Surface-plasmon microscopy with a two-piece solid immersion lens: bright and dark fields

Surface-plasmon microscopy with a two-piece solidimmersion lens: bright and dark fields

Jing Zhang, Mark C. Pitter, Shugang Liu, Chungwah See, and Mike G. Somekh

We report bright-field and dark-field surface-plasmon imaging using a modified solid immersion lens anda commercial objective of moderate NA in the epi configuration. The contrast and resolution are extremelygood, giving well-resolved images of protein monolayers both in air and in water. We also describe atwo-part solid immersion lens that allows the sample to be moved without degrading the image qualityin any observable way. The merits of the two-part lens are discussed and compared to commerciallyavailable microscope objectives. Finally, we introduce a simple Green’s function model that illustrates thekey features of both bright-field and dark-field surface-plasmon imaging. © 2006 Optical Society ofAmerica

OCIS codes: 240.6680, 110.0180, 110.0110.

1. Introduction

Surface-plasmon (SP) microscopy has attracted a con-siderable amount of interest in the past few years be-cause it is extremely sensitive to the properties of thematerials attached to a surface. SP techniques are rou-tinely used for point measurement of submonolayercoatings; these methods find considerable applicationin biological assays such as antigen or antibody bind-ing studies.1 If, however, the potential for using SPs forhigh-resolution microscopy and compact, high-density,label-free biochips is to be realized, it is necessary tocombine high lateral resolution with excellent surfacesensitivity.

SP microscopy has traditionally been performedusing the so-called Kretschmann configuration asshown schematically in Fig. 1. In this arrangement,SPs are excited by oblique illumination through aglass prism. The prism supports the gold layer andalso provides the high refractive index necessary tooptically excite SPs. The conventional method forhigh-resolution SP imaging is to use bright-field re-flection microscopy as shown in Fig. 1. Here the illu-mination beam is incident at or close to the optimumangle for SP excitation, and the reflected light (both

zero and diffracted orders) is imaged through lensesA and B. Note that for bright-field imaging the “zero-order stop” shown in Fig. 1 is not used.

For dark-field imaging, the specularly reflected orzero-order light is excluded and an image is formedwith only diffracted or scattered light. The advan-tages of dark-field imaging are the potential forhigher contrast, which leads to a better sensitivity tosmall changes in the thickness or roughness of ad-herent layers and the improved lateral resolution ob-tained by imaging only the scattered light. Thesefeatures would fulfill the two principal aims of SPmicroscopy.

Dark-field SP microscopy has been implementedin two main ways. The first technique, described byGrigorenko et al.,2 operates by reflection and simplyuses a zero-order stop to block the undiffractedbeam so that the image is formed entirely withscattered light. Images with noticeably differentfeature contrast from the conventional bright-fieldtechnique have been obtained with this method.The second technique is to view the light scatteredinto propagating waves with an objective lens (C inFig. 1) placed in medium ns. This method is sensitiveonly to scattered light because the angle of incidencethat excites surface plasmons is above the criticalangle. In the absence of scattering sample features,the illumination is totally internally reflected or ab-sorbed as plasmons in the gold layer, and there is nopropagating light in medium ns. The two methods forproducing dark-field images have different disadvan-tages. A problem with the reflection dark-field systemusing the zero-order stop is that the collection optics

The authors are with the Applied Optics Group, School of Elec-tronic Engineering, University of Nottingham, Nottingham N672RD, UK. J. Zhang’s e-mail address is [email protected].

Received 22 March 2006; accepted 18 May 2006; posted 14 June2006 (Doc. ID 69012).

0003-6935/06/317977-10$15.00/0© 2006 Optical Society of America

1 November 2006 � Vol. 45, No. 31 � APPLIED OPTICS 7977

is limited to a low NA. This is because the geometryof the optical arrangement causes aberration and re-quires a large working distance; only a small region ofthe oblique prism surface can be kept in focus at ahigh NA. The second method, where light is scatteredinto the upper medium, suffers from a different draw-back. Although it allows good resolution because theNA of the collection optics (C) can be high, the ar-rangement requires the microscope objective and im-aging optics to be located in the upper medium andthis can be disruptive to the experimental system onewishes to monitor. Additionally, simultaneous bright-field SP microscopy is not possible without a separateimaging path. For these reasons, a dark-field systemcapable of incorporating high NA optics, but that alsoleaves the upper half-space free, is necessary for manybiological applications. We describe such a “prismless”high NA dark-field SP microscope system.

