Around-the-objective total internal reflection fluorescence microscopy

Around-the-objective total internal reflectionfluorescence microscopy

Thomas P. Burghardt,1,2,* Andrew D. Hipp,1 and Katalin Ajtai1

1Department of Biochemistry and Molecular Biology, Mayo Clinic Rochester,Rochester, Minnesota 55905, USA

2Department of Physiology and Biomedical Engineering, Mayo Clinic Rochester,Rochester, Minnesota 55905, USA

*Corresponding author: [email protected]

Received 17 July 2009; revised 30 September 2009; accepted 2 October 2009;posted 6 October 2009 (Doc. ID 114410); published 2 November 2009

Total internal reflection fluorescence (TIRF)microscopy uses the evanescent field on the aqueous side of aglass/aqueous interface to selectively illuminate fluorophores within ∼100nm of the interface. Applica-tions of the method include epi-illumination TIRF, where the exciting light is refracted by the microscopeobjective to impinge on the interface at incidence angles beyond critical angle, and prism-based TIRF,where exciting light propagates to the interface externally to the microscope optics. The former has high-er background autofluorescence from the glass elements of the objective where the exciting beam isfocused, and the latter does not collect near-field emission from the fluorescent sample. Around-the-objective TIRF, developed here, creates the evanescent field by conditioning the exciting laser beamto propagate through the submillimeter gap created by the oil immersion high numerical aperture ob-jective and the glass coverslip. The approach eliminates background light due to the admission of thelaser excitation to the microscopic optics while collecting near-field emission from the dipoles excited bythe ∼50nm deep evanescent field. © 2009 Optical Society of America

OCIS codes: 260.6970, 180.2520, 180.4243, 170.2520, 170.0180, 110.0180.

1. Introduction

Fluorescence microscopy can detect low light levelsand measure spatial distribution and dynamics (ro-tational and translational) of small ensembles, orsingle biomolecules, in biological samples. The meth-od has uniquely contributed to cell biology studiesrequiring real-time monitoring of biomolecule dy-namics. Much of the progress is related to develop-ment of high-quantum-efficiency fluorescent probesthat can undergo many excitation/emission cycles be-fore irreversible photobleaching [1]. Optical micro-scopy hardware has likewise evolved to promotelight collection and transmission efficiency over awider optical spectrum. Analysis techniques for high-precision localization of isolated single fluorescent

molecules have advanced spatial resolution beyondthe diffraction limit [2–4]. New microscope config-urations have likewise improved spatial resolutionto below the diffraction limit by using evanescentfield excitation [5], surface plasmon coupled emission(SPCE) [6,7], supercritical angle fluorescence (SAF)[8], structured illumination (SI) [9], and the stimu-lated emission depletion (STED) of peripheral ex-cited molecules in a diffraction limited spot [10].Back focal plane (BFP) emission pattern imagingof isolated probes determined emitter dipole orien-tation by inspection [11] and lifted some of the de-generacy in orientation determination normallycharacteristic to dipole emitters [12].

Evanescent field excitation in total internal reflec-tion fluorescence (TIRF) microscopy takes placewhen an excitation laser beam impinges on a glass/aqueous interface from the higher refractive index(glass) side at an angle greater than critical angle,

0003-6935/09/326120-12$15.00/0© 2009 Optical Society of America

6120 APPLIED OPTICS / Vol. 48, No. 32 / 10 November 2009

θc, for total internal reflection (TIR). The beam to-tally reflects at the interface while producing an eva-nescent field in the lower refractive index (aqueous)side that decays exponentially with distance from theinterface. TIRF is used extensively in cell biology ap-plications, where it preferentially illuminates speci-men thin sections within ∼100nm of the interface[13,14]. Two common TIRF microscopy configura-tions introduce the exciting laser beam to the inter-face either through the microscope objective [15] orvia a prism external to the microscope in optical con-tact with the interface [16]. Through-the-objectiveTIRF (oTIRF) uses a coverglass and oil immersionobjective with numerical aperture (NA) significantlylarger than the water refractive index of 1.334. Alaser beam propagating parallel to the optical axisbut displaced laterally and focused at the BFP isrefracted by the objective and impinges on thecoverglass/aqueous interface at angles beyond θc.Propagating exciting light in the objective producesunavoidable background autofluorescence. Samplefluorescence emission necessarily occurs near theglass/aqueous interface, providing substantial SAFintensity collected in the objective. Through-the-prism TIRF (pTIRF) has the excitation laser beamtotally reflecting at a coverglass/aqueous interfacewith the coverglass optically coupled to an externalprism. Sample fluorescence is collected from the aqu-eous side or from the glass side with a water immer-sion objective to avoid frustrating the TIR. AlthoughSAF is emitted, it cannot be collected in the objective.A new TIRF setup, around-the-objective TIRF (ao-TIRF), combines through-the-prism excitation andSAF light collection. We demonstrate aoTIRF on up-right and inverted microscopes using artificial andbiological samples.In aoTIRF, the oil immersion objective (NA ¼ 1:45)

is focused on the coverslip/aqueous interface. A nar-row exciting laser beam at near skimming incidenceto the interface is focused at the objective focal pointby a low aperture lens. In the upright microscope, thefocused excitation beam propagates into the immer-sion oil, separating the interface from the tip of theobjective using a Littrow prism optically coupled tothe oil. The incidence angle is 87°–88° within theoil medium to assure that the focused beam clearsthe objective tip. After TIR at the intersection ofthe objective focal plane and optical axis, the excitingbeam exits the immersion oil using a second Littrowprism. In the inverted microscope, propagating theexciting beam into the immersion oil is more compli-cated because the beam must approach the objectivetip from below the microscope stage. We accom-plished this with an additional reflection of the beamat a glass/air interface as the beam approaches orretreats from the objective.We show that the upright and inverted microscope

versions of aoTIRF produce an evanescent field withpenetration depth of∼50nmby observing fluorescentnanosphere diffusion through the excitation field.BFP imaging of fluorescent nanosphere emission

detects the SAF emission from the object and de-monstrates the lower background characteristicsof aoTIRF excitation. Images from a green fluore-scent protein (GFP)-tagged myosin light chain ex-changed into cardiac papillary muscle fibers indicatethe favorable contrast of aoTIRF compared to epi-illumination in the wide-field imaging microscope.

