Total Internal Reflection Fluorescence (TIRF) Microscopy

Abstract

Total internal reflection fluorescence (TIRF) microscopy (TIRFM) is an elegant optical technique that provides for the excitation of fluorophores in an extremely thin axial region (‘optical section’). The method is based on the principle that when excitation light is totally internally reflected in a transparent solid (e.g., coverglass) at its interface with liquid an electromagnetic field, called the evanescent wave, is generated in the liquid at the solid-liquid interface and is the same frequency as the excitation light. Since the intensity of the evanescent wave exponentially decays with distance from the surface of the solid, only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. This unit will briefly review the history, optical theory, and the different hardware configurations used in TIRFM. In addition, it will provide experimental details and methodological considerations for studying receptors at the plasma membrane in neurons.

INTRODUCTION

TIRFM is a powerful technique for selectively imaging fluorescent molecules (e.g., GFP, membrane dyes, fluorochromes attached to antibodies, …) in an aqueous environment that are very near a solid substance with a high refractive index (e.g. coverglass). Depending on the excitation wavelength and objective numerical aperture, the thickness of the excitation depth, which is called the evanescent field, can be less than 100 nm from the solid surface. In comparison, the thickness of a confocal image section is approximately 500 nm. The advantage of such a small illumination volume is three fold: (1) the background is greater than 2,000-fold lower than when imaging by normal epifluorescence microscopy (Funatsu et al., 1995), which results in a high signal-to-background ratio; (2) there is virtually no out-of-focus fluorescence collected; (3) cells are exposed to a significantly smaller amount of light (Fig 1). The following are just a few of the many applications of TIRFM in cellular microscopy.

Figure 1.

Information revealed by TIRF microscopy. (A-B) HeLa cells were cultured on 18 mm round 1.78 RI coverslips. Post fixation with 4% paraformaldehyde, rhodamine-conjugated phalloidin was used to visualize F-actin. The cell was imaged in HBSS using a 100X 1.65 NA objective and 1.78 RI immersion liquid. Both images were taken at the same plane with the same exposure settings.

Receptors at the PM

• TIRFM can be used to measure the kinetics of receptor endocytosis in response to ligand binding, receptor channel opening and closing, receptor clustering, and the lateral movement of receptors (Drenan et al., 2008; Fan and Jin, 2007; Fowler et al., 2007; Hellen and Axelrod, 1991; Riven et al., 2006; Yudowski et al., 2006). For these types of studies TIRFM is sometimes combined with other advanced microscopy techniques (e.g., fluorescence recovery after photobleaching). Receptors at the PM can be visualized using fluorescently tagged ligands, antibodies, or small molecules. Alternatively, the receptor of interest can be fused to a fluorescent protein (e.g., GFP).

Exocytosis

• Numerous investigators have taken advantage of the utility of TIRFM for studying exocytosis events. By using TIRFM and styryl dyes such as FM1-43 (Zenisek et al., 2002), the loading of vesicles undergoing exocytosis with fluorescent dyes (Pangrsic et al., 2007; Pryazhnikov and Khiroug, 2008), or cargo fused to a fluorescent protein (Toomre et al., 2000) one can observe single exocytic events (Beaumont, 2003; Betz et al., 1996; Pollard and Apps, 2002). When visualizing events of exocytosis, fluorescently loaded vesicles appear bright as they enter the evanescent field. As the vesicles fuse with the PM the signal rapidly diffuses as the fluorescently labeled contents spills into the extracellular space or enters the PM (Steyer and Almers, 2001).

Proteins of the Endocytic Pathway

• TIRFM has been extensively used to qualitatively and quantitatively describe the roles different proteins play in exocytosis/endocytosis. For example, TIRFM was used to show that Rab3A and Rab27A cooperatively regulate docking steps of dense-core vesicles to the PM (Tsuboi and Fukuda, 2006) and to visualize the dissociation of Rab3A from vesicle during exocytosis (Lin et al., 2007). The function of clathrin and adaptor proteins have also been extensively studied using TIRFM (Keyel et al., 2004; Merrifield et al., 2002; Saffarian and Kirchhausen, 2008).

Cell-Substrate Contacts

• TIRFM can be used to observe the size, movement, and distance apart of the regions of contact between a cell and the solid substrate (e.g., coverglass) it is bound to (Axelrod, 1981). For these types of experiments, fluorescently labeled molecules (e.g., dextran) can be microinjected into the cell. Alternatively, GFP fusion proteins that localize to focal adhesions can be exogenously expressed. 5) Long term fluorescence live cell imaging–Because of the very thin optical section in TIRFM, which is localized to the cell-substrate contact region, cells tend to survive substantially longer than when imaged by standard epifluorescence techniques. In addition to low cell cytotoxicity, since in TIRF imaging photobleaching is occurring in such a small region, in most experiments there is a continuous flux of new fluorophores into the imaging field. Thus, optimal signal-to-noise ratios can be achieved with the same minimum exposure settings.

