TIRF Microscopies
prism-, lightguide-, and objective-based TIRFM geometries
In pTIRF and lgTIRF, the excitation lightpath is independent from the emission channel, enters the prism or lightguide from the outside, reflects at the TIRF interface, and exits at the opposite end. The excitation light does not interfere with the emission channel, which results in a superior signal-to-background ratio and clean TIRF effect.
In oTIRF, the excitation light shares the same optics with the emission channel, which results in large intensity of stray light and deteriorated TIRF effect.
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 the phenomenon of Evanescent Wave (EW) with maximum of intensity at the interface and exponential decay with the distance (panel at the left). Since the depth of EW penetration ~10 nm is less than the wavelength, the EW phenomenon allows for circumventing the diffraction limit. The opportunity to circumvent has opened a number of exciting areas based on the capability of visualizing and resolving single molecules with sizes much less than the wavelength. Due to this fortunate paradox, TIRF and EW became popular tools for a number of super-resolution microscopies, single molecule biology methods, and other single molecule studies that earlier seemed impossible.
TIRF Theory and Practice. In theory, the intensity of the EW exponentially decays with distance, as shown in the left panel. In practice, however, autofluorescence in lenses and imperfections of other optical elements, scatter, reflections, and refractions produce undesirable rays of light, collectively termed “stray light.” Stray light contaminates the exponential decay of the EW, excites the bulk of specimen, and deteriorates the TIRF effect, as shown in the right panel. This is an especially serious problem in the case of objective-TIRF. Fig. 3 below shows why stray light cases more problems in the case of objective-TIRF. All optical materials, to a certain extent, auto-fluoresce and scatter light. All interfaces between optical materials 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, its intensity is too large to be neglected. In the case of prism- and ligthguide-TIRF the number of sources is minimal in comparison with objective-TIRF. One can easily observe and visually estimate the amount of stray light in o-TIRF geometry by shining a 405 nm, <5 mW laser beam at the back of a microscopy objective, similar to that shown in Fig.3 of White Paper "Selecting TIRF Geometry"
objective-TIRFM
Objective-type TIRF Geometry shown in Fig. 3, at the first glance, is an elegant solution, which uses principles similar to epifluorescence microscopy. The excitation light is delivered to the glass/water interface via the emission channel. In the case of oTIRF it uses angles of incidence greater than 63 degrees - the critical angle for glass / water interface. Microscope objectives with NA smaller than 1.38 do not support such angles. Therefore, the o-TIRF scheme relies on specialized high-NA objectives, which imposes a number of rigid requirements and is associated with larger background.
Sources of stray light in o-TIRF. Fig. 3 illustrates numerous potential sources of stray light in o-TIRF, which generates the background signal. Significant interferences of stray light have been reported for the o-TIRF geometry - up to 10-15% of the evanescent wave at the surface. The ratio background / EW increases exponentially with the distance. 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 changes unpredictably. Two major sources of stray light were identified as originating from: (i) the TIRF objective, and (ii) the rest of the microscope optics [40 - 47]. 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 [40 - 47]. The first group of sources is related to undesirable auto-fluorescence, scatter, and reflections inside the objective. The intense excitation light travels through multiple lenses and interfaces on its way to and from the TIRF surface (Fig. 3). The quality of the optical glass (which should be minimally fluorescent and scattering) and the surface quality are critically important to the minimization of the intensity of stray light. The systematic comparison of TIRF objectives from the standpoint of the intensity of stray light has not been performed yet. Our own tests on a small number of TIRF objectives, analysis of the literature, and reports from our customers indicate that all TIRF objectives demonstrate a significant intensity of stray light due to autofluorescence and scatter. The front lens, which is in direct contact with the specimen, appears to be the most contributing factor. The second group of stray light originates from the optics inside the microscope, including the dichroic mirror. Even a high-quality dichroic mirror scatters and transmits a certain portion of light, which, in a perfect world, would be ideally reflected and blocked. The leaking of excitation light through the dichroic mirror, as well as through the emission filter, results in an increased background signal when it arrives 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 the EW excitation decreases, while the average 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. 10-15% of stray light at the surface is typical for a high quality optical system [42-47]. In certain cases, the intensity of stray light is even larger – comparable to that of the EW. In such cases, the depth of penetration calculated using eq. (3) does not describe the intensity profile anymore and can mislead the interpretation of biological TIRF images [43 - 48]. In fact, since the EW decays exponentially, the error caused by stray light increases exponentially with the distance. The dichroic mirror is the central element of o-TIRF geometry; its quality is critically important for the objective-type TIRF. The 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 in 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 made significant progress in improving technical performance of dichroic and bandpass filters. The companies have increased the thickness of dichroic mirrors to keep their flatness, which is necessary for precision focusing in o-TIRF, minimized surface roughness, and the density of pinholes [48, 49]. Even still, the improved filters are not perfect.
