Total Internal Reflection Fluorescence
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Objective-type TIRF Microscopy
Among three popular geometries for TIRF microscopy: prism-, lightguide-, and objective-based, only the latter geometry is contaminated with large intensity of undesirable stray light. The stray light excites the bulk of the specimen far beyond the thin layer of the evanescent wave (EW). Since the intensity of EW decays exponentially with the distance, the relative error exponentially increases. While typical level of stray light in objective-TIRF is 10-15% relatively to the intensity of EW at the interface [Mattheyses A, Axelrod D. Direct measurement of the evanescent field profile produced by objective-based TIRF. J Biomed Opt, 2006, 11: 014006A.], at the distance ~100 nm from the interface the error amounts ~40%. Such large error cannot be neglected. Not surprisingly, several authors have reported about severe deviations from theoretical predictions in the case of objective-TIRF geometry [cilck here for references].
High numerical aperture of microscope objectives is indeed advantageous for certain applications; they collect larger portion of the emitted fluorescence than low NA lenses. However, if you wish to TIRF and should use a high NA objective from the standpoint other than TIRF, your better option is of using the high-NA objective with prism- or lightguide-based TIRF geometries. It is not a good idea to use the low light emission channel for the delivering of intense excitation light to the TIRF interface. Moreover, the totally internally reflected excitation light travels back into the low light emission channel, which multiplies the problems. Let us consider in details why o-TIRF geometry fails to provide clean TIRF effect. See also TIRF Microscopy web page.
In theory, the intensity of the evanescent wave (EW) exponentially decays with distance, as shown in Fig. 1, TIRF Microscopy web page. In practice, however, autofluorescence, scatter, undesirable reflections, and refractions produce in case of o-TIRF undesirable rays of light, collectively termed as “stray light.” In the case of o-TIRF, stray light contaminates the exponential decay of the evanescent wave, excites the bulk of specimen, and deteriorates the TIRF effect, as shown in Fig. 2 TIRF Microscopy web page.
Sources of Stray Light. All optical materials, to a certain extent, auto-fluoresce and scatter light. 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; in certain cases the amount of stray light is too large to be neglected. 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.
For a number of reasons, Nikon, Olympus, Zeiss, and Leica have aggressively marketed only o-TIRF geometry, which depends on expensive high NA objectives. A typical o-TIRF microscopy system costs ~$80,000 or more. Ironically, in the case of TIRF high price does not imply high quality. Large deviations from theoretically predicted exponential decay of the evanescent wave have been well-documented in the literature. See, for example, Literature Cited at TIRF Microscopy web page. One should take into account the fact that researchers are reluctant to publish negative results about failed attempts. Such results cannot win grant funding or attract other sources of funding. Therefore, if a negative result is published, it suggests an iceberg of problems underwater. In many instances, the intensity of stray light is comparable with the intensity of the evanescent wave; oTIRF users frequently deal with an evanescent wave contaminated with over 50% of stray light.
On the other hand, TIRF geometries that demonstrate “clean” TIRF effect and superior signal-to-background ratios, namely p-TIRF and lg-TIRF, 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 [see Literature Cited at TIRF Microscopy and Applications web pages].
In objective-based TIRF, the excitation lightpath and the emission channel share the same optics, which results in large intensity of stray light. Unwanted stray light (typically 10-20%) excites the bulk of the specimen far beyond the penetration depth and deteriorates the TIRF effect.
Objective-type TIRF Geometry. Fig. 3 shows the schematics of o-TIRF system. The main 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 came from epi-fluorescence scheme. 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 [see Literature Cited at TIRF Microscopy web page Refs. 3-8]. The stray light excites fluorophores in the bulk of the specimen, therefore, 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 [see Literature Cited at TIRF Microscopy web page Refs. 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 [see Literature Cited at TIRF Microscopy web page Refs. 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 autofluorescent and 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 TIRF objectives with minimal autofluorescence demonstrate the smallest intensity of stray light. Quick assessment of autofluorescence can be performed with a laser pointer 405 nm or 450 nm. Examination of front, back and inside lenses with the laser pointer allows to visualize the sources of stray light.
The second group of stray light sources 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 [see Literature Cited at TIRF Microscopy web page Refs. 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. Chroma and Semrock recently improved 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 [see Literature Cited at TIRF Microscopy web page Refs. 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, pay close attention to the design of the filter cube, and the performance of emission filter. 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 external contaminations.
In summary, the problems of stay light, poor signal-to-background ratio, and compromised quality of the evanescent wave are 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 [see Literature Cited at TIRF Microscopy web page Refs. 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 naturally independent from the emission channel. Published articles show that p-TIRF and lg-TIRF geometries are well-suited for single molecule detection, 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 contact TIRF Labs: firstname.lastname@example.org
TIRF Labs offers objective-TIRF system embedded into uTIRF Cube, which also implements prism- and lightguide-based scheme. The latter can be taken as TIRF accessories to be used with a microscope. TIRF objective and the objective-TIRF illuminator module also can be taken from uTIRF to be used with a fluorescence microscope. See brochures and contact TIRF Labs for more information.