2. System Configuration

Figure 2 shows a schematic of the optical system usedin our studies. The key element is an aplanatic solidimmersion lens (SIL) that provides a convenient andpowerful means of increasing the NA of an opticalsystem.3 The enhanced NA afforded by a SIL has beenexploited in several applications, including improvinglateral resolution4 and light-gathering power. Our mo-tivation for using a SIL, however, is to increase therange of accessible incident k vectors to allow SPs to beexcited when even a modest NA microscope objective isused. In our experiments we used a 0.42 NA Mitutoyoobjective with an aplanatic SIL made of S-LaH79 glass(refractive index of n � 1.995 at 0.6328 �m, surfacequality 40–20, sphericity 2 �m) to form a combinedSIL objective lens with a theoretical NA of 1.67. Toexcite SPs in air a �1.05 NA is required. This is wellwithin the aperture of the SIL objective and eveninexpensive oil immersion lenses. The full advan-tages of the SIL objective become apparent when op-erating with samples in an aqueous environment.Here the required NA is �1.44, and this is at orbeyond the range of most commonly available micro-scope objectives. An objective with a NA of 1.65 isavailable from Olympus (Apo 100�, 1.65 NA) andsimilar images to those described in this paper can beobtained. Indeed we have used this objective for aque-ous SP imaging. However, we report the SIL ap-proach because it can potentially provide even higherNAs, should these be required for the excitation ofhigh k-vector wave modes. Moreover, the couplantused with these objectives (diiodomethane) is some-what volatile and renders stable, quantitative, long-term measurements rather difficult. Finally, thecapital costs of such objectives lenses are very high,but more of a problem is the extremely high cost of

Fig. 1. Schematic showing bright-field and dark-field imagingconfigurations in a Kretchmann-based SP microscope.

Fig. 2. Schematic of a solid immersion lens based system and the SIL. (a) System setup. (b) Diagram of a two-piece ball showing the raypaths for aplanatic configuration.

7978 APPLIED OPTICS � Vol. 45, No. 31 � 1 November 2006

the sapphire coverslips, with each costing more than1000 conventional glass coverslips.

The sample is Köhler illuminated with a laser beamthat has been passed through a rotating ground-glassdiffuser. Mask 1 [shown in Fig. 2(a)] placed in a planeconjugate with the back focal plane (BFP) of the SILobjective defines the source, and it is designed so thatit allows only incident angles that generate SPs topass. If all incident angles are used to illuminate thesample, it results in greatly reduced image contrast inthe bright field and renders dark-field operation im-possible. It is worth noting that the BFP or Fourierplane of the combined SIL objective does not corre-spond to the BFP of the microscope objective alone,but may be determined by passing a parallel beamthrough the base of a SIL (before the gold film isevaporated). The imaging arm of the microscope alsocontains a mask (mask 2 in Fig. 2), which is comple-mentary to mask 1. This ensures that only diffractedand scattered light is passed into the imaging system,thus ensuring dark-field operation. There is a secondimaging arm in the system, and this is used to observethe BFP distribution of the SIL objective. This channelprovides a good check of system alignment, the qualityof the sample, and the successful excitation of SPs.

It is necessary to discuss the SIL itself in moredetail. As mentioned above, we use the so-calledaplanatic or Weierstrass configuration in our exper-iment. Here the thickness of the ball is greater thana hemisphere by an amount equal to the ball radiusdivided by the refractive index.

For our application, this configuration has two ma-jor advantages over a simple hemisphere. The first isthat the NA of the microscope objective is increasedby a factor of n2 rather than the factor of n thatapplies to the hemispherical configuration. Thisarises from the additional refraction of the incidentbeam [shown in Fig. 2(b)] as well as the higher re-fractive index of the SIL. This large “amplification” ofthe microscope objective NA means that a relativelylong working distance lens of modest NA can be usedin conjunction with the SIL, thus simplifying align-ment and increasing the working space.

The second advantage of using the aplanatic ar-rangement is that for a given level of combined NAand aberration, the field of view is approximatelytwice that of the hemispherical configuration. Table 1summarizes our simulations of the aberration prop-erties of the two SIL configurations. We can see thatthe dominant aberration for the hemisphere is astig-matism, whereas field curvature is predominant forthe aplanatic arrangement.