2. Methods

A. Chemicals

Carboxylate-modified fluorescent microspheresare from Molecular Probes (Eugene, Oregon). ATP,Na-Azide, dithiothreitol (DTT), phenylmethyl-sulfonyl fluoride (PMSF), trans-1,2-Diaminocyclo-hexane-N,N,N,N-tetraacetic acid (DCTA), ethyleneglycol-bis (2-amino-ethylether)-N,N,N,N-tetra-aceticacid (EGTA), Triton X100, phosphocreatine, andcreatine phosphokinase are from Sigma (St. Louis,Missouri). Recombinant human cardiac troponin C(TnC) is from Life Diagnostic (West Chester, Pennsyl-vania). Leupeptin is from Roche Applied Sciences(Indianapolis, Indiana). All chemicals are reagentgrade or ultrapure, if available. The GFP-taggedmyosin regulatory light chain (RLC-GFP) was con-structed as described [17].

B. Solutions

The washing solution contains 4mM KCl, 1:8mMCaCl2, 1:0mM MgCl2, 1:8mM NaHPO4, 5:5mM glu-cose, 50mM imidazole buffer, and 140mM NaCl, pH7.4. Skinning solution A contains 1% Triton X100,50% glycerol, 10−8M Ca (pCa8), 1mM Mg, 7mMEGTA, 5mM MgATP, 15mM phosphocreatine,15U=mL creatine phosphokinase, 20mM imidazolepH 7.0, 1mM DTT, and ionic strength is adjustedto 150mMwith potassium propionate. Fiber Storagesolution is Skinning Awithout Triton X100. Relaxingsolution is Skinning A without Triton X100 and gly-cerol. Dissecting solution is Relaxing plus 15% gly-cerol. Extracting solution contains 5mM CDTA,50mM KCl, 0:2mM PMSF, 10 μg=mL leupeptin,0:5mM Na azide, 1mM DTT, 40mM tris pH 8.4,and 1% Triton X100. Skinning solution B is Relaxingplus 1% Triton X100. Reconstitution solution isRelaxing plus 40 μM RLC-GFP and 15 μM TnC. Con-tracting solution is Relaxing except that the Caconcentration is raised to 10−4 M (pCa4).

C. Sample Preparations

Clean #1 or #2 glass coverslips were sonicated for10 min in ethanol then plasma cleaned (HarrickPlasma, Ithaca, New York) for 15–30 min. All experi-ments were conducted at room temperature.

Red-orange carboxylate-modified 40nm diameterfluorescent spheres had excitation/emission maximaat 565=580nm. The yellow-green 100nm diameterfluorescent spheres had excitation/emission maximaat 505=515nm. Sphere concentrations were com-puted using the formula from the manufacturerand diluted in distilled water. We used 104–106 folddilutions from stock, giving a sphere concentration of

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1:4 × ð1011–109Þ spheres=mL for the 40nm diam-eter spheres or 3:6 × ð109–107Þ spheres=mL for the100nm spheres.Nanospheres with 106 fold dilution were flowed

into the sample chamber and allowed to dry. Afterdrying, some spheres are strongly attached to thesubstrate surface in a sparse random spatial distri-bution. The more concentrated sample (104 fold dilu-tion from stock) was then flowed into the samplechamber. All fluorescence nanosphere experimentswere conducted with sparsely surface attached nano-spheres in contact with aqueous solution containinga high concentration of nanospheres. These sampleswere investigated with epi-illumination or aoTIRFexcitation.Cardiac muscle fibers were from the porcine left

ventricular papillary muscle. The porcine heart leftventricle was opened and washed thoroughly withWashing solution, then bathed in Dissecting solu-tion. Papillary muscle was dissected into bundlesof ∼200 single fibers each, then placed into Storagesolution on ice for 1h. The fiber bundles were thentransferred to the Skinning A solution and kept at4 °C for 24h. The fibers were then transferred backto the Storage solution and kept at −20 °C until used.Fibers used for the RLC-GFP exchange were madefrom stored fiber bundles dissected down to smallerbundles containing ∼10 fibers. The small fiber bun-dle was mounted on a microscope slide and glued

down at the ends. A water resistant resin was ex-truded from a pipette tip to create a channel openat both ends containing the fibers. A coverslip wasplaced over the fiber. The fibers were washed withRelaxing (three times, 1 min each), then incubatedin Skinning B solution for 60 min (three solution ex-changes, 20 min each). The fibers were washed againwith Relaxing solution (three times, 1 min each). Thenative regulatory light chain was extracted by incu-bating the fibers in Extracting solution for 60 min(three solution exchanges, 20 min each). The fiberswere washed with Relaxing solution (three times,1 min each), then reconstituted with Reconstitutionsolution for 1h (four solution exchanges, 15 mineach). Finally, fibers were washed with Relaxing so-lution (three times, 1 min each) and transferred tothe aoTIRF setup on the inverted microscope (Fig. 1).