THE THEORY BEHIND THE TECHNIQUE

The basic concept of TIRFM in cellular microscopy is simple: an excitation light beam needs to travel at a high incident angle through a glass coverslip upon which cells are cultured. The refractive index (RI; see Table 1 for the refractive indexes of some common reagents) differences between the glass (n₂) and intact cell interior (n₁) regulate how light is refracted or reflected at the interface as a function of incident angle. At a specific critical angle (θ_critical) the beam of light is totally reflected from the glass/water interface, rather than passing through and refracting (Equation 1; Fig 2A). The reflection generates a very thin electromagnetic field (the evanescent wave) on the cell side of the coverslip that has an identical frequency to that of the incident light. The depth of the evanescent wave is a function of the incident illumination angle, wavelength, and refractive index differences (Equation 2) and its intensity exponentially decays with distance from the coverglass (Equation 3; Fig 2B). Usually, only fluorophores within 100-200 nm of the coverslip are efficiently excited.

$${\mathrm{\theta }}_{\mathrm{critical}}={\sin }^{­1}({\mathrm{n}}_1/{\mathrm{n}}_2)$$ Equation 1

$$\begin{array}{l}\mathrm{d}={\mathrm{\lambda }}_0/4\mathrm{\pi }{({\mathrm{n}}_2^2{\sin }^2\mathrm{\theta }-{\mathrm{n}}_1^2)}^{­1/2};\\ [\mathrm{alternatively},\mathrm{d}={\mathrm{\lambda }}_0/(4\mathrm{\pi }{\mathrm{n}}_2)\times {(({\sin }^2\mathrm{\theta }/{\sin }^2{\mathrm{\theta }}_{\mathrm{critical}})-1)}^{­1/2}]\end{array}$$ Equation 2

(note that d is a decreasing function of the angle of incidence and for θ - θ_critical ~0/1° d is of the order of the excitation wavelength λ₀)

$$\begin{array}{l}{\mathrm{I}}_{\mathrm{z}}={\mathrm{I}}_0{\mathrm{e}}^{­\mathrm{z}/\mathrm{d}}\\ ({\mathrm{I}}_{\mathrm{z}}=\mathrm{intensity\; at\; depth\; z};{\mathrm{I}}_0=\mathrm{initial\; intensity};\mathrm{note\; that\; at\; z}=0,{\mathrm{I}}_{\mathrm{z}}={\mathrm{I}}_0)\end{array}$$ Equation 3

Table 1.

Refractive Indices of Commonly Encountered Materials

Material	Refractive Index
Water	1.33
Air	1.0003
Glass	1.517
Coverglass	1.523
immersion oil	1.516
cell cytosol	1.38
Mount	**variable

^**Mounting media for fluorescence microscopy can vary substantial. To my knowledge none of the standard commercially available mounts are suitable for fixed sample TIRFM

Figure 2.

The evanescent wave. (A) The incident angle can be described using a coordinate system arranged to display all three orthogonal directions. The x-y plane represents the interface between the coverglass and the cell cytosol. The plane of incidence is the x-z plane, which is parallel to the excitation light beam. (B) The evanescent field intensity decays exponentially with increasing distance from the interface according to Equation 3. The penetration depth, which is usually between 50 and 300 nm, decreases as the reflection angle grows larger and is dependent on the refractive indices at the interface and the illumination wavelength. Illustrations were adapted from ones provided by Michael W. Davidson, Florida State University.

There are two common methods for producing the evanescent wave for TIRFM. One configuration uses a prism to direct laser light toward the interface between the coverglass and media/cells at an incident angle sufficient to cause total internal reflection. Although the majority of prism based TIRF microscope designs are centered on inverted microscope bases, upright microscopes can also be used (for review, see (Axelrod, 2001)). The prism based configuration has been applied to cellular microscopy for nearly 30 years (Axelrod, 1981). The use of TIRF in cellular microscopy increased once an objective-based TIRF (or prismless TIRF; Fig 3A) system was introduced to the scientific community (Stout and Axelrod, 1989) and increased even more once a commercial solution became available (Kawano et al., 2000). In objective-based TIRFM, one can easily switch between standard epifluorescence and TIRF by changing the off-axis position of the beam focus at the objective’s back focal plane. Several ways have been developed to change the position of the beam. For example, an actuator can be used to change the position of a fiber carry the laser light in relation to a fluorescence illuminator that attaches to the microscope (Fig 3A). The following lists the basic characteristics of these two different methods for performing TIRFM.

