Fluorescence Microscopy. Super-Resolution and Other Novel Techniques PDF
Preview Fluorescence Microscopy. Super-Resolution and Other Novel Techniques
FLUORESCENCE MICROSCOPY SUPER-RESOLUTION AND OTHER NOVEL TECHNIQUES Edited by A C P. M C NDA ORNEA AND ICHAEL ONN AMSTERDAM(cid:129)BOSTON(cid:129)HEIDELBERG(cid:129)LONDON NEWYORK(cid:129)OXFORD(cid:129)PARIS(cid:129)SANDIEGO SANFRANCISCO(cid:129)SINGAPORE(cid:129)SYDNEY(cid:129)TOKYO AcademicPressisanImprintofElsevier Thecovershows3Dreconstructionofneuronalnetworksintheunsectionedmousespinalcordobtainedby3DISCO method.TheimagescourtesyofDr.AliErtu¨rk.TopimageoriginallypublishedinThree-dimensionalimagingofthe unsectionedadultspinalcordtoassessaxonregenerationandglialresponsesafterinjury.AliErtu¨rk,ChristophP Mauch,FaridaHellal,FriedrichFo¨rstner,TaraKeck,KlausBecker.NatureMedicine13:1(166-172)NaturePublishing Group,January2012. AcademicPressisanimprintofElsevier 32JamestownRoad,LondonNW17BY,UK 225WymanStreet,Waltham,MA02451,USA 525BStreet,Suite1800,SanDiego,CA92101-4495,USA Firstedition2014 Copyright(cid:1)2014ElsevierInc.Allrightsreserved. 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Becauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationofdiagnosesand drugdosagesshouldbemade BritishLibraryCataloguing-in-PublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress ISBN:978-0-12-409513-7 ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com TypesetbyTNQBooksandJournals www.tnq.co.in PrintedandboundinUnitedStatesofAmerica 14151617 10987654321 Preface Microscopy has long been a cornerstone in our emphasis on super-resolution techniques, light-sheet knowledge of the living world, but the last decade has microscopy,samplepreparation,newlabels,andanaly- seentremendousadvancesthatchallengedearlierrules sistechniques. andlimitations.Opticalimaginghasevolvedintoaval- Authorswereselectedbasedontheirresearchcontri- uable, albeit complex, analytical and quantitative tool. butions in the area about which they have written and At one end ofthe size spectrum, thetheoretical limit of on their ability to describe their methodological contri- opticalresolution was broken,bridging the gapto elec- butions in a clear and reproducible way. They have tron microscopy. At the other end, whole organs and beenencouragedtomakeuseofgraphicsandcompari- evenorganismsmaynowbeimagedatthecellularlevel, sons to other methods and to provide tricks and revealingathree-dimensionalarchitecturethatwasdif- approaches not revealed in prior publications that ficult to grasp in traditional two-dimensional sections. make it possible to adapt methods to other systems. In Progressinmicromechanics,opto-electronics,computer some cases, the reader will see helpful video to guide power, molecular biology, and chemistry has allowed their work. advanced concepts in physics to provide commonly The editors want to express appreciation to the con- used technologies in the service of biomedical sciences. tributors for providing their contributions in a timely This volume reviews some of the most influential fashion and to the editors and staff at Academic Press recent advances in fluorescence microscopy with an forhelpful input. vii List of Contributors Daniel Axelrod Departments of Physics, Biophysics, and John P. Nolan La Jolla Bioengineering Institute, San Diego, Pharmacology,UniversityofMichigan,AnnArbor,MI,USA CA,USA MelanieBokstad BenGurionUniversity,Be’erSheva,Israel FelipeOpazo EuropeanNeuroscienceInstituteandUniver- Martin J. Booth Department of Engineering Science and sityofGo¨ttingen, Go¨ttingen, Germany Centre for Neural Circuits and Behaviour, University of Chan-Gi Pack RIKEN, Wako,Japan Oxford,United Kingdom Brian R. Patton Department of Engineering Science and Julian Borejdo University