D. aoTIRF Microscopy

Upright and inverted microscope configurations foraoTIRF are indicated schematically in Figs. 1(a)and 1(b). The exciting beam (dashed line) entersand exits the vicinity of the objective at incidenceangles of 87°–88 ° in both configurations. The30°–60°–90° Littrow prisms (Edmund Scientific,Barrington, New Jersey) are needed for couplingthe laser beam into and out of the coverslip. Totalinternal reflection at the glass/aqueous interface oc-curs at the focal point of the objective. The exciting

Fig. 1. Schematic aoTIRF diagrams for (a) upright and (b) inverted microscope configurations. Dashed lines show the beam path throughthe prisms to the glass/aqueous interface where the exciting beam totally internally reflects. The inverted configuration has two other totalinternal reflections at glass/air interfaces. Coverslips and Littrow prisms are optically coupled by immersion oil. The upright microscopeconfiguration also shows the beam conditioning elements consisting of a beamwaist reducer from 10× and 20×microscope objectives and alow-aperture focusing lens. Beam conditioning is needed in both upright and inverted microscope configurations but is shown only for theupright.


beam in the inverted microscope configuration alsototally internally reflects at the glass/air interfaceformed by the #2 coverslips. Prism size is larger inthe inverted microscope configuration to accommo-date the triple reflection. Exciting beam conditioningby the beam reducer and focusing lens is performedfor both upright and inverted configurations.Figure 2 shows an expanded view of the upright

microscope configuration. The flat surface on the ob-jective containing the lens has radius R ≈ 3:8mm.The working distance W ≈ 0:1mm, coverglass thick-ness C ≈ 0:15mm, gives L ≈ 0:25mm and arc lengthA ≈ L. The maximum value for θ2 (the complementof the incidence angle at the glass/aqueous interface)avoiding the objective edge (where segment A meetsthe objective) is ∼3:8 deg, but we wanted to bring inthe exciting beam nearer the midpoint of A, whereθ2 ≈ 2:3 deg. In this case, refraction at the air/glassinterface of the Littrow prism requires the incidenceangle for the laser exciting beam, θ1, to be ∼3:5 deg.Figure 3 shows an expanded view of the inverted

microscope configuration. Quantities R, W, L, C, θ1,and θ2 are equal to their counterparts in Fig. 2. The

bevel on the objective is θ3. Where the excitationbeam enters the Littrow prism is critical to assureit will leave the prism to enter the #1 coverslip. Withincidence at K ≈ 1mm, transition into the #1 cover-slip occurs at distance P ≈ 27mm well within theprism dimension of 37:9mm. The #2 coverglasshas thickness B ≈ 0:21mm, and is positioned to pro-vide the glass/air interface for the first reflection ofthe exciting laser beam and to support the oil retai-ner. The first reflection position is at Sþ R ¼ ðBþCÞ= tan½θ2� from the optical axis, giving S ≈ 5:2mm.We made the oil retainer from a 2mm thick stripof double sticky tape where ε ≈ 1:5mm.We find, then,that H ¼ 3:5mm and H þ F ¼ 37:4mm.

The argon ion laser beam has a Gaussian intensityprofile and a beam diameter of ∼1:5mm. It is condi-tioned for insertion into the aoTIRF setup using abeam diameter reducer and focusing lens (Fig. 1).The beam must fit into the ∼250 μm diameter ap-proach-limiting aperture (Fig. 2 or Fig. 3) at distanceR from the objective focal point. The exciting beamfocus is at the objective focal point where TIR occurs.The beam diameter is reduced to 0:75mm by a

Fig. 2. Detailed schematic aoTIRF upright microscope configuration. Symbols defining distances or angles are used in the text. DistanceC is ∼0:15mm for a #1 coverslip. The approach-limiting edge requires θ2 ≤ 3 deg and is the strictest constraint on laser beam shape. Theupright configuration works with both small and large base Littrow prisms. Part of the objective is shown with the incident laser beampath. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.

Fig. 3. Detailed schematic aoTIRF invertedmicroscope configuration. Symbols defining distances or angles used in Fig. 2 are equivalentlydefinedhere.Numeric distances are given inmillimeters.DistanceB is∼0:21mmfor a #2 coverslip.Oil is confinedby the objective, the loweredge of the #1 coverslip, the short edges of the #2 coverslips, and the oil retainers. The inverted configuration requires the large base Littrowprisms. The exiting laser beam pathway is a mirror image of the incident path with reflection through the objective symmetry axis.


diameter reducer and then focused by a 12:7mm ×150mm (aperture diameter x focal length) lens.We computed the intensity profile near the excit-

ing beam focus for a Gaussian laser intensity profilewith β ¼ ðaperture diameterÞ=ðbeam diameterÞ ≈ 17,as described previously [18–20]. It is represented asthe rippled surface in Fig. 4(a) or Fig. 4(b), with in-tensity plotted on the yp axis or the xp axis in arbi-trary units, respectively. The xp and yp axes definethe focal plane of the exciting light with zp in the di-rection of light propagation and yp in the incidenceplane at the glass/aqueous interface. Exciting lightis linearly polarized along yp in the p-polarization.The intensity profiles are practically identical in xpand yp and depend only on radial distance fromthe zp axis. The planes in Figs. 4(a) and 4(b) shownslicing through the intensity profile at focus repre-sent the objective field of view (FOV). The FOV isin the objective focal plane or, alternatively, the inter-face plane. The exciting beam reflects and refracts atthe interface plane, but this is not depicted in the fig-ure and does not affect the intersection of the inten-sity profile with the FOV. The FOV is approximatelythe size of the field of view for the 60×, NA ¼ 1:45objective. The FOV plane subtends a short distanceon yp (�2 μm) because the beam strikes the FOVplane at a glancing angle. The beam intensity profilein the xp dimension has an ∼70 μm full width in thefocal plane and fits entirely within the FOV.The approach-limiting edge (Fig. 2 or Fig. 3) forms

a barrier of∼250 μm full width in the yp dimension ofthe propagating beam coordinates at zp ¼ 3:8mm.

The beam intensity zp-dependence changes gra-dually, with 76% of its maximum peak height atthe origin retained at zp ¼ 3:8mm, where the beamintensity profile in xp or yp has ∼80 μm full width. Atzp ¼ 10mm, the beam intensity profile in xp or yp has∼120 μm full width. Figure 5 shows the beam crosssections in yp for various positions along zp and indi-cates that the majority of the exciting light energywill transmit the approach-limiting edge.

Figure 6 shows close-up photographs of the upright[Fig. 6(a)] and inverted [Fig. 6(b)] configurations. Theexcitation laser pathway shows up in the Littrowprisms as a thin red line due to autofluorescence.