Figure 3.

TIRFM System calibration. (A schematic drawing of a TIRF microscope that uses a micrometer to position a fiber that delivers laser light to the microscope. (B) Schematic drawing of how to use a hemicylindrical glass prism to determine the angle of incidence. (C) Schematic drawing of how to empirically determine the penetration depth of the evanescent wave using 1 μm diameter fluorescent microspheres. Note that in the example 370 equals the measured radius of the microsphere and that a is calculated using the Pythagorean theorem and 500-a = the empirically determined penetration depth. (D) Demonstrates how the objectives numerical aperture and the illumination wavelength affect the penetration depth of the evanescent field. Illustration A was provided by Michael W. Davidson, Florida State University.

Characteristics of Prism Method

• Prism used to attain critical angle

• Purer evanescent wave

• Easier to set up than prism-less system

• Laser focused to spot size about equal to field of view

• Access to sample can be restricted depending on prism position

• No commercially available system

Characteristics of Prism-less Method

• Beam must pass through periphery of high NA objective

• Higher NA will allow confinement closer to surface

• Laser focused off-axis at back focal plane of objective

• Decreased field of illumination

• Specimen is fully accessible

• Not as pure an evanescent wave as prism

• Increased light scattering within objective (decreases signal to noise ratio)

• Turn-key systems commercially available

Although a prism based system is easier to build than an objective based one, since the latter is what the average user will find in most core facilities the rest of this unit will focus on performing TIRFM using an objective based system.

TIRF OBJECTIVES

There are a variety of objectives designed specifically for TIRF imaging available today. TIRF objectives vary substantially in both magnification and NA. Listed are the specifications of a few: 60X 1.45 NA, 60X 1.49, 100X 1.45 NA, 100X 1.65 NA, 150X 1.45 NA. Some objectives also come with compensation collars used to control for varying temperature and coverslip thickness. The objective’s specifications will determine the experimental design, and thus the critical angle. For example, below is a comparison between the critical and maximum angles (Equation 4) of different objectives.

Critical angle

60X 1.45 NA objective:

$${\mathrm{\theta }}_{\mathrm{critical}}={\sin }^{­1}({\mathrm{n}}_1/{\mathrm{n}}_2)=65.22°$$

[calculated using n₂ = 1.52 (RI of coverglass and immersion liquid) and n₁ = 1.38 (cell cytosol)]

100X 1.65 NA objective:

$${\mathrm{\theta }}_{\mathrm{critical}}={\sin }^{­1}({\mathrm{n}}_1/{\mathrm{n}}_2)=50.83°$$

[calculated using n₂ = 1.78 (RI coverglass and immersion liquid) and n₁ = 1.38 (cell cytosol)]

Maximum angle of incident light that can be generated by the objective

$$\begin{array}{l}\mathrm{NA}={\mathrm{n}}_2\sin {\mathrm{\theta }}_{\mathrm{maximum}}\\ 60\mathrm{X}1.45\mathrm{NA}{\mathrm{\theta }}_{\mathrm{maximum}}=72.54°\\ 100\mathrm{X}1.65\mathrm{NA}{\mathrm{\theta }}_{\mathrm{maximum}}=67.97°\end{array}$$ Equation 4

A comparison of the two objectives using the above equations reveals that when aligning a system that uses a 60X 1.45 NA objective to produce the evanescent wave there are only 7.32° for which total internal reflection can occur. In contrast, using the 100X 1.65 NA objective one has 17.14° to work with. Thus, as objective NA decreases the ability to align a system for TIRF imaging becomes more difficult. To my knowledge, the smallest NA objective used for objective based TIRFM has been a 60X 1.4 NA objective, which only has ~1.9° of freedom.

As mentioned above, the depth of the evanescent wave is a function of the incident illumination angle, wavelength, and refractive index differences. Considering Equation 2, as the angle of incident increases penetration depth decreases. Choosing an angle of incidence for which internal reflection will occur for both the 60X 1.45 and 100X 1.65 objectives there performance in regards to penetration depth can be compared (Fig 3D). For example, using an angle of incidence of 66.5°, a wavelength of incident light (λ₀) of 488 nm, and n₂ = 1.38 (cytosol), the penetration depth of the evanescent wave for the 60X 1.45 NA objective would be ~197.6 nm, while that using the 100X 1.65 NA objective would be ~44.5 nm. The > 4X difference between the penetration depths between these two objectives under the same conditions would result in drastic differences in the excitation of fluorophores (see below). Thus, it is crucial that when designing TIRFM experiments one fully considers all hardware and experimental parameters.