of North Texas, Health Science Centre for Neural Circuits and Behaviour, University of Center, FortWorth, TX, USA Oxford,United Kingdom Alessandra Cambi Radboud Institute for Molecular Life Daniel A. Peterson Chicago Medical School, Rosalind Sciences, Radboud University Medical Center, Nijmegen, Franklin University of Medicine and Science, North TheNetherlands Chicago,IL,USA Julien Colombelli Institute for Research in Biomedicine, Jeremy Pike The University of Birmingham, Birmingham, Barcelona, Spain UnitedKingdom AliErtu¨rk Genentech,Inc., SanFrancisco,CA, USA TarlW.Prow TranslationalResearchInstitute,Woolloongabba, EugenioF.Fornasiero EuropeanNeuroscienceInstituteand Queensland,Australia Universityof Go¨ttingen, Go¨ttingen, Germany Joshua Z. Rappoport The University of Birmingham, Sung-SikHan DepartmentofLifeScience,KoreaUniversity, Birmingham, UnitedKingdom Seoul,Republic ofKorea E. Hesper Rego Harvard School of Public Health, Boston, Samie R. Jaffrey Weill Medical College, Cornell University, MA,USA NewYork, NY, USA YasushiSako RIKEN, Wako,Japan Min-Kyo Jung Department of Life Science, Korea Univer- Lin Shao Janelia Farm Research Campus, Howard Hughes sity,Seoul, RepublicofKorea MedicalInstitute, Ashburn,VA, USA Sandra de Keijzer Radboud Institute for Molecular Life AlexSmall CaliforniaStatePolytechnicUniversity,Pomona, Sciences, Radboud University Medical Center, Nijmegen, CA,USA TheNetherlands Mi-Roung Song BITERIALS Co., Ltd., Seoul, Republic of Jun-Sung Kim BITERIALS Co., Ltd., Seoul, Republic of Korea Korea Shane Stahlheber California State Polytechnic University, LynleeL.Lin TranslationalResearchInstitute,Woolloongabba, Pomona,CA, USA Queensland,Australia Rita L. Strack Weill Medical College, Cornell University, ErLiu LaJollaBioengineeringInstitute,SanDiego,CA,USA NewYork, NY, USA Corinne Lorenzo University of Toulouse and Centre Jennifer A. Thorley The University of Birmingham, Nationalde laRechercheScientifique, Toulouse, France Birmingham, UnitedKingdom Ohad Medalia Ben Gurion University, Be’er Sheva, Israel; Miko Yamada Translational Research Institute, Universityof Zurich,Zurich, Switzerland Woolloongabba,Queensland,Australia Marjolein B.M. Meddens Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen,The Netherlands ix C H A P T E R 1 Evanescent Excitation and Emission Daniel Axelrod Departments ofPhysics, Biophysics, and Pharmacology, University of Michigan, Ann Arbor, Michigan, USA INTRODUCTION (cid:129) TIRF intensity vs. incidence angle on film-coated surfacescandisplayaresonancebehaviorthatmay Someofthevarioussuper-resolutionmicroscopytech- measurethe thickness, refractive index and niques share a common feature in that they attempt to possiblelateralheterogeneities ofsurface- exceed the standard light microscope resolution limit supported multilayer lipid or protein coatings. by employing “evanescent” light that decays in at least (cid:129) TIRF on film-coated surfacescan enhance the one direction in a distance much shorter than the evanescent intensity by atleast anorderof wavelength.Thisgroupincludestotalinternalreflection magnitude. fluorescence microscopy (TIRFM; covered in another (cid:129) Polarized excitation TIRF can highlight chapter in this book), near-field scanning optical submicroscopic irregularities inthe plasma microscopy(NSOM),andvirtualsupercriticalanglefluo- membrane of living cellsand orientationof single rescence (vSAF) microscopy.1e4 In some cases, evanes- molecules. cence is in the excitation light, in othercases it is in the (cid:129) Intersecting TIRF beams can extend the super- emission,andinsomeitisinboth.Thischapterexplores resolution ofstructuredillumination. the physical concepts that these techniques share and (cid:129) Radially polarized ring TIR illumination