The final point we wish to discuss with respect to theSIL concerns the mounting of the sample. In an earlierpublication that describes bright-field SP microscopywith a SIL arrangement,5 the sample was mounteddirectly onto the gold film deposited on the flat surfaceof the ball. This is clearly inconvenient because only avery limited area of the sample around the center ofthe ball can be viewed without introducing unaccept-able aberration. To address this issue, coverslips of

0.6 mm in thickness were specially prepared usingthe same S-LaH79 glass as for the balls. The cover-slips were then mounted on a truncated aplanaticSIL so that the combined thicknesses of the ball andthe coverslip corresponded to the thickness requiredfor aplanatic operation. This is a further advantage ofusing the aplanatic configuration because the greateroverall thickness better lends itself to combining withthe coverslip. To attach the coverslip to the ball it isnecessary to use a high index fluid between the twoelements. The index of the glass used means that nonontoxic matching liquid was available. We thereforeused a small amount of diiodomethane with a refrac-tive index of 1.78. Although this is the immersionfluid used with the commercially available 1.65 NAobjectives, its volatile nature did not present a seri-ous problem here because the fluid was placed be-tween two flat surfaces and was less exposed to theair. It was found that the refractive index mismatchof the fluid and the glass did not result in a substan-tial degradation of optical performance, presumablybecause of the thinness of the liquid layer. Moreover,the NA of the SIL objective was less than the refractiveindex of the liquid. The advantage of using the cover-slip was that it allowed the sample to be moved orremoved easily without misaligning the SIL objective.

3. Results

Figures 3(a) and 3(b) show experimental and simu-lated BFP distributions obtained from a bare goldfilm sample in air with masks 1 and 2 removed. Wenote the familiar dips corresponding to SP excitationfor p-incident polarization. Particularly notable is thegeneral quality of the BFP distribution, indicatingthat the fluid coupled coverslip did not noticeablydegrade the optical performance.

Figures 4(a) and 4(b) show bright- and dark-fieldimages of 5 nm thick stripes of protein deposited on agold-coated coverslip. The width of the protein stripesis 5 �m. Figures 4(c)–4(e) show images of the samesample taken with complementary techniques: con-ventional bright-field microscopy using the SIL mi-croscope (1.6 NA), differential interference contrast(DIC) microscopy (0.6 NA), and tapping mode atomicforce microscopy (AFM). Note that the images ob-

Table 1. Summary of SILs and the Principal Aberrations in DifferentSIL Configurations

Features Hemispheric SIL Aplanatic SIL

System NAeff

increasedn n2

Field of View��machieved (imagedegraded by�2compared todiffraction limit)

60 110

Principal aberration Astigmatism(�10 timeslarger thanthat ofaplanatic SIL)

Field curvature(�n2 timeslarger than thatof hemisphericSIL)


tained with the complementary optical techniquesinvolve illumination from above the sample, whereasthe SP images are taken through the coverslip, whichis clearly a major advantage for biological imaging.The AFM images confirm the thickness of the proteinlayer to be 5 nm, and the contrast in the complemen-tary optical images is rather poor. Both the bright-and dark-field SP images show excellent contrastcompared to the other optical techniques. We notethat the texture of the SP images is different owing toits differing spatial frequency content; for example,as we might expect, the dark-field image is highlysensitive to edges. There are also features that are

visible in the dark-field image that cannot be seen inthe bright-field image; these features are indicated byarrows in Figs. 4(a) and 4(b). This is a region wherethe protein coverage is rather poor; it appears belowthe limit of detection in the bright-field image but canstill be observed in the dark-field image.

The BFP distributions and images shown in Fig. 5demonstrate the large NAs that can be readily ob-tained with the modified SIL objective. Figure 5(a)shows the BFP distribution of gold in air; we can seethat the resonance dip (dashed curve) corresponds toa NA of approximately 1.05. This is well within theaperture of the SIL objective. The corresponding im-

Fig. 3. BFP distribution obtained from plane gold sample using the split ball with air backing; the incident light is horizontally polarized.(a) Experimental distribution with the split ball. (b) Simulated distribution (NA of 1.65 used for simulation).

