Annual Reviews www.annualreviews.org/aronline Ann. Rev. Biophys. Bioen9. 1984.13 : 24748 Copyrioht © 1984 by Annual Reviews Inc. All riohts reserved TOTAL INTERNAL REFLECTION FLUORESCENCE Daniel Axelrod Biophysics ResearchDivision and Departmentof Physics, University of Michigan, AnnArbor, Michigan48109 Thomas P. Bur~thardt CardiovascularResearch Institute, Universityof California, San Francisco, California 94143 Nancy L. Thompson Departmentof Chemistry,Stanford University, Stanford, California 94305 INTRODUCTION Total internal reflection fluorescence(TIRF) is an optical effect particularly well-suited to the study of molecularand cellular phenomenaat liquid/solid interfaces. Suchinterfaces are central to a widerange of biochemicaland biophysical processes: binding to and triggering of cells by hormones, neurotransmitters, and antigens; blood coagulation at foreign surfaces; electron transport in the mitochondrial membrane;adherence and mo- by University of Illinois - Urbana Champaign on 02/02/09. For personal use only. bility of bacteria, algae, and culturedanimal cells to surfaces; and possible enhancementof reaction rates with cell surface receptors uponnonspecific Annu. Rev. Biophys. Bioeng. 1984.13:247-268. Downloaded from arjournals.annualreviews.org adsorptionand surface diffusion of an agonist. Liquid/solidinterfaces also haveimportant medical and industrial applications: e.g. detection of serum antibodies by surface immobilized antigens; and the manufacture of biochemical~aroducts by surface-immobilizedenzymes. Total internal reflection spectroscopyfor optical absorption studies (called attenuated total reflection or ATR)was developed somewhat earlier than the fluorescence applications and has been widely used in surface chemistrystudies (17). Thebasic bookin this area is lnternal Reflection 247 0084-6589/84/0615-0247502.00 Annual Reviews www.annualreviews.org/aronline 248 AXELROD, BURGHARDT & THOMPSON Spectroscopyby Harrick (18). Total iffternal reflection optics have also been combined with Ramanspectroscopy (24, 32), X-ray fluorescence (2), infrared absorption spectroscopy (16), and light scattering (3, 33). As a technique for selective surface illumination, total internal reflection fluorescence was first introduced by Hirschfeld (21) for solid/liquid interfaces, Tweetet al (39) for liquid/air interfaces, and Carniglia & Mandel (13) for high refractive index liquid/solid interfaces. TIRF has been combined with a variety of other conventional fluorescence techniques (polarization, Ffrster energy transfer, microscopy,spectral analysis, photo- bleaching recovery, and correlation spectroscopy) for a variety of purposes (detection of molecular adsorption; measurement of adsorption equilib- rium constants, kinetic rates, surface diffusion, adsorbate conformation; and observation of cell/substrate contact regions). This r~viewis organized according to those purposes. We begin with a summary’of the relevant optical theory. THEORY Evanescent Intensity Whena light beam propagating through a transparent medium of high index of refraction (e.g. a solid glass prism) encounters an interface with mediumof a lower index of refraction (e.g. an aqueous solution), undergoestotal internal reflection for incidence angles (measuredfrom the normalto the interface) greater than the "critical angle." The critical angle 0c, is given by 0c = sin- 1 (n2/nt), 1. where n2 and nl are the refractive indices of the liquid and the solid, respectively. Althoughthe incident light beamtotally internally reflects at the interface, an electromagnetic field called the "evanescent wave" penetrates a small distance into the liquid mediumand propagates parallel to the surface in the plane of incidence. The evanescent waveis capable of by University of Illinois - Urbana Champaign on 02/02/09. For personal use only. exciting fluorescent molecules that might be present near the interface. This effect has been regarded as experimental proof of the existence of the Annu. Rev. Biophys. Bioeng. 1984.13:247-268. Downloaded from arjournals.annualreviews.org evanescent wave (44). The evanescent electric field intensity I(z) decays exponentially with perpendicular distance z from the interface: I(z) = Ioe-~/a, where 20 2 d = ~ In1 sin2 0- n~2] - 1/2 2. Annual Reviews www.annualreviews.org/aronline TOTAL REFLECTION FLUORESCENCE 249 for angles of incidence 0 > 0c and light wavelengthin vacuum2 0. Depthd is independent of the polarization of the incident light and decreases with increasing 0. Except for 0 -~ 0c (where d ~ ~), d is on the order of 20 smaller. The intensity at z = 0, Io, depends on both the incidence angle 0 and the incident beam polarization. Io is proportional to the square of the amplitudeof the evanescent electric field E at z = 0.1 (These expressions are given in the next subsection.) For incident electric field intensities J" ’± with polarizations parallel and perpendicular, respectively, to the plane of incidence, the evanescent intensities I~,± are I~ = ,~11 4 cos2 0(220--2) sinn 3. n~ cos~ 0 + sin~ O-~ n and 2 ~ ±’ 4- cos 0 Io = dl_n 2 , 4. where n = n2/nl < 1. 