at the points toward some established and more speculative back focal plane(BFP) can producea uniquely possible directions for future work in evanescence- smallillumination volume, possiblyuseful for based super-resolution. The effect of evanescence on fluorescence correlationspectroscopyandhigh- the use and detection of polarization, in bothexcitation resolution scanning. andemission, iscovered inconsiderabledetail. (cid:129) TheevanescentfieldatanNSOMtipfacilitatesthe Evanescence in both excitation and emission can be mapping ofdistances to fluorophoresand surface understood as a response to geometrical compression, topology. or “squeezing,” of wavefront spacing in at least one (cid:129) On the emission side dimension. Evanescent light can be converted to or (cid:129) The emissionintensity pattern in the supercritical from propagating light traveling at supercritical angles zone of the BFP reports thefluorophore relative to a nearby interface. Evanescence has concentration profileas a function of distanceto numerous applications in fluorescence microscopy. the surface. Here is a preview of those to be discussed in this (cid:129) Theratioofemissionpowerinthesupercriticalvs. chapter: subcritical BFP zonecan sensitively report absolutedistanceofafluorophoretothesurfaceto (cid:129) On the excitation side an accuracy of tens of nanometers. (cid:129) Supercritical excitation (TIRF) is commonlyused (cid:129) Taking into account theinteraction of the for selective excitationof surface-proximal fluorophorenear field with asurfacealters the molecules, cell/substratecontact regions,and predicted depolarizationinduced by high- membrane-proximal cytoplasmic organelles. apertureobservation. (cid:129) Variable angle TIRF has been used to deduce the (cid:129) For a film-coated surface (such as a lipid concentration of fluorophoresas a functionof multilayer), the emission intensity pattern inthe distancefromthesubstrate. FluorescenceMicroscopy 1 http://dx.doi.org/10.1016/B978-0-12-409513-7.00001-4 Copyright©2014ElsevierInc.Allrightsreserved. 2 1. EVANESCENTEXCITATIONANDEMISSION supercriticalzone ofthe BFP isuniquelysensitive Forfreelypropagatinglight,thespacingbetweenwave- to film thickness. fronts along any directionx, y,z of propagation is (cid:129) O(cid:129)nBbyotchomexbciitnaitniogntahnedveSmAisFsieomnissisdieosn image protocol lmx;my;mz ¼2p=kmx;my;mz (1.3) with standardTIRF excitation, aneven higher The position-dependent part of the plane wave electric degreeof surfaceselectivity should be attainable field has the form exp(ik $r), where position vector m than fromeither individually, with much less r¼xbxþybyþzbz. Therefore, the spatial variation of the scattering background intensity. electricfieldin,say,thezdirectionisthesinusoidalfunc- tion exp(ik z). mz TherelevantfeatureofEquation(1.2)isthatthesum of the squares of the components k , k , and k in EVANESCENCE IN GENERAL mx my mz any medium must exactly equal k2 for that medium. m What happens if the geometry of the optical system For propagating light, the shortest spacing (l ) be- m somehow forces the sum of two of the components tween each traveling wavefront (i.e., the periodic locus to have squares greater than k2dfor example, ofpointsofequalphase)forpropagatinglightissimply m givenasl ¼l /n ,wherel isthelightwavelengthin ðk2mxþk2myÞ>k2m? This might happen if the wavefronts m o m o vacuum, and n is the refractive index of medium m in the xey direction are forced by geometry to become m (m ¼ 1, 2, 3 will be used here for a system of stratified closer together (i.e., squeezed). Equation (1.2) would planar layers). The wavefronts propagate through me- then demand that k2mz be negative and kmz thereby be dium mwith a speed of c/n . imaginary. The electric