Fig. 4. Two views of the exciting laser beam intensity profile(rippled surface) and (FOV plane for the microscope objective nearthe focal plane for the exciting light. The xp and yp axes lie in thefocal plane of the exciting light with zp in the direction of light pro-pagation and yp in the incidence plane at the glass/aqueous inter-face. The vertical axis is intensity in arbitrary units for theintensity profile and micrometers for the (a) yp or (b) xp spatialdimensions.

Fig. 5. The exciting laser beam cross section for various zp values,where the zp axis is parallel to the direction of light propagation,and the 1=e2 intensity level. The beam full width at zp ¼ 3:8mmmust be <250 μm for the exciting light to propagate past the ap-proach-limiting edge without producing excessive light scattering.

Fig. 6. Close up photographs of the (a) upright and (b) invertedaoTIRF configurations. The slightly red pencil of light inside theprisms is the 488nm laser beam exciting autofluorescence.


Scattered lightdimly illuminates thebackground, butthe excitation beam pathway can be seen as it ap-proaches and exits the sample area at the objectivefocal point. The sample region is clearly visible inthe inverted configuration, where excitation lightscatters as the excitation beam propagates betweenthe objective and the glass/sample interface. The ex-citing light scattering is larger just at the edges of theobjective lens, where there is amaterial discontinuityfrom the brass of the objective body to the glass ofthe lens.Excited fluorescence collected by the objective is

spectrally filtered and formed into an image by themicroscope tube lens at the light sensitive area ofa 12 bit CCD camera (Hamamatsu Orca ER) onthe upright microscope (Zeiss Axioplan) or a 16 bitEMCCD camera (Hamamatsu C9100) on the in-verted microscope (Olympus IX71). The emitted lightpathway is altered for BFP imaging (see Subsec-tion 2.E). We used the Olympus 60× NA ¼ 1:45(TIRFM) objective for all measurements.

E. Back Focal Plane Microscopy

BFP imagingwasdone on the uprightmicroscope.Ex-citation 488nm light from an argon ion laser excitesthe sample under aoTIRF. The p-polarized incidentlight has electric field polarization in the incidenceplane and produces an elliptically polarized evanes-cent electric field. Evanescent intensity is ∼78 or22% polarized normal or parallel to the interface [21].Single 100nm diameter fluorescent spheres immo-

bilized on the TIR interface are in diffusive contactwith identical spheres, as described in Subsec-tion 2.C. Excited fluorescence was collected by the ob-jective and spectrally filtered using a fluoresceinfilter set. Figure 7 shows the emission pathway op-tical setup, where OP is the object plane, OBJ isthe objective, TL is the tube lens with focal lengthf TL, RL is a removable lens, and CCD is the imageplane for the camera. Light emitted by the sample(black arrow at OP) emerges from the back of the ob-jective in parallel rays. An object at the BFP (light

double arrow) is imaged onto the CCD camera withinsertion of RL. The BFP is telecentric, implying thatan object displaced laterally in the focal plane doesnot change the position of its intensity pattern. RLis positioned to form a real image of the BFP atthe CCD plane by placing a translucent ruler atthe BFP and adjusting the RL position until its realimage is formed at the CCD plane. RL is corrected forchromatic and spherical aberrations and is remova-ble to facilitate conversion between BFP and normalimaging.

F. Emission Near an Interface

When a dipolar is near a dielectric planar interface,the radiated fields are significantly altered [22]. Anemitting fluorophore has a dipolar emission fieldthat is the superposition of propagating transversewaves forming the far field and nonpropagating long-itudinal (evanescent) waves forming the near field.The near field is not detected unless perturbed bya nearby interface creating detectable propagatingtransverse waves. Converted near-field propagatingwaves appear in the glass medium at angles beyondθc and are referred to as SAF [8]. Oil immersion ob-jectives with NA ≳ 1:3 capture SAF. Subcritical anglefluorescence (UAF) is from far-field emission.

The interface also affects total radiated power. Hel-len and Axelrod pointed out that, for a fluorophoreunder steady illumination, the dissipated powermust equal the absorbed power, implying that thefixed power, rather than the fixed amplitude, dipoleradiator is the appropriate model for probe emissionnear an interface [22].

The high-NA collection of light in a microscope ob-jective mixes light contributions from emitter dipoleCartesian components due to the collection of lightfrom a large solid angle surrounding the dipole. TheCartesian dipole moments produce differently polar-ized light that is combined by the objective such thattheir individual contributions cannot be separated byusing an analyzing polarizer. Separating the contri-butions into the Cartesian components of the dipolemoment is the basis for fluorescence polarizationmeasurement of dipole orientation. Axelrod de-scribed the solution to the problem in a homogeneousmedium [23]. We addressed the probe emission per-turbation by the interface and the complications forpolarized light collection by a high-NA objective inthe dipolar emission intensity from single probes.Our computations gave the two-dimensional (2D) in-tensity pattern in the objective BFP for comparisonwith experiment and showed the effect of probe or-ientation on the pattern as described previously[12]. BFP intensity pattern computation and dis-play was performed with Mathematica 7 (WolframResearch, Champaign, Illinois).