EMPIRICALLY DETERMINING INCIDENT ANGLE/PENETRATION DEPTH

When using TIRFM for performing qualitative measures just knowing that the system is in TIRF imaging mode is sometimes good enough. For these types of experiments, one can use 0.1 μm TetraSpeck fluorescent microspheres (Invitrogen, Carlsbad, CA) to confirm the microscope is in TIRF imaging mode. First, setup the microscope with a live cell chamber (see below). Then add 100 μl of water containing 5 μl of the TetraSpeck solution. When visualizing some of the beads will be sitting directly on the coverglass and not moving, while others will appear and disappear rather rapidly. In TIRF mode there will be a very high signal to background ratio and all you will see are events at the coverglass. In epifluorescence mode there will be a substantial amount of blur from out of focus beads and beads at and in relatively close proximity to the coverglass will always be visible.

When performing quantitative fluorescence TIRFM it is important to know that your system is performing as expected. Although the software that accompanies several commercially available turnkey systems reports on incident angle and penetration depth, don’t take for granted the values you see are correct. Particularly since the penetration depth is most likely being calculated using Equation 2. To determine the angle of incidence you will need a hemicylindrical glass prism (BK-7, which has a RI of 1.52; note this prism will not work for the 100X 1.65 NA objective). Add immersion oil to the top of the objective then place a highly polished coverslip on the stage and carefully focus on the upper surface of the coverslip. The prism needs to be centered (flat side down) directly over the objective, making contact with the coverslip through a layer of immersion oil (Fig 3B). Since the RI of the coverslip and prism are the same, the light will not be reflected and will pass through the prism. From this point forward, laser safety goggles should be worn. First adjust the beam so that laser light is shinning directly above on the room ceiling (note: you will probably need to reposition some of the microscope hardware in order to see the beam on the ceiling). Mark the position on the micrometer and then start very slowly walking the beam across the ceiling and eventually onto the wall. At a specific position the beam will disappear because you have exceeded θ_maximum and the internal aperture of the objective is blocking the laser light from exiting the objective. Mark the position on the micrometer. Move the beam back (decreasing the angle of incidence) until you clearly see the laser light on the wall again. To confirm that total internal reflection is occurring move the objective to an area of the coverslip that is not under the prism. Here, all most no light will emerge if in TIRF mode. There are two different ways to determine the angle of incidence. One method is to mark where the light leaves the prism and measure the radians starting from the bottom edge of the prism. The radians can then be directly converted into degrees. Alternatively, others have projected the light leaving the prism on a screen that rests perpendicularly on a surface level with the top of the objective (Zenisek et al., 2002). By measuring the distance from the center of the prism to the screen and the distance from the bottom of the screen to where the light from the prism hits one can calculate the incident light angle (Fig 3B). You can build a jig so that the screen is always positioned in the same place in relation to the objective/prism. By doing this you can place marks on the screen for specific incidence angles and then when setting up the system position the beam to the mark you want for the given experiment. In order to calibrate a system, the incident angle at different positions of the fiber, or mirror in some systems, will need to be measured.

As stated above, software packages determine the penetration depth of the evanescent wave using Equation 2. However, in that equation d is only the depth at which the intensity of the evanescent wave is 37% of the initial intensity. Thus, for all quantitative studies the experimental depth needs to be determined. There are several different ways to empirically determine the evanescent wave penetration depth (Keyel et al., 2004; Saffarian and Kirchhausen, 2008; Sarkar et al., 2004). I usually direct new users towards the method used by Keyel, Watkins, and Traub for measuring penetration depth (Keyel et al., 2004). Briefly, image 1.0 μm TetraSpeck fluorescence microspheres by TIRFM as described above for the 0.1 μm diameter microspheres. Image several beads that are clearly resting on the coverslip. Since the diameter of the 1.0 μm microspheres is substantially greater than the evanescent wave penetration depth, only a portion of the bead (< ½) will be imaged if the system is actually in TIRF mode. The next step is to determine the average measured diameter of the bead, followed by the use of the Pythagorean theorem to determine the experimental penetration depth. For example, using a 60X 1.45 NA objective with an incident light angle of 68.2° the calculated penetration depth for an excitation wavelength of 488 nm in aqueous medium (RI = 1.33) is 82.3 nm. Using the method above to empirically determine the penetration depth, 1 μm fluorescence microspheres had an average diameter of 740 nm (Fig 3C). Thus, the measured evanescent wave penetration depth was 164 nm, which is twice the calculated depth. It is important to note that both the calculated and empirically determined depth of penetration would be different if the fluorescence microspheres were within a cell. In order to get beads into cells one can use particle bombardment or microinjection. Depending on wavelength and objective, under these conditions I have found anywhere from 1.4 to 2.12 times difference between calculated (Fig 3D) and empirically determined penetration depths. Others have used cellular structures to help extrapolate measurements using beads in aqueous solution to experimental conditions (Keyel et al., 2004). Also important to mention here is that the physical limits inherent in the optical system and the diffraction limit of light make it impossible to empirically determine the exact penetration depth of the evanescent wave. With that said, it is clear by the differences in calculated and empirically determined penetration depths that it is imperative to perform some form of experimental measure for the accurate analysis and interpretation of data.

