRF. Large area of the evanescent  wave up to 20 mm X 50 mm can be used in lightguide-TIRF  geometry  for real-time microarrays to detect proteins, nucleic acids, and metabolites.

Use the uTIRF as a turnkey station, or take prism-, lightguide-, or objective-TIRF units to use as add-on accessories for your microscope

  uTIRF Cube -  ultimateTIRF microscopy turnkey station

Introducing uTIRF - the ultimate TIRF microscopy turnkey station

To compare TIRF microscopy geometries click here

To minimize stray light in TIRF microscopy click here

uTIRF Brochure.pdf

White paper: Selecting Optimal TIRF Geometry.pdf

Application notes and published papers

uTIRF turnkey station consists of prism-, lightguide-, and objective-based TIRF,  infinity objective 20X, 40X, 60X, or 100X,  low light CMOS camera, and a multicolor fiber-coupled illuminator. uTIRF is available in several configurations with open perfusion and closed flow chambers, optional electrochemical and dielectrophoretic control. Prism-, lightguide-, and objective-TIRF units can be taken from uTIRF to be used as add-on accessories for fluorescence microscopes. Inquire about other options.

Main Pros / Cons of  TIRF geometries  (for details click here)






The cleanest TIRF effect - stray light<0.01%. Can be used with dry, water-, and oil-immersion objectives.

No open perfusion for prism-down geometry.



Clean TIRF effect - stray light <0.1%.

Can be used with dry, water-, and oil-immersion objectives.

TIRF area needs to be aligned with the objective.



TIRF area stays with the objective.

Evanescent wave contaminated by 10-15% of stray light.

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