Lightguide-based TIRF Microscopy

Lightguide-based Total Internal Reflection Fluorescence  Microscopy – lgTIRF

lgTIRF  is a novel powerful tool for single molecule detection, super resolution methods, plasma membrane studies, real-time microarrays, combination of TIRF with AFM, multi-photon, patch clamp single ion channel, dielectrophoresis, electro-chemi-luminescence, and other applications that require the excitation of fluorescence confined in space.

  • lgTIRF features the excitation path, which is naturally independent from the emission channel
  • lgTIRF yields a superior signal-to-background ratio
  • lgTIRF can be used with dry-, water-, and oil-immersion objectives
  • lgTIRF is a factory-aligned system that is well-suited for multicolor TIRF experiments
  • lgTIRF uses glass or silica coverslips, or Petri dishes with optical bottom
  • lgTIRF platform also implements Shallow Angle Fluorescence Microscopy (SAFM)
  • lgTIRF uses fiber-coupled illuminators connected by ~2-m optical fiber cable
  • lgTIRF provides a reproducible intensity of the evanescent wave
  • lgTIRF can be used with UV excitation, which is not available in objective-TIRF
  • lgTIRF allows for precision control of the penetration depth by using optical traps
  • It takes no time to install/uninstall lgTIRF, and to switch between TIRF, SAFM, micro-spot excitation, epi-fluorescence, transmittance, and other methods.

This web page describes the principles and features of the lightguide-based Total Internal Reflection Fluorescence (lgTIRF) system. The lgTIRF system is designed as an add-on accessory for inverted microscopes and can be reconfigured for upright microscopes. The excitation light is delivered to the lgTIRF unit via a 2-meter fiber optics cable. The FC-PC outlet of the cable is connected to the lgTIRF unit, and the inlet is connected to a multicolor or a single color illuminator.

lgTIRF is a powerful and versatile analytical tool, which implements at the same platform TIRF effect, Shallow Angle Fluorescence Microscopy (SAFM), and Micro-Spot Excitation (MSE). The latter method, MSE, is well-suited for the probing of organelles, single ion channels, and other objects with sizes from 1 to 100 microns. There are three versions of TIRF excitation light launchers that differ by the geometry of coupling light: (i) from the end of the coverslip; (ii) from the top, and (iii) from the bottom of the coverslip. Different light launchers are used for TIRFing Petri dishes, rectangular coverslips, and other formats of specimen substrate. There are two “flavors” of the (i) geometry (from-the-end): stationary and mobile. The former is fixed on the K-Frame stainless steel platform for the convenience of quick alignment, the latter can be moved around the coverslip and use any side surface to couple light into the coverslip.

TIRF is used for single molecule detection (SMD), super resolution microscopies and other studies that require the excitation of fluorescence confined in space [1-4]. In SMD, spatial confinement is necessary for minimizing the background signal and obtaining the contrast sufficient for detecting single molecules. lgTIRF provides a superior spatial confinement – it excites only a ~100 nm layer of the specimen. In comparison, the confocal scheme excites ~1,000 nm. In TIRF, the intensity of excitation light is maximal at the surface and exponentially decays with the distance.  Only molecules that are at the TIRF surface and in a ~100 nm proximity to the surface are excited and fluoresce; the bulk of the specimen is not excited and does not fluoresce. The surface selectivity of TIRF is also well-suited for cell membrane studies, the analysis of biomolecular interactions, and other areas. 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., Ref. 5].

The TIRF effect can be achieved in different optical schemes, including prism-, objective-, and lightguide-based geometries. Each of the geometries has its own set of advantages and limitations. The prism-based scheme provides the best signal-to-background ratio [1], but is difficult to use with open perfusion chamber on inverted microscopes. The objective-based scheme collects the maximal amount of emitted fluorescence, but TIRF effect is compromised due to large intensity of stray light (~15%), which excites fluorophores located in the bulk of specimen, outside of the evanescent wave. Only in objective-TIRF the excitation light travels through the emission channel and generates large intensity of stray light. It has been documented in the literature that only in objective-TIRF geometry stray light deteriorates the TIRF effect by illuminating the bulk of specimen [1-3]. Lightguide-based geometry (lgTIRF) offers superior signal-to-background ratio and is exceptionally well-suited for multicolor TIRF, including FRET for single molecule detection, cell membrane, real-time microarray, and other studies.  lgTIRF can be used with dry, water-, and oil-immersion objectives. It provides a reproducible intensity of the evanescent wave in one experiment and between experiments. lgTIRF can be used with UV excitation, a feature which is not available in objective-based TIRF.

Fig. 1 above illustrates the principles, and Fig. 2 shows a photo of the lgTIRF system. The system is mounted onto a 110x160mm K-frame, which is a standard for motorized XY translation stages. In Fig. 2,  K-frame is shown nested into a larger platform 200x220mm suitable for manual XY stages. The base model of  lgTIRF system is equipped with four TIRF excitation light launchers: two side-end launchers, a mobile launcher for coupling light from the top surface, and a mobile launcher or the bottom-entrance. The launchers are schematically shown in Figs. 3-6.Two versions of the side-end launchers SEL-1 and SEL-7 differ by the width of the TIRF area generated at the surface. SEL-1 produces a narrow band – up to 1-mm wide. SEL-1 is recommended for single molecule detection and other applications that require high intensity of the evanescent wave.  SEL-7 launcher generates a wider band of the evanescent wave up to 20-mm wide, which is well-suited for real-time microarray applications that require measuring of the response of the entire microarray printed at ~20×20 mm area (Fig. 4a).