TIRF IMAGING OF PLASMA MEMBRANE RECEPTORS IN NEURONS

Neurons are highly polarized cells that have three distinctive compartments: somatic, axonal, and denritic. In order to define the unique features of each compartment it is usually necessary to use microscopy to perform an equivalent of biochemical analysis. The following describes two examples of using TIRFM to study the endocytic pathway in primary hippocampal neurons.

Materials

• Microscope: Olympus IX81 inverted microscope equipped with a TIRFM illuminator [multispectral (488, 568, and 635) single fiber], 60X 1.45 NA objective, Hamamatsu C4742-98 CCD camera, and high precision Prior motorized XY stage, and is controlled by SlideBook 4.2 software (Intelligent Imaging Innovations, Inc; Denver, CO).

• Humidified environmental chamber: For maintaining cell viability during imaging, the glass coverslips on which neurons were grown (see below) were placed in an imaging chamber (model RC-41LP) that was inserted into a micro-incubator chamber (Model DH-40i). A perfusion valve controller (model VC-6) was used to exchange media throughout the experiment and temperature control was performed using a dual channel heater controller (Model TC-344B; stage and objective) and an in-line solution heater (SH-27B) all from Warner Instruments.

• Expression vector for vesicular stomatis viral G protein fused to green fluorescent protein (VSVG-GFP; Dr. M. Aridor, University of Pittsburgh, PA)

• High affinity NPY-Y1 receptor agonist Leu 31, Pro 34 NPY1-36 covalently labeled with Alexa 488 (NPY-488; Tamas Bartfai; The Scripps Research Institute, La Jolla, CA)

• Sprague Dawley E18 time pregnant rat (Harlan)

• Imaging solution (HEPES-buffered Tyrode’s solution): 119 mM NaCl, 5 mM KCl, 25 mM HEPES buffer, 2 mM CaCl₂, 2 mM MgCl₂, 6 g/liter glucose (pH to 7.4 with NaOH)

Culturing Primary Rat Hippocampal Neurons

For both studies, neurons were cultured at low density from embryonic day (E) 18 rats as previously described (Aridor et al., 2004; Fish and Krucker, 2008; MacLaurin et al., 2007). A detailed protocol of the method has been previously published (Goslin and Banker, 1991). Briefly, hippocampal neurons were plated onto poly-L-lysine-coated glass coverslips (#1.5 18 mm round) that are inverted over a monolayer of glial cells after 2 hr incubation. Cells were plated at a density of 2700 cells per cm² to achieve low-density cultures. Paraffin dots attached to the coverslips were used to keep the neurons separated from the cells making up the glial feeder. Neuron cultures were fed every few days by replacing ½ the media with fresh media. Most neurons, >90%, developed the characteristic mature morphology of spiny neurons between 16-21 days in vitro (DIV).

Microscope System Alignment and Setup

In both examples listed below the same basic microscope setup routine and alignment were performed before TIRF imaging began. Thus, the step-by-step set of instructions listed here are for all three examples.

1. Prior to starting the experiment determine the angle of incidence and penetration depths of the illumination wavelengths that will be used following the instructions under the section entitled Empirically determining incident angle/penetration depth.

2. Pre heat all hardware components (objective, imaging chamber, micro-incubator chamber) to 38.5 °C ~1 hr prior to starting the experiment. Since in the system used here the imaging chamber, which will hold the coverslip, and micro-incubator are two separate components, I insert the micro-incubator into the stage and heat the objective/micro-incubator using a dual channel heater controller and place the imaging chamber in the incubator that is housing the neurons.

3. For long term experiments or those that require the media to be exchanged setup a perfusion device. For Example 2 below a gravity fed perfusion system equipped with a valve controller was used to exchange media. An in-line solution heater was used to heat the solution to 37° C and was placed just before the imaging chamber.

4. Approximately 10 min before moving the neurons to the microscope insert a coverslip on which the neurons are grown into the imaging chamber, cover with imaging solution, and return to the incubator.

