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Total Internal Reflection Fluorescence (TIRF) has established itself as an indispensable tool for single molecule detection, super resolution microscopy, and other areas of life sciences. [1-8, 11-15]. TIRF selectively excites only a ~100-nm layer of the specimen next to the surface, while the bulk of specimen is not illuminated and does not fluoresce. This superior spatial selectivity (in comparison, the confocal scheme excites ~1,000 nm) allows for the minimization of the background signal and providing the contrast sufficient for the detection of single molecules. 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” .
There are three popular schemes to realize the TIRF effect: (i) prism-, (ii) lightguide-, and (iii) objective-based geometries, as shown in the schemes above. Each geometry has its own set of advantages and limitations. This web page analyzes errors caused by the stray light in TIRF microscopy and their interference with the TIRF effect. It compares the advantages and limitations of prism-, lightguide- and objective-type TIRF microscopy, discusses the sources of stray light in each of the geometries, and suggests ways for stray light mitigation. This web page will help you to choose a TIRF geometry, which is suited for your studies.
In pTIRF and lgTIRF, the excitation and emission channels are independent.
The excitation light enters the prism or lightguide, reflects at the TIRF interface,
and exits at the opposite end. It does not use and does not enter into the emission
channel, which results in “clean” TIRF effect and superior signal-to-background ratio
Total Internal Reflection Fluorescence
TIRF Principles. The phenomenon of total internal reflection occurs at the interface between two optical media with different refractive indices, e.g. glass/water. If the angle of incidence is greater than critical, the incident light reflects back into the glass and generates a profile of exponentially decaying intensity at the interface, termed the Evanescent Wave (EW) (Fig. 1). The critical angle is given by eq. (1) (Fig.1). For the glass/water interface the angle is 62 degrees. EW intensity is maximal at the surface and decays exponentially with distance - eq. (3) (Fig.1) The depth of penetration depends on the wavelength, the angle of incidence, and the ratio of refractive indices eq. (2). For a typical TIRF experiment with a glass/water interface, the depth of penetration is ~100 nm or less.
TIRF Theory and Practice. In theory, the intensity of EW exponentially decays with distance, as shown in Fig. 1. In practice, however, scatter, reflections, and refractions produce undesirable rays of light, collectively termed “stray light.” Stray light contaminates the exponential decay of EW, excites the bulk of specimen, and deteriorates the TIRF effect, as shown in Fig. 2. This is especially serious problem in the case of objective-type TIRF.
Sources of Stray Light. All optical materials, to a certain extent, scatter light, and all surfaces and interfaces between optical parts reflect, refract, and scatter light. Due to the combination of these factors, the undesirable stray light is present in all practical systems. The intensity of stray light and its interference with the TIRF effect critically depend on the optical scheme used to implement TIRF. It also depends on the quality of the optical elements used in the system, including the quality of the materials themselves, the quality of manufacturing, especially surface quality, and the accumulation of dust particles and other external contaminations.
TIRF Geometries. As mentioned above, there are three popular geometries that are used for TIRF microscopy: through-objective, prism-, and lightguide-based optical schemes (o-TIRF, p-TIRF, and lg-TIRF, respectively). For a number of reasons, rather of monetary nature than the technical performance, Nikon, Olympus, Zeiss, and Leica have aggressively marketed only o-TIRF geometry. Objective-TIRF employs expensive high NA objectives and other sophisticated optics. A typical o-TIRF microscopy system costs ~$80,000 or more. On the other hand, TIRF geometries that demonstrate “clean” TIRF effect and superior signal-to-background ratios, namely p-TIRF and lg-TIRF, that are cost-efficient and flexible, were not offered commercially, until recently. In the past, each research group built p-TIRF and lg-TIRF systems on their own, until 2010, when TIRF Labs started to market p-TIRF and lg-TIRF systems. Since 2010, our customers generated unique TIRF data and demonstrated superior advantages of p- and lg-TIRF geometries on a number of applications, including single molecule detection [1, 2]. The sections below describe potential sources of stray light in each of the geometries and outline ways to mitigate the stray light interferences.