o-TIRF stray light mitigation. First, it appears to be rational to explore the opportunity of using alternative TIRF geometry. If your study dictates the use of the o-TIRF scheme, select a TIRF objective with the smallest amount of stray light. Use a 405 nm laser pointer for a quick assessment and visualization of autofluorescence and scatter in TIRF objectives and coverslips. Always use safety goggles and longpass filters to inspect the objective and coverslips. Select the best quality objectives, coverslips, dichroic mirror and the emission filter. Use an additional excitation filter to block undesirable lines in the excitation light. Make sure that the dichroic mirror, emission filter, and other accessible optical parts are free from dust particles and contaminations. Contact TIRF Labs for more information: info@tirf-labs.com.
prism-TIRFM
Prism-based TIRF Geometry has been shown to provide the best signal-to-background ratio and the cleanest TIRF effect [42-44]. p-TIRF is a geometry where the excitation and emission channels are naturally independent, as it is illustrated in Fig. 4. This separation of lightpaths provides several evident advantages to TIRF. Since the excitation light travels through the prism, reflects at the glass/water interface, and escapes through the opposite facet – the excitation stays away from the emission channel. Indeed, potential sources of stray light are a few reflections at the prism facets, prism/slide interface, autofluorescence and scatter in the prism and slide/coverslip. These few sources are located away from the EW region, thus, minimally interfering with the detection channel. Crisp, high-contrast TIRF images have been reported for p-TIRF [40 - 44]. If your application permits to enclose your specimen into a closed flow cell, prism-TIRF geometry is the best choice. For demanding experiments, the surface of the TIRF prism and TIRF slide should be of high quality to prevent scatter. There are many different configurations of prism-TIRF for inverted and upright microscopes. Contact TIRF labs for details. For example, for live cell studies an upright microscope with a prism-down geometry and water-immersion objective is a geometry well-suited for use with open perfusion chamber. It is difficult to arrange open dishes for working with live cells an inverted microscope. However, several versions of p-TIRF geometry with open perfusion chambers on inverted microscopes is also available from TIRF Labs. These versions use objectives with working distance 2 mm or more. Contact TIRF Labs for more information info@tirf-labs.com.
Disadvantages of prism-TIRF. Among limitations of the prism-TIRF geometry is the necessity to align the excitation light beam (and, therefore, the EW area) with the microscopy objective. Optional arm which holds the excitation beam collimator provides XY-axes alingment. s
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. Download the prism-TIRF.pdf brochure and contact TIRF Labs for more information. Our pTIRF systems are designed as an add-on accessories for inverted and upright microscopes. The most popular puTIRF system is supplied on a platform of nested design, which can be used with manual or motorized XY translation stages, or inserted into the round 4-inch windows of microscopes, Gibraltar platforms, or rectangular frames with the footprint of 96-well SBS plate. Optional arm provides travel of the excitation spot and EW together with objective. pTIRF systems are compatible with dry, water- and oil-immersion objectives. For excitation light, the prism and the slide represent continuous optical medium. In the case of puTIRF system a thin layer of aqueous solution and an optical window separate the TIRF surface from the objective.
Embedded Microfluidic Channels and fluidics cartridge create planar low-volume TIRF flow cell encompassing the TIRF surface and provides high share rates at small volumetric flow rates, which allows one to measure 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. Alternatively to the embedded fluidics, one can use reusable fluidics cartridges that create 20-40 microliter flow cell around the TIRF surface. Virtually any shape of slide or cover slip with sizes larger than 20 mm can be used with the cartridge, including 1-inch x 3-inches slides (25 mm x 75 mm), half-slides ~25 mm x 38 mm, or round slides or cover slips with diameter larger than 20 mm. An external pump, or gravity flow (the latter is always by hand) can be used with TIRF fluidics system for kinetic experiments.