5. Add immersion oil to the top of the objective, place the imaging chamber in the micro-incubator, and carefully focus on the upper surface of the coverslip. It is essential that the coverslip bottom be thoroughly cleaned before the imaging chamber is transferred to the microscope.

Example 1: Visualizing VSVG-GFP Transport to the PM

In this example TIRFM was used to visualize the fusion of VSVG-GFP exocytic vesicles with the PM (Fig 4). For the expression of exogenous vesicular stomatis viral G protein fused to green fluorescent protein (VSVG-GFP) plasmid DNA was transported into neurons by electroporation prior to plating using a Nucleofector II device (Amaxa Inc. Gaithersburg, MD) and the manufacturer’s rat neuron kit (VPG-1003). VSVG-GFP trafficking was imaged in cultures at 7 days in vitro. The images were captured at 11.32 frames per second with 2×2 binning. Using a 60X 1.45 NA objective, illumination wavelength of 488 nm, and a 70° angle of incidence, the empirically determined evanescent field penetration depth was 191 nm. When visualizing an exocytosis event (the arrival of a VSVG-GFP vesicle at the PM), the vesicle first becomes brighter as it enters the evanescent field. As the vesicle fuses with the PM the signal rapidly diffuses as VSVG-GFP enters the PM (Fig 4).

Figure 4.

Visualizing the arrival of VSVG-GFP to the PM. VSVG-GFP plasmid DNA was transported into neurons by electroporation prior to plating. At 7 days in vitro the trafficking of VSVG-GFP vesicles to the PM was imaged by TIRFM using a 60X 1.45 NA objective at 11.32 frames per second. The panels labeled 1-10 are sequential frames. As a VSVG-GFP vesicle nears the PM it becomes brighter. Upon fusing with the PM the signal rapidly diffuses. Bar = 2 μm.

Example 2: Imaging NPY-Y1 Receptor at the PM

In this example a fluorescently labeled high affinity Y1r agonist Leu31, Pro 34 NPY_1-36 (NPY-488) was used to detect the arrival of new receptors at the PM (Fig 5). An advantage to using NPY-488 is that once the labeled agonist binds Y1r the complex is stable and when endocytosed is degraded (Bernet et al., 1994). Therefore, the appearance of new NPY-488/Y1r complexes at the PM represents the arrival of new receptors. The empirically determined penetration depth of the evanescent field was 192 nm. Since the thickness of the optical section is very thin and located at the interface between the coverslip and neuron, imaging could be performed in the presence of NPY-488 in the culture media without free NPY-488 interfering with bound NPY-488. In this experiment NPY-488 was added to the culture media 10 min after step 5 above. Although we have previously found that the Y1 receptors at the PM in cultured hippocampal neurons are functional as high affinity binding sites (1.65 picomoles/mg protein), we added a large excess of NPY-488 to the media (10^-6 M) to make sure all NPY receptors at the PM would be bound to NPY-488. Again, having a substantial amount of free fluorescently labeled ligand in the media does not interfere with our imaging. Imaging was performed at 2 frames/min without camera binning. Shortly after adding NPY-488 to the media the presence of NPY-Y1 receptors on the PM of some hippocampal neurons was revealed by NPY-488 receptor binding (fluorescent puncta in Fig 5A; note the high signal to background ratio). Using timelapse imaging the removal of NPY-Y1rs from the PM and the arrival of new receptors can be easily followed (Fig 5B).

Figure 5.

Visualization of single Y1r arrival at the PM by TIRFM. (A) Taken shortly after NPY-488 was added to the culture media. The signal to background ratio is not affected by the presence of free fluorescently labeled agonist in the media. (B) Captured 10 minutes after the image in A. Note the loss of 488 fluorescence along the dendritic branches and in some cases (open arrows) the detection of new receptors at the PM. Bar = 2 μm.

MULTI-WAVELENGTH TIRFM

There is an increasing demand for multi-wavelength TIRFM. Depending on hardware, multi-wavelength TIRFM can be performed in three general ways: (1) different channels are collected sequentially either using the same or different incident angles for each wavelength; (2) different channels are collected simultaneously using the same angle of incidence; (3) multi-angle illumination combined with simultaneous capture. In configuration 1 an actuator is needed to change the angle of incidence during imaging. In methods 2 and 3, the systems need to be able to pass multiple excitation wavelengths simultaneously. In configuration 2, this can be accomplished by using either a single multiline laser, also known as a mixed-gas laser (e.g., Argon/Krypton ion laser), or a laser combiner that houses several single line lasers that can exit the combiner simultaneously. In configuration 3, each wavelength remains independent from the others until it passes a unique set of hardware used to adjust the angle of incidence. In configurations 2 and 3 there also needs to be a mechanism for the simultaneous capture of the different emitted wavelengths. This can be accomplished using matched cameras connected to the microscope using a multi camera adaptor. Alternatively, simultaneous multi-wavelength imaging can be performed on a single camera using devices like the DV2 and Quad-view from Mag Biosystems (Tucson, AZ). These devices connect to the camera port and use a beamsplitter(s) to split the emission light from the microscope into independent channels that are simultaneously projected onto different quadrants of the same CCD.