Objective-type TIRF Geometry. Fig. 3 shows the schematics of o-TIRF system. The main peculiar feature of o-TIRF, which might appear elegant at first glance, is the use of the emission path for delivering the excitation light to the glass/water interface. This feature has been inherited from epi-fluorescence. The o-TIRF scheme uses large angles of incidence, greater than the critical angle for glass/water interface, > 63 degrees. Microscope objectives with a Numerical Aperture (NA) smaller than 1.38 do not support such angles. Therefore, the o-TIRF scheme depends on specialized high-NA objectives to deliver the excitation light at angles greater than critical. Fig. 3 illustrates numerous potential sources of the stray light in the o-TIRF scheme.
Sources of stray light in o-TIRF. Not surprisingly, significant interferences of stray light have been reported for o-TIRF geometry [3-8]. Fluorophores in the bulk of the specimen are excited by the stray light and the TIRF effect is compromised. In many instances, the intensity of stray light is large and changes unpredictably. Two major sources of stray light were identified as (i) originating from the TIRF objective, and (ii) originating from the rest of the microscope optics [4, 5]. Only minor contributions were detected due to the scatter at the TIRF glass/water interface and at refractive-index boundaries within the specimen, including live cells [4, 5].
The first group of sources (i) is related to undesirable scatter and reflections inside the TIRF objective. The intense excitation light travels through multiple lenses and interfaces of the objective on its way to and from the TIRF surface (Fig. 3). The quality of the glass itself, which should minimally scatter light, and the surface quality of optical parts manufactured from the glass, are critically important for minimizing the intensity of stray light. To our knowledge, the systematic comparison of TIRF objectives from the standpoint of the intensity of stray light has not been performed yet. Our own tests on small number of TIRF objectives, the analysis of the literature, and reports of our customers and colleagues indicate that Nikon TIRF objectives demonstrate the smallest intensity of stray light. Perhaps, the better quality of Nikon lenses is related to the fact that Nikon makes its own high-quality optical glass, while other firms acquire glass from external sources.
The second group of stray light sources (ii) originates from the optics inside the microscope. A significant amount of stray light is generated at the dichroic mirror. Even a high-quality dichroic mirror scatters and transmits certain portion of light, which, in a perfect world, would be ideally reflected and blocked. The leaking of the excitation light through the dichroic mirror, as well as through the emission filter, results in increased background at the photodetector. In o-TIRF, the intensity of stray light changes unpredictably with the angle of incidence and XYZ coordinates. It increases with the amount of imperfections located on the path of the excitation light. Certain types of imperfections are distributed randomly, while other types exhibit more systematic patterns of their occurrence. If the angle of incidence increases, the depth of penetration and the intensity of EW excitation decreases, while the intensity of stray light remains the same or increases. If you are performing variable angle TIRF experiments using o-TIRF geometry, the effect of stray light should be carefully taken into account. In certain cases, the intensity of stray light is comparable with that of EW. In such cases, the depth of penetration calculated using eq. (3) (Fig.1) does not describe the intensity profile anymore and can mislead the interpretation of biological TIRF images [3-8].
Dichroic mirror and emission filter. The dichroic mirror is the central element of o-TIRF geometry; its quality is critically important for the objective-type TIRF. The beam of excitation light reflected from the dichroic beamsplitter must be focused at the back focal plane of the objective. A significant focal shift or a change in the focal spot size caused by a bend of the dichroic mirror can make it difficult to achieve TIRF, especially if the microscope has a limited ability to adjust the collimation of the excitation beam. Credit should go to Chroma and Semrock engineers, who recently made great progress in improving technical performance of dichroic and bandpass filters. The companies increased the thickness of dichroic mirrors to keep their flatness, which is necessary for precision focusing in o-TIRF, minimized surface roughness, as well as the density of pinholes [9, 10].
o-TIRF stray light mitigation. First, it appears to be rational to explore the opportunity of using alternative TIRF geometries for your study. If your study dictates the use of o-TIRF scheme, select a TIRF objective with minimal amount of stray light. Use the best quality dichroic mirror and the emission filter. Pay close attention to the design of the filter cube; it should provide flatness and no-bend to dichroic and filters. If your excitation light is not monochromatic, use an additional excitation filter to block undesirable lines in the excitation light. Finally, make sure that the dichroic mirror, emission filter and other accessible optical parts are free from dust particles and other contaminations.
Prism-based TIRF. p-TIRF geometry has been shown to provide the best signal-to-background ratio [1-3]. Indeed, p-TIRF is a scheme, where excitation and emission channels are naturally independent, as is illustrated in Fig. 4. The excitation light travels through the prism, reflects at the glass/water interface, and escapes through the opposite facet of the prism. Prism-TIRF has become the most popular geometry for single molecule studies and other applications that require TIRF effect of the highest quality.