Fig. 4. Different imaging modalities used to image5 nm protein stripes on a gold surface. (a) Bright-field SPimage of the protein grating. Image width � 90 �m. (b)Dark-field SP image of the protein grating. Image width� 90 �m. (c) Conventional bright-field image of theprotein grating. Image width � 90 �m. (d) Differentialphase image of the protein grating. Image width �70 �m. (e) AFM image of the protein grating. Imagewidth � 35 �m. Images (c), (d), and (e) were obtainedwith the protein facing the imaging head, whereas the SPimages were taken “through” the gold layer.


age of Fig. 5(b) shows high contrast. When the sampleis immersed in water, the NA required for SP excita-tion increases dramatically. This is shown in Fig. 5(c),where SP-induced dips in the BFP distribution cor-respond to a NA of approximately 1.44 and the outeraperture of the SIL objective combination is close to aNA of 1.65. Note that the objective used prior to theSIL has a NA of only 0.42, so larger NAs can bereadily obtained if necessary. Figure 5(d) is the cor-responding image of the protein stripes immersed inwater taken with the modified SIL objective.

4. Simple Model Relating Bright- and Dark-FieldSurface-Plasmon Imaging

Here we present a model that describes the key fea-tures of SP imaging in both bright and dark fields. The

model is based on a simple Green’s function and ex-plains the essential difference between bright- anddark-field imaging. The virtue of the model is that itprovides an intuitive framework that can be used tounderstand the performance of SP and other surfacewave microscopy.

Consider a plane wave incident on a sample asshown in Fig. 6(a). The parallel lines in the incidentmedium n0 represent plane wavefronts propagatingalong the direction indicated by the dashed arrow.We consider that each incident point on the wave-front gives two contributions to the reflected light.The first will be a direct specular reflection at thepoint of incidence, marked “spec” in Fig. 6(a). Thesecond will arise from light that is coupled into SPsand then reradiated back into the incident medium

Fig. 5. BFP distributions and images of the protein grating (5 �m a in width and 5 nm in thickness) in air and water conditions. (a) BFPdistribution of the protein stripes in air backing; the circle indicates a NA of 1.05. (b) Corresponding image of protein stripes (image widthof 70 �m). (c) BFP distribution with aqueous backing, circles indicate a NA of 1.44 (dashed lines) and a NA of 1.65 (dashed-dot lines). (d)Corresponding image of protein stripes (image width 80 �m).


with a characteristic decay length (indicated as “plas”).Note that this second contribution is distributed overthe sample since the plasmon decay length defines anaverage propagation length rather than a specific po-sition of reradiation. The plasmon contribution fromeach point can be considered to be independent of theangle of incidence and propagates in both directionsalong the interface. However, the contribution to theoverall reflection coefficient is sensitive to the angle ofincidence because efficient excitation of SPs relies onthe contributions from each point adding in phase.This, of course, occurs when the component of the kvector parallel to the interface matches that of thesurface wave.

We develop a simplified Green’s function based onan approximate reflection coefficient. It is quite possi-ble to develop a more accurate Green’s function basedon the true reflection coefficient calculated from theFresnel equations. However, the loss of mathematicalsimplicity obscures the essential physics and thereforethe simplification is more useful.

Again consider a plane-wave incident from me-dium n0, whose k vector along the plane of the sampleis kx and is represented as exp�ikxx�. In the spirit ofthe approximations discussed in the preceding para-graph, we consider the reflection coefficient corre-sponding to the specular contribution to be �1. TheSPs are excited on the sample from a point such as x inFig. 6(a) and propagate to point x0 before reradiating.The propagation constant for the SP, kp, is given by

kp � k� � i�kc � ka�. (1)

This propagation constant consists of the real partk� and two imaginary terms that describe the atten-uation of the wave as it propagates. The kc termcorresponds to the attenuation due to SP coupling outinto propagating photons in the medium n0, and ka

represents ohmic (thermal) losses in the metal layer.We can then write the contribution to the field at x0owing to the plane wave incident to the left, ul�x0�, as

ul�x0� � 2kc ��

x0

exp�ikxx�exp ikp�x0 � x�dx. (2)

In physical terms, this means that the contributionfrom x0 arises from a summation of sources along theincident plane wave multiplied by the complex phasefactor arising from propagation from x to x0. The fac-tor of 2kc accounts for the efficiency of coupling andreradiation of the SP. This is analogous to the cou-pling factor used for surface acoustic wave excitationand may be calculated by expanding the reflectioncoefficient around the pole as a Laurent series andevaluating the field contribution by contour integra-tion.6,7