There is some debate over if it is necessary to use wavelength specific angles of incidence when performing multi-wavelength TIRFM. The answer can be reached by performing a simple experiment, but first it is important to understand how excitation wavelength affects evanescent wave penetration depth, which can easily be visualized by performing a few calculations using Equation 2 [d = λ₀ / 4π (n₂² sin²θ − n₁²)^-1/2]. The below calculations are based on a 60X 1.45 NA objective for which θ_critical = 65.22° and θ_maximum = 72.54° (using n₂ = 1.52 and n₁ = 1.38).

$$\begin{array}{llll}\mathrm{If}& d=162.13& \mathrm{If}& d=120.66\\ & \mathrm{and}{\mathrm{\lambda }}_0=488\mathrm{then}\mathrm{\theta }=67.14°& & \mathrm{and}{\mathrm{\lambda }}_0=488\mathrm{then}\mathrm{\theta }=68.79°\\ & \mathrm{and}{\mathrm{\lambda }}_0=568\mathrm{then}\mathrm{\theta }=67.86°& & \mathrm{and}{\mathrm{\lambda }}_0=568\mathrm{then}\mathrm{\theta }=70.18°\\ & \mathrm{and}{\mathrm{\lambda }}_0=647\mathrm{then}\mathrm{\theta }=68.69°& & \mathrm{and}{\mathrm{\lambda }}_0=647\mathrm{then}\mathrm{\theta }=71.86°\\ \mathrm{If}& \mathrm{\theta }=68.79°& & \\ & \mathrm{and}{\mathrm{\lambda }}_0=488\mathrm{then}d=120.66°& & \\ & \mathrm{and}{\mathrm{\lambda }}_0=568\mathrm{then}d=140.44°& & \\ & \mathrm{and}{\mathrm{\lambda }}_0=647\mathrm{then}d=159.97°& & \end{array}$$

There are a few important things to notice about the above calculations. First, as the wavelength increases the penetration depth increases. Second, the shorter the wavelength the less the incident angle needs to change for the same change in penetration depth. Next, a simple experiment was performed to demonstrate how differences in penetration depth affect experimental outcome.

Materials

• 0.1 μm fluorescent microspheres: make a 1:1 solution of 488 ex and 625 ex microscopheres in water.

• Microscope coverglass bottom chamber (e.g., Warner Instrument DH-35i culture dish chamber)

• TIRFM system: [e.g., Olympus IX81 equipped with TIRF illuminator, a laser combiner (488, 568, and 635), and 60X 1.45 NA objective]

Experimental steps

1. Setup microscope to empirically measure the angle of incidence using the 488 nm laser line. In our experiment the determined angle of incidence = 68.1°. Thus, the calculated evanescent wave penetration depth for 488 nm = 82.78 nm, and for 635 nm = 107.72 nm (using n₁ = 1.33 and n₂ = 1.52).

2. Following the procedures above, empirically determine the evanescent wave penetration depth. I found an experimental penetration depth for 488 nm = 161.2 nm and for 635 = 201.8 nm.

3. Dry down10 μl of the 488 nm / 625 nm fluorescence microsphere mixture onto the coverglass and empirically determine the 488 nm / 625 nm microsphere ratio of the mixture (I had a ratio of 1 / 0.96).

4. Change the coverglass and add 200 μl of the microsphere mixture and quantify the number of microspheres in each channel. Per imaging field, I had an average ratio of 1 (488 nm) / 1.22 (625 nm).

5. Since in this experiment we can’t change increase the incident angle enough so that the 635 nm line has a calculated penetration depth of 82.78 (it would need to be 74.31°, but the maximum angle for the experimental conditions is 72.54°) we will change the incident angle such that the evanescent wave produced by the 488 nm excitation line equals that of the one produced by the 635 nm line. Using Equation 2 [θ = sin^-1(((λ₀ / d4π)² + n₁²) / n₂²)^-1/2] estimate the new angle of incidence. I used a new angle of 65.04°, which is above the θ_critical (θ_critical = 61.05°).