Stray light in p-TIRF. Potential sources of the stray light are a few reflections at the prism facets, prism/slide interface, and scatter in the prism material. These few sources of stray light are located away from the EW region and the detection channel. In p-TIRF, stray light minimally interferes with the evanescent wave generated at the glass/water interface. Only a small portion of stray light enters the emission channel. Crisp, high-contrast TIRF images have been reported for p-TIRF [1-3, 8].
There are different configurations of prism-TIRF geometry, including optical schemes that are suited for inverted and upright microscopes, with open perfusion chamber and closed flow cell, fixed or variable angle of incidence, for TIRFing 1-mm slides or 150-micron thick coverslips, for using plain slides, coverslips, or Petri dishes with optical bottom. Visit web page p-TIRF Microscopy and contact TIRF Labs for more information.
p-TIRF stray light mitigation. If your application permits to enclose your specimen into a closed flow cell, as shown in Fig. 4, p-TIRF geometry is the best choice for your studies. Typically, in the case of the p-TIRF scheme, the intensity of stray light is negligibly small, provided that high-quality optical materials were selected to minimize the scatter and autofluorescence inside the prism and the TIRF slide. Additionally, for demanding TIRF experiments, the surface quality of the TIRF prism and TIRF slide should be high enough to ensure minimal scatter. At the emission channel, the selection of an efficient emission filter, which blocks the stray light, will reduce the background.
A limitation of p-TIRF is related to the fact that experiments with live cells in open dishes is difficult to perform with p-TIRF on an inverted microscope. An upright microscope with a prism-down geometry and water-immersion objective is a geometry well-suited for live cell studies.Visit www.tirf-labs.com for additional information.
CCD camera. The TIRF signal is inherently low. Therefore, a low light CCD camera is a necessary module for TIRFing. Electron-multiplying CCD cameras, for example an iXon EMCCD by Andor Technology, is a rational choice. Scientific-grade low light CCD and sCMOS cameras, and modest performance cameras available from Thorlabs and other vendors are sufficiently sensitive for many TIRF applications, including single molecule detection .
UV-absorbing-blue-light-emitting fluorophores. p-TIRF and lg-TIRF systems can be used with UV excitation, a feature not available in o-TIRF. There is a new class of fluorescent probes with large Stokes shift that are excited by UV light and emit fluorescence in the range 360-450 nm. These fluorophores, including modified nucleic and amino acids possess great potential for life science and material science applications. p-TIRF and lg-TIRF systems are well suited for TIRFing such fluorophores.
Selecting microscope objective. Unlike in rigid o-TIRF scheme based on high-NA specialized objectives, p-TIRF and lg-TIRF systems can be used with high and low NA objectives, dry, water- and oil-immersion lenses. This freedom causes good problem of choice. For TIRFing with prism- and lightguide-based geometries, you have freedom to chose an objective, which is suitable for your application, not just for generation of the evanescent wave. Many TIRF applications, e.g. real-time microarrays deal with relatively large amount of fluorescence; low-NA objectives with low magnification will be good for such studies. Large numerical aperture objectives collect more light (equation (4)). If your application, such as single molecule detection, involves detection of low light, it is advantageous to use a high numerical aperture objective. However, you do not have to pay $10k-$15k for a specialized high-NA objective anymore. If budget constraint is a concern, it will be rational to buy an NA 1.0-1.2 objective at $1k-$2k, and invest the remaining budget into low light camera. Rapid progress of CCD and especially scientific CMOS cameras resulted in a situation that cost-efficient CMOS cameras exhibit quantum yield only two-three times less than that of expensive EMCCD cameras. The intensity of excitation light is one more parameter to take into account for low light TIRF experiments. The intensity of the evanescent wave should be large enough (~1kWatt/cm2 for single molecule detection) to provide emission detectable by the camera. When you are choosing the objective, take into account that the efficiency of collecting light by a microscope objective, G, is proportional to the square of numerical aperture of the objective, NA, and reverse proportional to the magnification, X:
G = k S (NA)2 (X)-1 (4)
where k- is a coefficient, constant for the given microscope, S- coefficient of the CCD camera, which is typically proportional to the area of single pixel and the quantum yield of the camera. The table below enumerates the relative efficiency of objectives with different numerical apertures and magnifications. For example, a 60X/1.20 objective, which is available as a cost-efficient water-immersion lens, provides 65% efficiency, relatively to the costly 60X/1.49 lens. The collection efficiency of inexpensive 60X/1.20 lens exceeds that of the pricey 100x/1.49 objective.