field’s spatial dependence in m There are situations, however, when the wavelength thezdirectionthenbecomesanexponentiallydecaying spacingcanbeforcedtobesmallerthanlo/nm.Suchspe- function exp((cid:2)jkmzjz), an evanescent field. cial geometrical situations include the lower refractive Thisgeneralmechanismofproducingaspatialexpo- index side of an interface at which total internal reflec- nentialdecayinatleastonedimensionissharedincom- tion (TIR) occurs; they also include confinement of the mon among the otherwise very different optics of TIR, light source to a region smaller than its wavelength, NSOM,andvSAF.Thedifferenceliesinhowthegeom- such as very near an excited molecule or the tip of a etry produces the “excess” k2 þk2 (or, equivalently, mx my fineopticalfiber.Inthesecases,lightcannotfreelyprop- squeezed wavelength spacing). These shared mecha- agateandinsteadbecomesexponentiallydecayinginat nisms and individual differences will be examined least one dimension. moreclosely in thefollowing sections. The fundamental physical processes are related among these situations and can be understood most easily by considering the electric field of plane waves EVANESCENCE IN EXCITATION: TIR and then generalizing to other wavefront shapes. Plane-wavelightpropagatinginamediumofrefractive In TIR, the wavefront spacing squeeze is a direct indexnmischaracterizedbyawavevector,km,pointing consequence of the geometry of refraction at an inter- in thedirection of its propagation: face. Plane-wave light approaching a planar interface k ¼ k bxþk byþk bz (1.1) fromahigherindex,n3,dielectrictowardalowerindex, m mx my mz n (say, in the xez plane, with z being the normal to 1 Theorientationofthe(x,y,z)axescanbearbitrarybutis the interface and no component in the y direction so chosenbasedonthegeometryofopticalsurfacesnearby. thatk ¼k ¼0;seeFigure1.1),cancreateanexponen- 1y 3y The amplitude ofkm,calledthe wavenumber, is tially decayingfield(ratherthanapropagatingfield)in (cid:2) (cid:3) 1=2 thelowerindexmedium,providedtheincidenceangle, km ¼ k2mxþk2myþk2mz (1.2) q3, in medium 3 (measured from the normal) is greater than a critical angle q . From the perspective of ray h2p=l ¼2pn =l ¼n u=c c m m 0 m optics, this critical angle is a straightforward whereuistheangularfrequencyoftheparticularcolor result of Snell’s law, which in general specifies that of the light, and c is the speed of light in vacuum. Fre- n1sinq1 ¼ n3sinq3, here evaluated for incidence angle quency u is equal everywhere in the optical system, q3whenthe angle of refraction q1 ¼90(cid:3), sothat (cid:4) (cid:5) regardlessof refractive index. Becauseoftheconstancyofuandc,thewavenumber, qc ¼sin(cid:2)1 n1 (1.4) n k , and its corresponding wavelength, l , are real 3 m m numbers that are fixed for any light in medium From the perspective of wavefronts as depicted in m, regardless of direction or proximity to interfaces. Figure 1.1, the spacing l of wavefronts along the 3x 3 EVANESCENCEINEXCITATION:TIR FIGURE1.1 WavefrontspacinginTIR.(Leftpanel)Subcriticalq <q.(Middlepanel)Criticalq ¼q.(Rightpanel)Supercriticalq >q. 3 c 3 c 3 c Wavefrontsareshownasheavysolidlines.Theplaneofincidenceisthexezplaneasshown.Thewavefrontspacinginthexdirection,l ,varies 1x andisprogressivelysqueezedbygeometryasq increases.Forq >q,l becomessmallerthanthespacingl demandedbyfreelypropagating 3 3 c 1x 1 lightinmedium1(shownaslightverticaldashedlines).Thissqueezingforcestheelectricfieldinthezdirectiontodecayexponentially.Theheavy dashedarrowsindicatepropagationdirection.Aphaseshiftexistsbetweenwavefrontsinmedium3vs.medium1forthesupercriticalcase,butit isnotshownhereinordertoclarifythedepictionofwavefrontspacing. interface (the x direction) just inside medium 3 is to the square of the electric field strength) in