Evaluating this term we obtain an expression forthe field above the sample due to SPs as

up�x0� � 2kc exp�ikxx0�r�kx, kp�, (3)

where

r�kx, kp� �i

�kp � kx��

�kc � ka� � i�k� � kx��kc � ka�2 � �k� � kx�2. (4)

We can see that this term reaches a maximum valuewhen k� � kx. As mentioned earlier, this maximumarises when the sources add in phase along the samplesurface. There will also be a contribution to the totalreradiated field from waves propagating from theright, and this is formed by changing the sign of kp.The size of this contribution will be negligible when k�

is close to kx owing to strong phase cancellation. Thismeans that for plane-wave excitation only those an-gles close to the phase-matching condition need to beconsidered. From the foregoing arguments we seethat the approximate reflection coefficient, R�kx�, cor-responding to SP excitation is given by

R�kx� � �1 � 2kcr�kx, kp�. (5)

Despite the simplifications in the foregoing arguments,the essential features of the reflection coefficient in thepresence of SPs can be obtained. Figure 7 shows theeffect of varying the ratio of kc to ka. We can see thatwhen the value of kc�ka is equal to 1, the reflectivitydips to zero. When the ratio is less than 1, we have“undercoupling,” which for SP excitation correspondsto when the metal layer is thicker than optimal.

Fig. 6. Schematic of the simplified Green’s function model. (a) Plane wavefronts creating a line of point sources. (b) Point source generatedin the sample and scattered at discontinuity.


When the ratio is greater than 1 we have a situationanalogous to a very thin gold layer, the so-called over-coupling case. Reducing the values of both ka and kc

produces a sharper resonance and allows one to ef-fectively model a change in the material properties,analogous to changing from a gold layer to a silverlayer.

From the point of view of microscopy, the merit ofthe model lies in the ability to consider the effect of aninhomogeneous sample, allowing us to determinehow a SP microscope will respond to different fea-tures. For instance, the Green’s function model can bedeveloped to help us understand what happens whenimaging a discontinuity in sample properties. To fullyunderstand the imaging behavior when there is ex-citation over a range of azimuthal angles, we need todevelop a Green’s model for a point source. This willbe necessary to explain why the spatial resolution ofbright-field SP microscopy through a microscope ob-jective is superior to that obtained with prism-basedexcitation. However, the essential differences be-tween bright- and dark-field operation can be readilyexplained with the point source model.

Let us consider the simplest inhomogeneous sam-ple consisting of regions of different SP k vectors kp1and kp2, as shown in Fig. 6(b). This corresponds to thesituation where the propagation of the SP is affectedby, for instance, the presence of an overlayer, as dis-cussed in the preceding experimental results.

The procedure discussed above can be easily ex-tended to account for the presence of two layers. Aplane wave with incident wave vector kx, close to k�,ensures that excitation of a SP propagating from leftto right dominates [see Fig. 6(b)]. This is given by aspecular reflection and three principal surface waveterms, namely, (i) a field generated in region 1 andreradiated in region 1, (ii) a field generated in region1 and reradiated in region 2, and (iii) a field generatedin region 2 and reradiated in region 2. In addition,there is a field generated in region 1 that is reflectedfrom the interface and reradiates back into region 1.

An incident field exp�ikxx� on the sample, depictedin Fig. 6(b), with the discontinuity between regions atx � 0, thus produces a field due to SPs as

ul�x0� � 2kc1 exp ikxx0H�x�r�kx, kp1� � 2�1 � H�x��kc1kc2t12 exp ikp2x0r�kx, kp1�� 4ikc2�1 � H�x��exp i�kx � kp2�

�x0

2 sin�kx � k2�x0

2 r�kx, kp2�

� 2kc1r12r�kx, kp1�exp � ikp1x0, (6)

where H�x� � 1 for x � 0, H(x) � 0 for x � 0, and t12and r12 are the transmission and reflection coeffi-cients for the surface wave at the interface:

t12 �2kp1

kp2 � kp1, r12 �

kp2 � kp1

kp2 � kp1.