G. Random Walk Simulation

Fluorescent spheres diffuse through the aoTIRFillumination volume, giving a time-dependent fluor-escence intensity depending on sphere diffusion

Fig. 7. The BFP microscopy emission pathway optical setupwhere OP is the object plane, OBJ is the objective, BFP is the backfocal plane, TL is the tube lens with focal length f TL, RL is thechromatic and spherical aberrations corrected removable lens,and CCD is the image plane for the camera.


constantD, detection volume (Vd) size and shape, andon constraints imposed on sphere position by the pre-sence of the planar glass/aqueous interface whereTIR occurs. We model sphere diffusion as a three-dimensional (3D) randomwalk subject to constraints,compute the time-dependent fluorescence intensity,and compare computed with observed signals todeduce the size and shape of Vd.The random walk simulation of diffusion with step

size δ, characteristic time between steps of τ, andwith constraints imposed by planar glass/aqueous in-terface was carried out as described previously [18].The Einstein–Smoluchowski relation gives the diffu-sion constant, D ¼ kT=ð6πSηÞ, where kT is thermalenergy, S is the sphere radius, and η is the fluid visc-osity. The diffusion constants for the 40 and 100nmdiameter spheres in water at room temperature,where η ¼ 0:01 g cm=s are 1:1 × 10−7 and 4:4×10−8 cm2=s, respectively. Wemeasured 2D sphere dis-placement in Brownian motion where the 3D move-ment was projected onto 2D using the CCD cameraon the upright microscope. Measurements were con-ducted on bulk diffusing spheres in the proximity(<1 μm) of the glass/aqueous interface. Resultsagreed with the Einstein–Smoluchowski relationfor the 100nm spheres, but gave a diffusion con-stant ∼½ that expected for the 40nm spheres. Themeasurements suggest the 40nm spheres were ag-gregates or their movement was inhibited by transi-ent adsorption to the glass interface.We chose a δ ¼ 2nm step size that is more than

20-fold smaller than any linear dimension in our sys-tem. Characteristic times, τ ¼ δ2=2D, are ≤1 μs and∼104 fold shorter than any time-dependent measure-ment we can make. The real sphere executes multi-ple random steps for each simulated step; however,the dimensions of the system and the time frameof our measurements prohibit any detectable effectfrom this computational short cut. Simulationsprovided time-dependent trajectories of the spherecenter coordinate ðx0ðtÞ; y0ðtÞ; z0ðtÞÞ.The evanescent field intensity, I, on the aqueous

side of the glass/water interface is given by

I ¼ I0 exp�zd

�where d ¼ λ

4πffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðng sin θÞ2 − n2

w

q ; ð1Þ

and for I0 the evanescent field intensity at z ¼ 0, ex-citing beam incidence angle θ, exciting light wave-length λ, and the refractive indices for glass andwater ng and nw. Values appropriate for our applica-tion are θ ¼ 87:7, λ ¼ 488nm, ng ¼ 1:516, andnw ¼ 1:334, giving d ≈ 54nm. Fluorescence intensity,F, emitted from a finite-size sphere is proportional toexcitation intensity [I in Eq. (1)] times the fluoro-phore spatial density integrated over the sphere vol-ume. Excitation intensity from the evanescent fieldvaries over the spatial dimensions of the sphere.When fluorophores are uniformly distributed onthe sphere surface

F ¼ F0 exp�−z0d

�; ð2Þ

where F0 is a geometric factor varying with spheresize and normalized arbitrarily to be 1 for a pointsource. Finite-size spheres have F0 ¼ 1:02 for S ¼20nm and 1.15 for S ¼ 50nm. Equation (2) definesthe z dimension of Vd as penetration depth d. Otherdimensions of Vd are defined by the resolution limitor the CCD camera pixel size, whichever is larger. Weperformed quantitative sphere diffusion measure-ments with the upright microscope, where the reso-lution limit for the objective matches 2 × 2 binnedpixels for the Orca ER corresponding to ∼220nm.Vd is a thin slab with height given by d and base di-mensions identically 220nm. The simulations showthat the characteristic time for sphere diffusionthrough Vd is highly sensitive to d.

We observed fluorescent nanospheres within Vdusing aoTIRF. We recorded and characterized thebrightest transient fluorescent events for a givensphere size and light collection interval as they dif-fuse through Vd. The brightest events characterizethe sphere position optimal for light collection thatoccurs when the sphere is adjacent to the interface.The simulated trajectories were selected to includeat least one instance where sphere position is adja-cent to the interface. We estimated the expectedintensity using Eq. (2), with z0ðtÞ, for x0ðtÞ and y0ðtÞwithin a single pixel, and F summed over a timesegment equal to the light collection interval.

3. Results

A. Sphere Diffusion Through Vd

Figure 8(a) shows three sequential images of 100nmdiameter fluorescent spheres under aoTIRF illumi-nation in the upright microscope. The images are20ms exposures of the CCD camera to light butare separated in time by 59ms due to overhead inthe imaging software (Slidebook, Intelligent ImagingInc., Denver, Colorado). The images show the transi-ent appearance of a diffusing sphere in the evanes-cent field. The fluorescent sphere in the lower partof each panel is fixed to the interface and indicatesthe fluorescence intensity for a sphere constantlyin the most intense region of the evanescent field.The brightest pixel for the stationary sphere has641 counts for the 20ms collection time. The bright-est pixel for the transient sphere in the upper rightcorner of the middle panel has 214 counts for thesame collection time. The transient events observedcharacterize the brightest events for the diffusingsphere. The average background is ∼16 counts. Ty-pically ∼20 single-sphere events like that depictedin Fig. 8(a) were observed over 250 exposures inthe time-lapse movie of spheres diffusing throughVd. Table 1 indicates the statistics for the eventsand their comparison to simulation. Similar ex-periments were carried out for 40nm diameter fluor-escent spheres, with results also summarized in


Table 1. Observed and simulated characteristicsagree favorably.Figure 8(b) shows three sequential images of

100nm diameter fluorescent spheres under aoTIRFillumination in the inverted microscope configura-tion. Exposure time was 100ms on the EMCCD cam-era and frames are separated in time by 135ms.Spheres in the upper left and lower portion of theframes are stationary. The transient event is inthe middle of the frame. The intensity of the transi-ent sphere somewhat exceeds that of the stationaryspheres, probably because the stationary spheres arephotobleached due to exposure to light or because thetransient is from an aggregate of spheres. Other sta-tionary spheres in the FOV (not shown) match thetransient sphere intensity. The inverted microscope

configuration would benefit from the routine use ofquartz glass prisms and coverslips, as is the practicewith conventional pTIRF [24].