medium 1 alwayslongerthanthenaturalpropagationwavelength has the z dependence exp(e2jk jz) ¼ exp(ez/d),where mz l ¼2p/k inmedium3,becauseofthenon-orthogonal characteristic depth d is (assuming k ¼ 0 without loss 3 3 1y angle with which the x-axis interface cuts the wave- ofgenerality) fronts.Thewavefrontspacingl ¼2p/k inmedium1 1x 1x 1 1 on the other side of the interface is always forced to be d¼ ¼ (cid:2) (cid:3) (1.7) exactly equal to l3x because of the requirement to 2jk1zj 2 k21x(cid:2)k21 1=2 match the periodic boundary conditions imposed by Maxwell’s equations. That common wavefront FromEquations(1.2),(1.4),and(1.6)wecanrewritethis spacingbecomessmallerwithincreasingq andisgiven as 3 by l l d¼ (cid:6) o (cid:7) ¼ o l1x ¼l3x ¼l3=sinq3 (1.5) 4p n23 sin2 q3(cid:2)n21 1=2 4pn3ðsin2q3(cid:2)sin2qcÞ1=2 Aslongasl1xislongerthanthepropagatinglightwave- (1.8) length l ¼ 2p/k in medium 1 the periodic boundary 1 1 For typical refractive indices, depth d ranges from wl condition at the interface can be matched if the light in for q zq þ2(cid:3), down to about l /10 for larger buot medium 1 propagates away at some acute refraction 3 c o still easily attainable q . This small d is the reason why angle(givenby Snell’s law).But,forasufficiently large 3 incidence angle q (i.e., greater than q ) the wavelength TIR excitation of fluorescence (TIRF) is useful for spacing l along3the x direction becocmes smaller than selectiveexcitationofsurface-proximalmoleculesinme- the natura1xl propagation wavelength l for medium 1. dium 1, cell/substrate contact regions, and membrane- 1 proximal cytoplasmic organelles, while minimizing The corresponding wavenumber k for that spacing l is 1x excitation of background fluorescence originating 1x deeperwithinthe sample. 2p 2p 2p 2pn k ¼ ¼ ¼ sinq ¼ 3 sinq (1.6) 1x l l l 3 l 3 1x 3x 3 0 For q > q , wavevector component k becomes larger 3 c 1x TIR Theory: Field Strength, Polarization, and than the k (¼ 2p/l ) permissible for propagating light 1 1 Intensity inmedium 1.Thissituation forcesk to becomeimagi- 1z nary, as seen fromEquation(1.2). Completeexpressionsforeachcomponent(inthex,y, TheTIRevanescentfieldhasfourfeaturesinteresting zdirectionsasdefined above) oftheevanescentelectric for experimental techniques: depth, intensity, polariza- fieldvectorE inmedium1atz¼0canbederivedfrom 1 tion, andphase. boundary conditions imposed by Maxwell’s Equations. Eachoftheseexpressionsassumesthattheincidentlight electric field E is linearly polarized with components TIR Theory: Depth 3 E and E in the p-pol direction (i.e., parallel to the 3p 3s Because the electric field strength z dependence is planeofincidenceandperpendiculartothepropagation givenbyexp(ejk jz),theintensity(whichisproportional direction) and the s-pol direction (i.e., perpendicular to mz 4 1. EVANESCENTEXCITATIONANDEMISSION boththeplanesofincidenceandpropagationdirection), phase and consequently the p-pol evanescent field is respectively. elliptically(not linearly)polarized inthe planeof E ¼E þE incidence. 1 1p 1s E1p ¼E1xbxþE1zbz (1.9) totThheeirerveaspneescctievnetsicnatleanrspitrioesduI1cptasnEd*$IE1s.aGreivpernotphoerctioornrea-l E1s ¼E1yby sponding incident intensities jE1pj2 and jE1sj2, the evanescent intensities at z ¼0 are where (cid:6) (cid:7)(cid:6) (cid:7) E1x ¼(cid:6)(cid:2)2icosq3(cid:6)sin4(cid:7)qccos2q3þsin2q3(cid:2)sin2qc(cid:7)(cid:2)1=2 I1pð0Þ¼(cid:8)(cid:8)E3p(cid:8)(cid:8)2sin44cqocsc2oqs32q32þsinsi2nq23q(cid:2)3(cid:2)sins2inq2cqc (1.13) sin2q3(cid:2)sin2qc 1=2e(cid:2)idpE3p (cid:6) (cid:7) 4cos2q E1y ¼2ccoossqq3e(cid:2)idsE3s (1.10) I1sð0Þ¼jE3sj21(cid:2)sin2q3c (1.14) c Theseintensitiesareproportionaltotherateofenergy E1z ¼2cosq3(cid:6)sin4 qc cos2 q3þsin2q3(cid:2)sin2 qc(cid:7)(cid:2)1=2 