5. Figure 1 illustrates IioI’± as functions of 0. Note several features: (a) the evanescent intensity I~ ’± is not weakand can be several times stronger than the incident intensity for angles of incidence within a few degrees of the critical angle; (b) the intensities of the two polarizations are different, with I~ somewhatmore intense for all 0; (c) Iio I’± both approach zero as 0~90°. Evanescent Fields The phase behavior of the E field is quite remarkable (7). The field componentsare listed below, with incident electric field amplitudes A II ,± and phase factors relative to those of the incident E and H fields’ phase at z = 0. (The coordinate system is chosen such that the x-z plane is the plane by University of Illinois - Urbana Champaign on 02/02/09. For personal use only. of incidence.) Annu. Rev. Biophys. Bioeng. 1984.13:247-268. Downloaded from arjournals.annualreviews.org Ex ,, / , 6. [(n2~_e-~s 2cOs0~_~ ~-n -~--~-~/z7(sin20--n2) l 1/2Aile l _,,, +~.:, ~:,, =l[ Lii-- 2coso ~-)-rnj A±e-"% 7. 1 The evanescent intensity that excites fluorescence is given by IEI2 (12, 13). Generally, the energy flux of an electromagnetic field is given by the real part of the Poynting vector, S = (c/4n)E x H, whereH is the magnetic field. For a transverse field, 181is proportional to IEI2. Note that for an evanescent wave, ISl is not proportional to IEI2. Annual Reviews www.annualreviews.org/aronline 250 AXELROD~ BURGHARDT & THOMPSON -I _~ [~n* 2 cos 0 sin 0 where (sin2 U2 ~11 -= tan-~F k ~-~-c--~-~O-- n2) J7 9. and 6z -- tan -~ L = -c-~s -0 J" 10. Notethat the evanescentelectric field is transverseto the propagation . direction (+x) only for the _k polarization. TheIt polarizationE field 5,0 -- I~,l vs. 0 4.54.0 - ,\~ n = 1’33A,50 =0.88-/ 3.5- \ \Io" " >" 3.0 \\~~ 2.0- by University of Illinois - Urbana Champaign on 02/02/09. For personal use only. ’"- ~.0 Annu. Rev. Biophys. Bioeng. 1984.13:247-268. Downloaded from arjournals.annualreviews.org 0.5- 0t;00c 65 70 75 80 85 90 INCIDENCE ANGLE O (deg.) Figure1 Intensiti¢s I~ ’z vs incid¢nc: an#¢e, for n = 0.887, correspondingto a critical angl¢ ofO~= 62.46°. Intensity is expressedas the ratio ofevanes~ntintensity at ~ = 0 to the incident intensity for eachpolarization. Annual Reviews www.annualreviews.org/aronline TOTAL REFLECTION FLUORESCENCE 251 "cartwheels" along the surface with a spatial period of 2o/(nl sin 0) as shownschematically in Figure 2. For absorbers with magnetic dipole transitions, the evanescent magnetic field H is relevant. Assumingequal magnetic permeabilities at both sides of the interface, the componentsof the evanescent field H at z = 0 are Hx t_ I’2cos 0~_1 ~nin2) -iTT(sin2 O--n2)1/2-] -i(t5 -~) 12. Hr =-- (n* COS2 0+sin 20--n2) ~/2’ A)le-i(atl-’~/2)’ H~ = (1 -n2) ~/2 A±e-i~" 13. The angular dependenceof the phase factors ~± and ~ II gives rise to a measurable longitudinal shift of a finite-sized incident beam, knownas the Goos-I-Ianehenshift (29). Viewedphysically, someof the energy of a finite- width beamcrosses the interface into the lower refractive index material, skims along the surface for a Goos-Hanchenshift distance ranging from a fraction of a wavelength(at 0 -- 90°) to infinite (at 0 = 0c), and then reenters the higher refractive index material. In manyTIRF experiments, an incident laser beam of Gaussian intensity profile passes through a converginglens, enters the side of a prism, and then focuses at a totally reflecting surface. The nature of the evanescent EVANESCENT ~" "~ field . +Z by University of Illinois - Urbana Champaign on 02/02/09. For personal use only. Annu. Rev. Biophys. Bioeng. 1984.13:247-268. Downloaded from arjournals.annualreviews.org :::::::::::::::::::::::i:i:i:.’i:i:i:i::::i¥i:i:i.’i:i:i:i:i:i:i:i:i:i:i:i.’:i:i:i:i:i:i:i:i:i:i:i:: ::::::::::::::::::::::::::: Figure 2 Electric field vectors of incident and evanescentlight for the II incident polarization, showingthe phase lag 6 II and the "cartwheel" or elliptical polarization of the evanescentfield in the plane of propagation. Both the incident and evanescentvectors refer to the z = 0 position ; they are schematically displaced a distance e (~0) below and above the interface along their constant phase lines for pictorial clarity only. Annual Reviews www.annualreviews.org/aronline 252 AXELROD, BURGHARDT & THOMPSON illumination produced by such a focused finite beam geometry has been investigated in general (12). For typical experimental conditions,