Figure 8 summarizes the intensity responses wewould expect from bright-field SP microscopy with awave incident to excite SPs from left to right. Thesolid curve in Fig. 8(a) shows a SP slightly off reso-nance in the left-hand medium. As the wave passesinto the region where the SP is in resonance, thesignal decreases with a characteristic decay lengthrelated to the strength of the coupling and the ohmiclosses. We note that the wave does not quite decay tozero since the case was chosen to match a 45 nm layerof gold, where kc and ka are not precisely equal asshown in the figure caption. The oscillations from theinterface are due to reflections forming standingwaves. These were not observed in our experiments,presumably because the interface is not sufficientlysmooth. In prism-based SP imaging the spatial band-width is insufficient to resolve these features. Thedashed curve shows the situation where the SP is onresonance in the left-hand region and passes to a re-gion where the SP is off resonance with a subsequentincrease in the signal level. Figure 8(b) shows the same

Fig. 7. Reflection coefficients produced with an approximate reflection coefficient: solid curve, ka � 0.006k� � kc; dotted curve,ka � 0.006k� � 2kc; dashed curve, ka � 0.006k� � 0.5kc. (a) Amplitude and (b) phase of reflection coefficient.


situation, except that coupling and attenuation areincreased. Here we see similar phenomena, except thatthe transitions are much more rapid, corresponding toimproved spatial resolution. On the other hand, wenote that the contrast is now lower because the dip inthe reflectance function becomes broader (see Fig. 7).The argument above provides a formal frameworkconsistent with explanations of the trade-off betweenresolution and sensitivity discussed throughout the lit-erature of prism-based SP microscopy (see, for in-stance, Ref. 8). Bright-field images of protein gratingswith 5 �m wide stripes are shown in Fig. 9. Figure9(a) shows the situation where the SPs propagateparallel to the grating, and Fig. 9(b) shows the casewhere the SPs propagate across the grating. We cansee that surface edges are somewhat less sharp in

Fig. 9(b) compared with Fig. 9(a) due to the effectsillustrated in Figs. 7 and 8. The experimental reso-lution with our microscope is considerably better,even when the surface waves are launched across thegrating. This is because surface waves propagate overa range of angles, unlike either the model or thenormal Kretschmann case.

Figure 10 shows the predicted response for dark-field imaging. We note that the width of the responseis independent of the SP propagation length as boththe strong and the weak coupling cases have similarfull width at half-maximum values of approximately1.4 wavelengths. The strong-coupling case that cor-responds to the wave traveling from the on-resonancecondition to the off-resonance condition is not shownbecause it almost overlays the dashed curve. We note

Fig. 8. Surface wave response across an interface on and off resonance for strong and weak coupling for a single incident angle kx.One unit of wavelength is the surface wave wavelength in the faster medium. (a) Weak coupling corresponding to ka � 0.0055k�,kc � 0.0065k�. Solid curve; left-hand medium k� � 1.01kx, right-hand medium k� � kx. Dashed curve, left-hand medium k� � kx, right-handmedium k� � 1.01kx. (b) Strong coupling corresponding to ka � 0.011k�, kc � 0.013k�. Solid curve, left-hand medium k� � 1.01kx, right-handmedium k� � kx. Dashed curve, left-hand medium k� � kx, right-hand medium k� � 1.01kx.

Fig. 9. Experimental SP images of the protein grating. The grating structure is parallel and perpendicular to the dominant SPpropagation direction. (Image width of 90 �m). (a) Dominant SP direction parallel to the grating. (b) Dominant SP direction perpendicularto the grating.


that, in general, the dark-field signal strength isgreatly reduced compared to the bright field, al-though the contrast is higher. We will discuss thisdifference in signal levels in more detail in the nextsection.

5. Discussion and Conclusion

We have presented a method for obtaining dark-fieldSP images in a modified, solid immersion lens cou-pled microscope. The images presented show radi-cally different features from the bright-field images,and we believe that it is important to regard thedark-field imaging mode as complementary to bright-field SP imaging, rather than replacing it.

We have presented a simplified Green’s functionmodel that formalizes the accepted view of image for-mation in the Kretschmann configuration SP micros-copy. That is, as the SP propagates from one region toanother there is a transition region as the signal levelchanges from a value appropriate for one region to avalue appropriate for the other. This transition regionis comparable to the decay length of the SP. This effectand the prism optics limit the lateral resolution ofconventional SP microscopy to a few micrometers. Asimilar mechanism still operates when we image withan objective lens although the range of incident azi-muthal angles is greater and the lateral resolution isconsiderably better because the SPs propagate inmany directions rather than in just one, as illustratedin the images of Fig. 9, The improved resolution can beexplained by extending the model described here, andsuch a development will be presented in future publi-cations. The other mechanism that operates in SP mi-croscopy is the scattering of SPs into propagating light.