aabresoprplotitotendbyvsa.flqu3orionphFoigreurine t1h.e2ev(saonliedscecnutrvweasv).eaTnhde sinq3e(cid:2)idpE3p smuopneorctorintiiccaalllyevaapnpersocaecnhteisnzteenrositays qof/eac9h0(cid:3)p.oSluabriczraittiicoanl 3 The angles inthe phase factors are angle intensities (corresponding to a propagating "(cid:6) (cid:7) # d ¼tan(cid:2)1 sin2q3(cid:2)sin2qc 1=2 (1.11) refracted ray) are also shown in Figure 1.2; they are p sin2q cosq c 3 "(cid:6) (cid:7) # d ¼tan(cid:2)1 sin2q3(cid:2)sin2qc 1=2 (1.12) s cosq 3 Several qualitative features are implicit in Equations (1.10) to (1.12): 1. Theevanescentdecaydepth(dforintensityand2dfor electricfield) isthe same foreach incident polarization. 2. The amplitudeof evanescent electric field E andits 1 polarization (i.e., the relative amplitudesof the vectorial components)arestrongfunctions of incidenceangleq . 3 3. GivenaparticularincidentE andq ,thecomponents 3 3 ofE dependontheratio ofrefractiveindicesn and 1 1 n but noton their separatevalues. 3 4. Ans-polincidentlightgivesrisetoapurey-polarized evanescent field. 5. Ap-polincidentlightgivesrisetoamixofanx-and z-polarized evanescent field, while its wavefronts propagatealong the x direction.This distinguishesa p-pol evanescent fieldfromfreelypropagating subcritical p-pol refracted light, whichhas no componentlongitudinaltothedirectionoftravel.The FIGURE1.2 Evanescentintensityatz[0vs.incidenceangleq. 3 longitudinal component (bx)of the p-pol evanescent Thebluelinesareforp-pol,andtheredlinesarefors-pol.Thesolid fieldiszeroat q ¼q but increasesmonotonicallyin linesareforbaresurfaces,andthedashedlinesareforsurfacescoated thesupercritical3rangce q > q . withanintermediatelayerof2.4-lthickness(wherelisthevacuum 3 c wavelength of the incident light). The ordinate axis is in multiples 6. TmheeaniminaggtihnaatrythfeacttworoicaopmppeoanrsenintsEa1rxeb9u0t(cid:3)noouttinofE1z, o1.f3t3h3e4i(nwcaidteern)t,ninte¼ns1i.t4y2j(Ein3ptje2romrejdEi3astje2.fiPlamra,mifeutesresd)a,sasundmend¼are1.n5215¼5 2 3 (glasscoverslip). 5 EVANESCENCEINEXCITATION:TIR completely continuous with their respective supercriti- evanescent field in medium 1. From the shape of the calintensities.Foreachpolarization,boththesubcritical fluorescence vs. q curve (perhaps even spatially 3 andsupercriticalregionsreachtheir(different)maxima resolved in a microscope) the thickness and refractive atthecriticalangle.Forsupercriticalanglesjustaboveq , index and possible lateral heterogeneities of the film c theevanescents-polintensityisfourtimesthes-polinci- may be inferred. This phenomenon, as yet unapplied dentlightintensityjE j2(andthep-polintensityismore in biophysics, may have applications, for example, in 3s than four times jE j2). This discrepancy is not a viola- characterizing a multilayer lipid coating supported on 3p tion of conservation of energy, as the energy in the asurface.Atparticularincidenceanglesq thatproduce 3 evanescent field in medium 1 does not escape away aresonanceinthefilm,theevanescentfieldinmedium1 from the interface and in fact flows back into medium can be enhanced in intensity by at least an order of 3.However,afluorophoreneartheinterfaceinmedium magnitude over what it would bewith no film. 1cantapintothatenergyandwill,infact,decrementthe evanescentintensityslightlybyitspresence,aswoulda fluorophoreinapropagatinglightfield.Intheimmedi- Polarized Excitation TIRF atesubcriticalrange,refractedlightdoesescapethesur- Polarized excitation TIRF is a technique which can faceregion,butthewidthofthebeam(whichisalways uniquely highlight submicroscopic irregularities in the finite) isnarrowedbythegeometryofrefraction,sothe plasma membrane of living cells10 and in supported increase in its intensity is compensated by its smaller lipid bilayers.11,12 For samples with multiple