Figure 8(c) is epi-illumination of the bulk solutionof 40nm diameter spheres observed in the uprightmicroscope configuration. The images are 10msexposures of the CCD camera to light, but are sepa-rated in time by 80ms. This sphere remained withinthe ∼600nm objective depth of focus for >400ms.The epi-illumination time-lapse images contrastsharply with the aoTIRF images, where sphereswere not observed in sequential frames.

B. Back Focal Plane Imaging of an InterfaceImmobilized Sphere

Single 100nm diameter fluorescent spheres immobi-lized on the TIR interface and in diffusive contactwith identical spheres are illuminated under aoTIRFwith emission spatial distribution observed at theBFP using the setup depicted in Fig. 7. As pointedout by Mattheyses and Axelrod, the microscope ob-jective maps a ray propagation angle from an in-focus source into off-axis radial positions at theBFP [25]. The BFP intensity pattern from a high-aperture objective separately maps far- and near-field emission. Patterns from emitting sources closeto the interface show an intense circular band wherecritical angle light intersects the BFP. Light fallingoutside the band is SAF, and light falling withinthe band is UAF [14]. We interpreted the BFP pat-tern, emitted from a dipole excited by oTIRF illumi-nation, in terms of fluorescent dipole orientation anddistance from the interface [12].

Figure 9 shows the BFP image from a 100nm di-ameter sphere emitting fluorescence excited by ellip-tically polarized aoTIRF illumination (top) and thepattern computed from a single emitting dipole lying50nm above the interface and pointing horizontallyalong the x axis direction (bottom). The 100nmsphere lies on the planar glass/aqueous interfacewith its center at 50nm above the interface. The com-puted BFP patterns have reflection symmetrythrough the x axis at the junction of the two images;hence, we can compare patterns with the half-planerepresentation. The aoTIRF excitation is p-polarizedin the incidence plane with the evanescent electricfield intensity polarized ∼78 or 22% normal or par-allel to the interface. Excitation light propagatesalong the interface parallel to the x axis with polar-ization either normal or along the x axis. The emis-sion pattern most closely resembles the patternexpected for dipoles aligned to the parallel electric

Table 1. Event Statistics Observed for Nanospheres Diffusing in Water at Room Temperature

Sphere Radius S (nm)

Mean Relative Intensity� SD

Light Collection Interval (ms) Event CountObserved Simulated a

20 0:42� 0:11 0:46� 0:02 10 5450 0:26� 0:04 0:25� 0:02 20 30

aSimulated using the random walk algorithm described in Section 2.

Fig. 8. (a) Sequential images of 100nm diameter fluorescentspheres under aoTIRF illumination in the upright microscope con-figuration. The images are 20ms exposures of the CCD camera tolight but are separated in time by 59ms. (b) Sequential images of100nm diameter fluorescent spheres under aoTIRF illuminationin the inverted microscope configuration. Exposure time was100ms on the EMCCD camera and frames are separated in timeby 135ms. (c) Sequential images of 40nm diameter spheres diffus-ing in bulk solution under epi-illumination and observed in the up-right microscope configuration. The images are 10ms exposures ofthe CCD camera to light but are separated in time by 80ms. Scalebars are shown for each panel.


field. Dipoles aligned normal to the interface wouldproduce a BFP pattern with circular symmetry.Critical angle emission occurs at the bright narrow

band identified in the computed pattern in Fig. 9 atthe arrow. The band is apparent but less distinctivein the observed BFP pattern, probably because mostof the emitting dipoles on the sphere are closer than50nm due to the narrow penetration depth of theexcitation evanescent field. Critical angle emissionseparates UAF and SAF emission. We see that thefluorescent sphere emits in both regions due to itsproximity to the interface. Artifacts are producedin the sphere emission pattern at 3 and 9 o’clockby scattered light. Figure 6 shows that laser lightscattering is larger just at the edges of the objectivelens, where there is a material discontinuity from thebrass of the objective body to the glass of the lens.This light enters the objective by propagatingroughly parallel to the optical axis and is focusedto two spots near the BFP edges but within theUAF emission region. Background subtraction toform the image at the top of Fig. 9 removes mostof the autofluorescence excited in the objective bythe focused scattered light, but the artifact is visible.The autofluorescence is smaller than that producedby oTIRF because emission is excited by a tiny frac-tion of the exciting light entering the objective in ao-TIRF compared to the total intensity of the excitingbeam focused at the BFP in oTIRF. The BFP is withina glass element of the Olympus objective, producinga very bright spot under oTIRF excitation in the SAFregion, prohibiting sample fluorescence detection.

C. aoTIRF Imaging of a Cardiac Papillary Muscle Fiber

The striated pattern characteristic to transmittedlight microscopic images of skeletal and cardiac pa-pillary muscle fibers comes from the interdigitatedmyosin and actin filaments, where thick filamentbands are due to myosin scattering of transmittedlight. The muscle sarcomere is the elementary con-tractile structure consisting of thick filaments andopposed actin filaments. The thick filament is bipo-lar, with myosins segregated to align similarly alongthe þz axis and in the reverse polarity along the −zaxis when the thick filament center is at z ¼ 0. Actinfilaments are unipolar but oppositely directed withinthe sarcomere, where they are connected to the Z-disks defining the sarcomere boundary. Actin andmyosin polarity is complementary within the sarco-mere, permitting the actomyosin interaction toshorten the sarcomere during contraction. Myosincontains the enzymatic ATPase activity in contract-ing fibers that isolates to the myosin head domain,usually called the cross-bridge in the fiber. Thecross-bridge also contains the actin binding site.

Epi-illumination fluorescence from myosin crossbridges labeled with a chromophore also appearsstriated. Contrast between light and dark regions de-pends on the depth of focus. Epi-illumination bright-field and confocal images of the striated muscle fiberhave lower and higher contrast levels between lightand dark regions. The shorter depth of focus in theconfocal image removes out-of-focus light, givingthe greater contrast. oTIRF images have a subdif-fraction limit depth of focus and surpass the contrastof the confocal images. We showed that inserting athin metal film at the glass/aqueous interface inoTIRF further narrows the detection volume depthfrom which fluorescence is detected [6].