This can be incorporated into the Green’s functionmodel and is observed as a discontinuity of the field orits gradient at the scattering interface. The lateralresolution associated with this mechanism is deter-mined primarily by the diffraction limit of the detec-tion optics rather than the propagation length of theSPs. Dark-field SP microscopy may therefore bethought to eliminate those spatial frequencies wherethe first mechanism predominates. It is clear fromboth experiment and modeling that dark-field imagescontain much lower light levels, although we believethat they are still sufficient to observe submonoloayerchanges in a time scale that is fast compared to typ-ical biological reactions. Dark-field methods shouldbe particularly useful when the bright-field methodsare limited by contrast and for small features wherethe signal level does not have sufficient propagationdistance to reach its final value.

It is worth pointing out a feature not brought out inour model: a large field enhancement corresponds toa long SP propagation length, and therefore the con-ditions that make SP microscopy more sensitive willalso be optimal in dark-field SP microscopy. The greatadvantage of a dark field, however, is that there is noassociated compromise in lateral resolution as shownin Fig. 10.

We now briefly discuss the issues of signal leveland contrast. The results we have presented showthat dark-field image contrast is higher than bright-field image contrast. A benefit of this is demonstratedby the observation of features in Fig. 4(b) that are notvisible in Fig. 4(a). On the other hand, the signallevels obtained with the dark-field configuration wereconsiderably lower. The bright-field images were ob-tained in 30 ms on a high-resolution cooled CCDcamera, whereas the dark-field images required in-tegration for 5 s using the same camera. There is aneed for a great deal of optimization of both bright-and dark-field imaging before the ultimate limits ofsensitivity can be established. In bright-field micros-copy, more precise control of the incident angles mayprovide better contrast without seriously degradingspatial resolution. In addition, it is likely that theoptimum substrates for dark-field imaging may notbe metals supporting SPs, as used here, but multi-layer dielectric structures supporting surface wavessuch as those described in Ref. 9. The reason is thatthe roughness of the metal film scatters light, whichadds background to the dark-field signal. It is consid-erably easier to prepare very smooth dielectric layersthan metal layers, so the obtainable contrast ispotentially better. Interestingly, the field enhance-ments from dielectric surface waves can be equal to oreven greater than those obtained with surface plas-mons, which is ideal for dark-field applications. Ingeneral, however, despite the large field enhance-ment, the position of the resonance angle is much lesssensitive to environment. These layers are thereforegenerally less suitable for bright-field applicationswhere the change of resonance conditions is the mainmeasurand, but they are ideal for dark-field applica-tions where strong scattering is required and sample-

Fig. 10. Dark-field surface wave response across an interface onand off resonance for strong and weak coupling for a single incidentangle kx. One unit of wavelength is the surface wave wavelength inthe faster medium. Solid curve, weak coupling corresponding toka � 0.0055k�, kc � 0.0065k�, left-hand medium k� � 1.01kx, right-hand medium k� � kx. Dashed curve, weak coupling left-handmedium k� � kx, right-hand medium k� � 1.01kx. Dotted curve,strong coupling, ka � 0.011k�, kc � 0.013k�, left-hand mediumk� � 1.01kx, right-hand medium k� � kx.


induced variations in the resonant k vector are, ifanything, undesirable.

In summary, we have presented a dark-field,epi-microscope configuration capable of detectingsubmonolayers with micrometer lateral resolution to-gether with a simplified but general model to describethe observed performance. We have also demonstratedthat splitting the SIL into a coverslip and truncatedsphere provides a practical solution for imaging biolog-ical samples in both bright and dark fields.

We acknowledge the support of the Engineeringand Physical Sciences Research Council and thank M.Denyer and A. J. M. Mahadi of Bradford University forpreparing the protein samples. J. Zhang thanks theUniversity of Nottingham for financial support. M. C.Pitter thanks the UK Research Councils for funding anacademic fellowship in functional imaging.

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Surface-plasmon microscopy with a two-piece solid immersion lens: bright and dark fields

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Transcript of Surface-plasmon microscopy with a two-piece solid immersion lens: bright and dark fields