   (note: for this step it is necessary that you have a calibrated system that allows you to accurately change the incident angle)

6. Perform a new estimate of the number of 488 nm microspheres / field. Using similar evanescent wave penetration depths, the new ratio was 1 (488 nm) / 1.03 (625 nm).

7. Optional: empirically determine the 488 nm evanescent wave penetration depth at the new angle of incidence. The new measured depth was 199.8 nm.

Author’s Recommendations

Clearly for this experiment using the same angle of incidence resulted in a misleading finding. The question is why. TIRF can be viewed as a probability curve: increase evanescent wave penetration = increase probability at any given spot of exciting a molecule. Thus, in experiments where the goal is to quantitate the number of different objects within the evanescent field using different fluorescent probes it is important that the penetration depths of the different illumination lines are matched. For experiments in which simultaneous imaging is required configuration 3 (multi-angle illumination combined with simultaneous capture) should be used. However, since the penetration depth for each line in a multi-angle illumination systems should be empirically determined at the onset of each experiment, which is time consuming, for experiments that do not require simultaneous imaging I recommend using a system in which different channels are collected sequentially using different incident angles for each wavelength. If the system is calibrated, one can use Equation 2 and an actuator to achieve very similar evanescent field penetration depths without using empirical measures to set the angle of incidence for each line. The use of an actuator provides high accuracy and the capability to switch rapidly (~ 50 ms) between wavelengths. Importantly, I have found that although the calculated and empirically determined penetration depths are always different, when using angles of incidence for different illumination wavelengths that have the same calculated penetration depth the empirically determined penetration depths are also the same. Thus, even without empirically determining the penetration depths one can be relatively assured that when using calculated angles of incidence the same penetration depth across illumination lines can be achieved.

It is important to point out that the above TIRFM imaging was performed in water rather than within the cell cytosol. Thus, θ_critical = 61.05°, rather than 65.22° (that for imaging in cytosol). Very small changes in n₁ have a big effect on differences in penetration depth when performing multi-wavelength TIRFM. For example, the calculated evanescent wave penetration depths for 488 nm and 635 nm are 82.78 nm and 107.72 nm, respectively, at an angle of incidence = 68.1°using n₁ = 1.33 and n₂ = 1.52. However, they are 133.53 and 173.76, respectively, if using n₁ = 1.38. Thus, in an aqueous environment there is a difference in evanescent penetration depth of 24.94 nm (calculated) between the two wavelengths, while in cytosol the difference jumps to 40 nm. These and other considerations covered in this unit are important to keep in mind when designing and performing TIRFM experiments, and analyzing/reviewing TIRFM data.

FINAL EXPERIMENTAL SUGGESTIONS

1. The coverglass (total internal reflection surface) should be highly polished.

2. Depending on the design of the system, photobleaching within the evanescent field may be greater than under epifluorescence conditions. Several precautions should be taken to reduce photobleaching. For example, minimize light needed, maximize photon detection and capture, and carefully selects dyes. In addition, reagents like Trolux (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma Aldrich cat# 238813), DABCO (1,4-diazabicyclo[2.2.2]octane), nPG (n-propyl gallate), ascorbic acid, OxyFluor (Oxyrase Inc.) all possesses antioxidant activity and can be added to the media to increases photostability. If studying protein and lipid mobility in the PM of red blood cell perform deoxygenation (Corbett et al., 1994).

3. Since objective based TIRFM uses high magnification/NA objectives there is a high propensity that focus drift, which can be caused by subtle temperature fluctuations and air circulation, will wreak havoc on time-lapse experiments performed at physiological temperatures and experiments in which the temperature needs to be changed. Thus, for these types of experiments I highly recommend using some form of microscope autofocus mechanism, which is available from nearly all the major microscope manufacturers and several third party suppliers.

CONCLUDING REMARKS

This Unit has touched on the history, optical theory, and hardware configurations of TIRFM. In addition, it provides useful tools for aligning a TIRF microscope and practical experimental protocols. Since the purpose of this unit was to provide useful information to both beginner and advanced TIRF microscopists, advanced methodologies were not covered. Thus, it is important to point out that advanced approaches for using TIRF microscopy to study biological problems are continuously being developed. For more than 20 years TIRF imaging has been combined with fluorescence photobleaching recovery (Burghardt and Axelrod, 1981) and fluorescence correlation spectroscopy (Thompson and Axelrod, 1983). In addition, atomic force microscopy and TIRFM have been combined to study protein unfolding (Sarkar et al., 2004). More recently, two new techniques (Differential evanescence nanometry and standing wave TIRF) have been used to perform superresolution fluorescence microscopy (Chung et al., 2006; Chung et al., 2007; Saffarian and Kirchhausen, 2008).