fluoro- cross-sectional area,andenergyisstill conserved. The fluorescence emission intensity vs. q profilehas phores, the effect requires the incorporation of fluoro- 3 phore into the membrane with a high degree of beenusedtodeducetheconcentrationofafluorophore orientationalorder.Therequirementforensembleorien- as a function of distance z from the substrate, because only the lower q angles, with their deeper evanescent tational order is eliminated in viewing singly labeled 3 single molecules rather than membranes with highly field depth, can reach out to fluorophores farther away oriented heavy labeling. Polarized excitation TIR has from theinterface.6,7But, becausetheevanescent inten- sity at z¼0 is itself a strong function of q that been successfully used to determine the orientation of 3 single molecules ofsparsely labeled F-actin.13 dependence must be taken into account when PolarizedexcitationTIRFdependsonthefactthatthe measuring z-dependent concentration profiles near a p-polevanescentfieldisdirectedpredominantlyinthez TIR surface. (As we shall discuss below, the emission direction(i.e.,normaltothesubstrate).NotefromEqua- gatheringefficiencyisalsoafunctionofz,amoresubtle tion (1.10) that E is typically much larger than E for effectthat must alsobe taken into account. 1z 1x all generally accessible supercritical q . If a membrane 3 lying flat upon the substrate is labeled with a fluoro- phore (such as the membrane probe diI-C -(3) or diI) 18 TIR Resonance in Films whose absorption transition dipole moment orients in the plane of the membrane (the xey plane), then a TheIvs.q solidlinecurvesofFigure1.2(forevanes- 3 p-polevanescentfieldwillnotefficientlyexcitethefluo- cence at a bare glass/water interface) become much rophores.But,ifthemembranehasanindentation(even richer if the substrate glass is coated with a thin film of a submicroscopic one), that indented region will not be refractiveindexn (seethedashedlinesinFigure1.2).8.9 2 parallel to the substrate and thereby can be excited by Ifthefilmisgreaterthanaboutl /2inthicknessandits o the p-pol evanescent field. This orientational sensitivity refractive index is intermediate (n > n > n ), the film 3 2 1 can be separated from local fluorophore concentration canactasalossyplanarwaveguide,completewithreso- changes by dividing the p-pol excited image with a nance modes that depend on q . If q is such that light 3 3 s-pol excited image of the same region. propagatesintothefilmfrommedium3butthentotally reflects at the interface with medium 1, the totally reflected light will subsequently partially reflect at the TIR-FRAP, TIR-FCS, and Two-Photon TIRF n :n interface, return to totally reflect again at the n : 3 2 2 n interface (and so on), and thereby set up a pattern Totalinternalreflectionhasbeencombinedwithother 1 of destructive and constructive interference in the film. fluorescence microscopy techniques, and the combina- Because the intensity of the light just inside the film in tions themselves have important uses and nontrivial medium 2 immediately near the n :n interface is theoreticaldescriptions.TIRexcitationcanbecombined 2 1 proportional to the intensity of the evanescent field withfluorescencerecoveryafterphotobleaching(FRAP) just inside medium 1, the resonance-like behavior in or fluorescence correlation spectroscopy (FCS) to the film should be evident in the observed q measure: (1) diffusion coefficients of surface-proximal 3 dependence of fluorescence intensity as excited by the fluorophores, in solution, in model or living cell 6 1. EVANESCENTEXCITATIONANDEMISSION membranes,incellcytoplasm,orevenwithinsubmicro- interference fringe pattern in the evanescent intensity scopic cellularorganelles;14 (2) chemical kinetic rates of (provided theyhavethesame