Precision Optical-Mechanical Design of pTIRF is an option, which provides high reproducibility of TIRF measurements within one experiment and between different TIRF sessions. Synthetic 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 at: info@tirf-labs.com. pTIRF add-on accessory is the state-of-the-art, but robust system, which combines optical, mechanical, and fluidics modules. Typically, a researcher is capable of TIRFing with pTIRF after reading the Quick Start Guide. On-site training by TIRF Labs’ personnel is available as an option. TIRF Labs offers pTIRF systems equipped with closed flow cells or open perfusion chambers, designed for upright or inverted microscopes. Most of our pTIRF accessories are factory aligned systems: the angles of incidence are fixed to provide reproducible intensity of the evanescent wave. We also offer variable-angle pTIRF systems. However, even with fixed-angle pTIRF, one can decrease the depth of penetration using special optical traps that extinguish low angles of incidence, which results in a decreased penetration depth. For more information contact TIRF Labs info@tirf-labs.com.
lightguide-TIRFM
Lightguide-based TIRF is a novel powerful tool for single molecule biology, super resolution microscopies, plasma membrane studies, real-time TIRF microarrays, combination of TIRF with AFM, electric field, electrochemical and dielectrophoretic control, multi-photon, single ion channel single molecule detection, electro-chemi-luminescence, and other applications that require the excitation of fluorescence confined in space.
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lgTIRF features the excitation path, which is naturally independent from the emission channel
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lgTIRF yields a superior signal-to-background ratio
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lgTIRF can be used with dry-, water-, and oil-immersion objectives
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lgTIRF is a factory-aligned system that is well-suited for multicolor TIRF experiments
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lgTIRF uses glass or silica coverslips, or Petri dishes with optical bottom
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lgTIRF uses fiber-coupled illuminators connected by ~2-m optical fiber cable
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lgTIRF provides a reproducible intensity of the evanescent wave
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lgTIRF can be used with UV excitation, which is not available in objective-TIRF
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lgTIRF allows for precision control of the penetration depth by using optical traps
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It takes no time to install/uninstall lgTIRF, and to switch between TIRF, SAFM, micro-spot excitation, epi-fluorescence, transmittance, and other methods.
Our most popular lgTIRF product - a platform for fluorescence microscopes 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. lgTIRF platform also implements Shallow Angle Fluorescence Microscopy (SAFM), and Micro-Spot Excitation (MSE). The latter method, is well-suited for the probing of organelles, single ion channels, and other objects with sizes from 1 to 100 microns. Download the lightguide-TIRF.pdf brochure to learn more. There are three versions of TIRF excitation light launchers that differ by the geometry of coupling light: (i) from the end of the coverslip or slide; (ii) from the top, and (iii) from the bottom of the coverslip/slide. Bottom-type light launchers are used for TIRFing Petri dishes, and other formats of specimen substrate. There are two “flavors” of the from-the-end light launchers: 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. The surface selectivity and multiplex format of lgTIRF is well-suited for the analysis of biomolecular interactions, measuring k-on, k-off, and Ka affinity constants. 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.” [59]. lgTIRF provides a reproducible intensity of the evanescent wave in one experiment and between experiments. 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 launcher for the bottom-entrance, well-suited for TIRFing with Petri dishes. Additional light launchers are available upon request. 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 for real-time TIRF microarray applications that require measuring of the response of the entire microarray printed at ~20×20 mm area. 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. 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.
Embedded Microfluidics. We offer lgTIRF with embedded microfluidics or microfluidic cartridges that create planar low-volume TIRF flow cells. An advanced microfluidic system embedded into lgTIRF creates closed flow cell encompassing the TIRF surface and provides high share rates at small volumetric flow rates, which allows one to measure 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.
lgTIRF platform also implements
Shallow Angle Fluorescence Microscopy (SAFM)
– the mode in which the excitation light propagates at shallow angles along the surface and illuminates fluorophores that are 1-5 microns away from the surface. For more information contact us: info@tirf-labs.com.