The proximity of the fluorophore to the surface of glass results in anisotropy of the fluorescence emitted in TIRF mode [14, 15]. The emission pattern of a fluorophore located closer than 50 nm to glass-water interface is shifted toward the coverslip, which is an additional rational for using high NA objectives. However, for most single molecule detection experiments a 60X/1.20 objective turns to be sufficient.
In summary, the problem of stay light, which contaminates the evanescent wave and causes poor signal-to-background ratio, is inherent only to objective-type TIRF geometry. Compromised quality of TIRF effect caused by undesirable excitation of the bulk of the specimen have been documented in the literature only for the case of o-TIRF geometry. [1-6]. These interferences are caused by the fact that the excitation and emission channels share the same optics. On the other hand, p-TIRF and lg-TIRF geometries demonstrate excellent quality of TIRF effect, yield minimum amount of stray light, and produce crisp and high-contrast TIRF images. Both p-TIRF and lg-TIRF geometries employ optical schemes in that the excitation light is separated and naturally independent from the emission channel. Published articles show that p-TIRF and lg-TIRF geometries are well-suited for single molecule detection, super resolution methods, living cell membrane studies, and other areas of lifesciences. For supersensitive TIRF experiments it is rational to use p-TIRF and lg-TIRF with microscope objectives of high or moderate NA numbers (>1.0 and more) to ensure that the objective collects sufficient portion of the emitted fluorescence. For additional information please visit www.tirf-labs.com or contact TIRF Labs: email@example.com.
Lightguide-based TIRF geometry. Lg-TIRF is a sensible alternative to o-TIRF. It is a flexible geometry available with open perfusion chambers on inverted microscopes, and closed flow cells . Lg-TIRF can be used with dry, water- and oil-immersion objectives, with UV and visible excitation. Novel method of Shallow Angle Fluorescence Microscopy, in which objects a few microns away from the surface are illuminated, can be implemented at the same platform . Lg-TIRF principles are close to that of p-TIRF: the excitation light is naturally independent from the emission channel. In lg-TIRF, the excitation light undergoes multiple reflections from the top and the bottom surface of glass or silica coverslip which serves as the TIRF lightguide. The excitation light escapes from the opposite end of the coverslip. Unlike in o-TIRF, it does not enter the emission channel. The optical scheme of lg-TIRF allows for using Petri dish chambers for experiments with live cells. Potential source of scatter and autofluorescence is the coverslip material. Additionally, the top, bottom, and end surfaces of the coverslip play role in lg-TIRF. For demanding TIRF experiments these surfaces should be of high quality.
Lg-TIRF stray light mitigation. To minimize the intensity of stray light and to maximize the signal-to-background ratio in lg-TIRF, it is recommended to use coverslips made from high-quality glass or silica with low autofluorescence. The use of coverslips polished to the highest optical surface quality reduces scatter from the surfaces. However, regular coverslips work well for many lg-TIRF applications. Higher quality coverslips as well as superior emission filters are necessary only for demanding TIRF applications.
Overall, it appears that it is not a good idea to use the emission path for delivering TIRF excitation in o-TIRF. Indeed, p-TIRF and lg-TIRF geometries provide superior signal-to-background ratios, because the excitation light is naturally separated from the emission channel. lg-TIRF performs all the functions that o-TIRF does. Additionally, it can be used with UV excitation. If your applications involve TIRFing of living cells in open chambers, lg-TIRF is the optimal system for this purpose. If you are TIRFing single molecules with bright fluorophores, an inexpensive objective 1.20 NA will be sufficient. Use of expensive >1.4 NA lenses will increase the amount of light collected by the objective, and the lg-TIRF geometry give you the freedom to select an objective, which is better fitted for your specific task. Visit www.tirf-labs.com for additional information.
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Table. Relative light-collecting efficiency of microscope objectives with different values of the numerical aperture (NA) and magnification (X).
Magnification / Numerical Aperture
Relative efficency of collecting light