evanescent polarization). binding/unbindingtothesurface;and(3)absolutecon- If the relative azimuthal angle f between the two TIR (cid:3) centrationsoffluorophores(inthecaseofTIR-FCS).The beams is 180 , then the node-to-node spacing s of the basic theory for these somewhat related techniques15 evanescent fringesis given by and a recent review of TIR-FCS applications and prac- l tical experimental protocols16 have been published. s¼ 0 (1.15) 2n sinq Two-photon TIRF17 in principle would cut the evanes- 3 3 cent characteristic distance d in half. But, because the Spacingsisnotdependentupontherefractiveindexn 1 infrared photons used as an excitation source in this of the medium (or cell) and can be smaller than the nonlinear technique have about double the wavelength Raleigh resolution limit of the microscope. These fine of those typically used in standard single-photon TIRF, stripes can be employed in structured illumination,19 that possible advantage is canceled. On the other thereby producing an even finer lateral resolution than hand,TIRFsometimessuffersfromalow-intensitynon- what is standard for that already super-resolution evanescentbackgroundduetoscatteredexcitationlight, technique. and two-photon TIRF gives a distinct advantage to the Interestingandpotentiallyusefulpatternscanbepro- brightest (and nonscattering) evanescent portion of the duced by interfering more than one pair of TIR beams. illumination. By further combining two-photon TIRF TwoorthogonalpairsofintersectingcoherentTIRbeams (cid:3) with TIRF-FRAP, an enhancement of signal can be (i.e.,fourazimuthalanglesspacedat90 )willproducea expected.18 checkerboard pattern in the evanescent intensity. In the limit of an infinite number of azimuthal angles (i.e., two-dimensional plane waves of equal strength all converging to a central spot where the phases are the TIRF Evanescent Field Phase and Interference same), we must integrate evanescent two-dimensional The evanescent field in medium 1 is sinusoidally plane waves propagating along the surface with wave- periodicinthexeyplane(theplaneoftheinterfacesup- vectors k from all azimuthal angles f (see surf portingtheTIR),withaslightphaseshiftrelativetothe Figure1.3A),eachwith an amplitude k given by surf incidentpropagatingplanewaveinmedium3thatisq3 2pn sinq dependent. The evanescent field phase has no z depen- k ¼ 3 3 (1.16) surf l dence, meaning that the wavefronts are perpendicular o to the interface and the field can be represented simply This is the same wavenumber as k in Equation (1.6) 1x asatwo-dimensional waveinxey.Because oftheclose exceptthatherethewavesareconvergingfromalldirec- spacingofwavefrontsintheevanescent field,illumina- tionsinthesurfaceratherthanjustpropagatingalongx. tionbyapairofintersectingandmutuallycoherentTIR The interference among this continuum of waves laser beams can produce a very closely spaced striped produces a small bright central spot of sub-wavelength FIGURE1.3 TIRfrommultipledirections.(A)Schematicviewof12two-dimensionalevanescentplanewavesconvergingtoanin-phase center.Aninfinitenumberofsuchtwo-dimensionalplanewavescreatesaconvergingcircularwave.(B)Thiscircularconvergingpatterncan beproducedexperimentallybyathinannulusofilluminationattheBFPofthemicroscopeobjectiveusedforTIRFexcitation.Thepolarizationat theBFPmustberadialsothattheevanescentfieldsfromeachplanewavecomponentwillbeallpredominatelyinthezdirection(normaltothe TIRsurface)sotheycanmutuallyinterfere.(C)Theevanescentintensitytheoreticallypredictedfromsuchannularilluminationaccordingto Equation(1.17).Thewidthofthecentralmaximumtothefirstminimumisw0.26l,assumingtheannularringattheBFPhasaradiuscorre- o spondingtoNA¼1.45,whichiseasilyaccessiblewitha1.49-NAobjective.
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