Figure 10 shows images from cardiac papillarymuscle fiber tissue preparations that have beenchemically skinned to permeabilize the membrane.Fluorescence is detected from RLC-GFP specifi-cally replacing native RLC on myosin cross bridgesin the muscle fibers. The muscle fibers are in therelaxed state where cross bridges spend the pre-dominant amount of their time detached from theactin filament. Figures 10(a) and 10(b) are the sameGFP-tagged cardiac papillary muscle fiber under epi-illmuniation and aoTIRF illumination. Figure 10(c) isfrom another cardiac papillary fiber under oTIRF. Allimages were taken with the inverted microscope.

Figures 10(a) and 10(b) differ due to the depth offocus. In epi-illumination, wide-field excitation lightpropagates from the objective and transmits thesample. Emitted fluorescence is collected fromexcited dipoles within the focal depth of the highNA objective and detected by the CCD camera.The∼600nm focal depth gives an image formed fromemitted light from a relatively thick section of tissue.Relative myosin filament lateral displacementsin shallow versus deep sections of the fiber tend tolower contrast between the thick and thin fila-ment regions. The ∼50nm deep aoTIRF evanescent

Fig. 9. The BFP image from a 100nm diameter sphere emittingfluorescence excited by aoTIRF illumination (top half) and the pat-tern computed from a single emitting dipole lying 50nm above theinterface and pointing horizontally along the x-axis direction (bot-tom half). The arrow at the bottom of the figure designates thebright ring due to critical angle emission.


excitation images myosin filaments in a thinner sec-tion, where they are in better register. The image haslarger contrast between the thick and thin filamentregions; however, the shallow illumination producesless total fluorescence because fewer dipoles are ex-cited. The signal-to-noise ratio is not as favorable asthe oTIRF image [Fig. 10(c)], where the evanescentexcitation intensity is∼100nm deep, but contrast be-tween light and dark bands is larger in the aoTIRFversus oTIRF images. The M-line is a myosin cross-bridge depleted part of the thick filament, wheremyosin tails reverse direction to enable myosin crossbridges to pull actin filaments toward the thick fila-ment center. It is a narrow dark line in the center ofthe thick filament image and subtly discernable inboth the oTIRF and aoTIRF images.The cardiac papillary muscle fiber specimen con-

tains more connective tissue than the similarlyprepared skeletal muscle fiber. Nonfluorescent con-nective tissue displaces the cardiac fiber from theglass/aqueous interface, making tight contact be-tween fiber and glass more spatially intermittent.

The brighter stripe in the center of the middle panelis from a small section of myosin containing speci-men in closer contact with the interface. Skeletalmuscle fibers have less connective tissue and makea tighter contact with the glass/aqueous interfaceover the entire field of view than can be seen in pre-viously published work [6].

D. aoTIRF with Other Infinity-Corrected Objectives

Diffusing spheres were observed under aoTIRF ex-citation using a Zeiss 100× NA ¼ 1:45 infinity-corrected objective on the upright microscope. Theobjective external dimensions are consistent withapplication of aoTIRF and the approach-limitingedge is more accommodating because distance R(Fig. 2) is smaller at 3mm and the objective workingdistance W is slightly larger at 0:11mm. Under ao-TIRF, we observed diffusing spheres as intense lightpulses surrounding steady intensity from stationaryspheres attached to the glass in the glass/water in-terface of the illuminated region. The phenomenamatched observations from the Olympus objective.

4. Discussion

TIRF imaging of biological samples is widely used totake advantage of the subdiffraction limit focal depth(z-dimension) resolution provided by the evanescentfield. This feature provides a substantial improve-ment over confocal fluorescence imaging z-dimensionspatial resolution. oTIRF is popular inmicroscopy ap-plications because modern high-NA objectives makeoTIRF simpler to implement than the older pTIRFsetup. The oTIRF and pTIRF setups are suitablefor both upright and inverted microscopy, but are dis-tinguishable by complementary advantages and dis-advantages. Practical application of oTIRF requiresthe highest NA objectives available (NA ≥ 1:4) thatare SAF collection capable (NA > 1:3). However, theNA restricts the maximum incidence angle of the ex-citation light to 73°–79° for NA of 1.45–1.49 objec-tives. In practice, incidence of the exciting laserbeam is less than themaximum to allow sufficient en-ergy to reach the sample. Autofluorescence in oTIRFis due in part to emission from glass in the objectivetransmitting the high-intensity focused excitationlight and is directly observable in BFP imaging fromoTIRexcited samples.ThepTIRFsetup isnot limitingfor exciting light incidence angle or by autofluores-cence that cannot be reduced with use of quartzprisms and coverslips [26]. Nonetheless, pTIRF asnormally implemented does not detect SAF becauseemission is collected via a water immersion objective.aoTIRF circumvents the loss of SAF in a prism-basedTIRF setup by delivering exciting light and collectingemitted light from the glass side of the interface in op-tical contact with the microscope objective. Excitinglight propagates to the interface for TIR externallyto the objective, thusavoiding the largest source of un-preventable autofluorescence. Implementation of ao-TIRF provides the minimum evanescent field depth(∼50nm), but does not permit a practical ability to

Fig. 10. Fluorescence images of cardiac papillary muscle fibers.Fluorescence is detected from GFP tagged myosin regulatory lightchain specifically replacing the native regulatory light chain onmyosin cross bridges. Images in (a) and (b) compare the same sam-ple but under epi-illumination and aoTIRF illumination. (c) is fromanother cardiac papillary fiber under oTIRF. All images weretaken with the inverted microscope and the scale bar applies toall panels.