Fig. 5 shows the principle of coupling light using a top-surface mobile fiber optics launcher. The latter can be used for coupling TIRF excitation from any site available at the top surface of the coverslip. Fig. 6 illustrates coupling from the bottom surface. This geometry is engineered for TIRFing Petri dishes equipped with optical bottoms. In this case, the light enters from the bottom of a Petri dish and undergoes multiple reflections as shown in Fig. 6. The fiber, which is embedded into the K-frame, and the coverslip are brought in optical contact by a droplet of immersion oil, so that the Pertri dish can “float” along the XY axes.  

Figs. 7 and 8 show optional Micro-spot Excitation (MSE) mobile probes. These mobile probes do Not provide TIRF effect. However, they turned to be convenient tools for many related applications. In particular, these probes are well-suited for confined in space excitation of live cell organelles  and  other objects of interest with sizes from 1 to 100 microns. The probe shown in Fig 8 was developed for single ion-channel/single molecule detection to study  TRPC ion channels at the level of single molecules [Asanov A et al, Cell Calcium. 2015 Jan;57(1):1-13]. This probe can be used with or without electrophysiology.

Superior Signal-to-Background Ratio. From the standpoint of signal-to-background ratio, lgTIRF is close to that of prism-TIRF geometry. Similar to pTIRF, the excitation path in lgTIRF is naturally independent from the emission channel. Light enters the TIRF lightguide through one of  its ends and escapes from the opposite end. Independence of the excitation from the emission channel provides superior signal-to-background ratio, which is 3-4 orders of magnitude better than that for objective-TIRF.

lgTIRF System is Compatible with Dry, Water- and Oil-immersion Objectives and, thus, can be used for broad range of studies – from TIRF imaging of cellular organelles to parallel detection of real-time response from microarrays and TIRF viewing of a group of cells.

lgTIRF can be used with Glass or Silica coverslips as TIRF lightguides. Rectangular or round coverslips, with mounted disposable or reusable temperature controlled open perfusion chambers and closed flow cells are available as options for lgTIRF. As mentioned above, there are several TIRF excitation light launchers: one stationary and three mobile. The stationary launcher couples the excitation light from the end of a coverslip, as shown in Fig. 4. Three mobile launchers couple light from the the end, from the top, and from the bottomof the coverslip, as shown in Fig. 4, 5, and 6, respectively. The latter launcher is well-suited for TIRFing with Petri dishes equipped with optical bottoms.

Silica Optics of the excitation channel comprises fiber optics cable, collimators, and optics of the excitation light launchers; they are made from UV silica. Respectively, the use of silica coverslips allows for TIRFing with excitation wavelengths 190-1000 nm, including UV,  a feature which is not available in objective-based TIRF.

 Turnkey TIRF Station. TIRF Labs offers the entire range of instruments, supplies, and services for a turnkey TIRF installations. The instruments include: uTIRF Cube – ultimate TIRF turnkey station, TIRF accessories, including prism-, lightguide-, and objective-TIRF, fluorescence illuminators, digital fluidics, electrochemical, dielectrophoretic, temperature control, and other accessories for simple and sophisticated experiments with TIRF systems, filter wheels, XY and XYZ motorized stages, and respective software to control the instruments. Using of electrochemical or dielectrophoretic control permits not only to image the cells under the microscope, but also interact with them. We supply a variety of chemically modified and bio-functionalized lightguides, TIRF slides and coverslips for cell biology studies. Application Notes illustrate the use of our TIRF products for single molecule detection, cell biology, lipid rafts, and real-time microarray studies.  

Reproducible TIRF Measurements. lgTIRF is a geometry with the intensity of evanescent wave reproducible within one experiment and between different experiments.  For this purpose, the angle of incidence is fixed. Switching between different excitation wavelengths does not require realignment. The depth of penetration can be changed by optical traps that extinguish light with small angles of incidence.  

SAFM. The lgTIRF platform also implements Shallow Angle Fluorescence Microscopy (SAFM) – the mode of excitation schematically shown in Fig. 9. In SAFM mode, a portion of excitation light propagates at shallow angles along the surface and illuminates fluorophores that are 1-5 microns away from the surface. lgTIRF is supplied with Micro-Spot Excitation (MSE) probes schematically shown in Figs. 7 and 8. In MSE mode, one uses a patch-clamp pipette as a lightguide which transmits the excitation light to the 1-micron tip of the pipette. Fig. 7 shows a version of MSE equipped with a microfiber 5-50 microns in diameter to selectively excite an organelle or a group of ion channels with sizes 5-100 microns. It takes no time to switch between TIRF, SAFM, and MSE mode, as well epi-fluorescence, transmittance, and other methods.  For more information see Application Notes and articles referenced at the URL: www.tirf-labs.com/applications

  Lightguide-TIRF.pdf
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. Asanov A, Zepeda A, Vaca L.  Platform for Combined DNA and Protein Microarrays Based on TIRF. Sensors, 2012, 12, 1800.
  5. Steyer JA, Almers W. A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol. 2001, 2(4), 268.