alter field depth by changing exciting light inci-dence angle.The aoTIRF setups were demonstrated for the

upright and inverted microscope configurations(Figs. 1–3). The upright microscope configurationis simpler to implement because it is more tolerantof variation in the geometric arrangement of the ap-paratus. The Littrow prisms can be the smaller orlarger variety (12:5–38mm base) and just one glasscoverslip thickness (#1) is needed. The inverted mi-croscope configuration is complicated by the triple re-flection, requiring two glass coverslip thicknesses (#1and #2). Care is needed to maintain the tolerances inthe geometric arrangement of the apparatus, asshown in Fig. 3. The inverted microscope setup iscarefully constructed, but everything can be done re-producibly by hand. For either configuration, wefound that 60–75mm long #1 coverslips (availablefrom Electron Microscopy Sciences, Hatfield, Penn-sylvania) were most practical because they enablethe two prism configuration, where the exiting laserbeam is coupled out of the coverslip in the same man-ner as the entering laser beam was coupled into thecoverslip. The exiting light produces the clean inten-sity profile of a totally internally reflecting beam on awhite card placed a few feet from the microscope.Achieving a clean exit intensity profile provides agood guide for correctly conditioning the enteringbeam with the beam diameter reducer and focusinglens (Fig. 1).On first implementing aoTIRF, it is desirable to

practice on the fluorescent spheres sample to demon-strate that the exciting beam is truly undergoingTIR. Stray scattering can contaminate the FOV withan improperly conditioned exciting beam. This prac-tical difficulty is easily identified and eliminated byimaging the stationary and diffusing fluorescentspheres (40 or 100nm diameter). The stationaryspheres are firmly adsorbed to the interface by allow-ing a very dilute bulk sample to evaporate within thesample chamber. The stationary spheres provide thetarget for focusing the microscope objective, and theycontrast with the diffusing sphere transients ob-served when the more concentrated bulk sample ofspheres is placed in the sample chamber. Under ao-TIRF, diffusing spheres are rapidly blinking lightssurrounding the stationary sphere images. Quan-titative measurements (like we did) can be done;however ,the diffusing sphere fluorescence transientsunder aoTIRF are obviously different from thoseproduced by stray light excitation.Light scattering and the autofluorescence it ex-

cites are a part of the background that we seek tominimize. Autofluorescence in pTIRF is mitigatedby use of quartz substrates and low-fluorescence im-mersion fluids. In oTIRF, intense propagating excita-tion light excites background autofluorescence fromthe glass elements in the objective. The aoTIRF set-up forgoes propagating excitation light through theobjective. Autofluorescence from excitation lightscattering collected by the objective was detected

while imaging the BFP under aoTIRF illumination;nonetheless, there was a significant reduction inautofluorescence when comparing BFP images in ao-TIRF versus oTIRF. In oTIRF, the focused excitationlaser light at the BFP excited autofluorescence thatwas too bright compared to the emitting sample topermit imaging. Figure 9 is the BFP image producedunder aoTIRF.

We tested aoTIRF on two high-aperture oil immer-sion objectives. They provided equivalent results de-spite their different external dimensions. We foundthat, for common oTIRF objectives, the chief diffi-culty in applying aoTIRF is accommodating theclearance to the objective focal point for the excitinglaser beam defined by the approach-limiting edge.The clearance affects two factors that are limitedby practical considerations. The distance R fromthe objective optical axis to the approach-limitingedge limits the allowable incidence angle of the excit-ing beam. The objective working distance W limitsboth incidence angle and the allowable width ofthe focused exciting beam. Permissive geometrieshave smaller R and larger W. The Olympus 60×NA ¼ 1:45 objective sets R ¼ 3:8mm and W ¼0:1mmas desirable minimums for these parameters.

A GFP-tagged cardiac papillary muscle fiber wasimaged with aoTIRF. Results compare favorably withoTIRF images of similarly prepared tissues. The ao-TIRF illumination has a somewhat higher contrastbetween light and dark bands, but has a lower sig-nal-to-noise ratio for the image. Both effects arelikely due to the lower evanescent field penetrationdepth under aoTIRF. The cardiac papillary fiber is achallenging tissue to image under TIRF because theconnective tissue associated with the preparationsoften tends to displace to fiber too far from theglass/aqueous interface to make contact with theevanescent field. Selected regions did make closecontacts and were excited by the evanescent field.

5. Conclusion

We demonstrated a near-field excitation/emissionTIRF microscopy technique that combines advan-tages of oTIRF with pTIRF. The technique has eva-nescent illumination of dipoles at a glass/aqueousinterface with excitation light external to the micro-scope objective and collection of supercritical anglefluorescence from the excited dipoles. The approacheliminates background light due to the admission ofthe high-energy laser beam to the microscopic optics,as in oTIRF, while collecting near-field emission fromthe excited dipoles, as is impossible with waterimmersion objectives used in pTIRF. We call the ap-proach around-the-objective TIRF (aoTIRF) becausethe evanescent field is created by a conditioned laserbeam that propagates through the submillimeter gapcreated by the oil immersion high-NA objective col-lecting emitted light and the glass coverslip. Setupswere developed for both upright and inverted micro-scope configurations. The aoTIRF technique wastested with 40 and 100nm diameter fluorescent


spheres in water that were diffusing into and out ofthe detection volume, with BFP imaging of single100nm fluorescent stationary spheres adsorbed tothe glass substrate of the interface, and with imagingof cardiac papillary muscle fibers labeled withexchanged myosin RLC-GFP. Results confirm thatthe evanescent field has penetration depth of∼50nm, that the background autofluorescence fromthe objective glass is significantly reduced, andthat the technique is useful for imaging biologicalsamples.

This work was supported by National Institutes ofHealth (NIH)-National Institute of Arthritis andMusculoskeletal and Skin Diseases (NIAMS) grantR01AR049277 and the Mayo Clinic Rochester. Wethank Ken Kilby and Kevin Duffy from Leeds Instru-ments (Minneapolis, Minnesota) for providing uswith the Olympus objective specifications.

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Around-the-objective total internal reflection fluorescence microscopy

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