Midinfrared surface-plasmon resonance: A novel biophysical toolfor studying living cells

M. Golosovsky,1,a� V. Lirtsman,1 V. Yashunsky,1 D. Davidov,1 and B. Aroeti21The Racah Institute of Physics, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel2Department of Cell and Animal Biology, The Alexander Silberman Institute of Life Sciences,The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

�Received 2 April 2008; accepted 20 June 2008; published online 19 May 2009�

We discuss the surface-plasmon resonance �SPR� technique based on Fourier transform infrared�FTIR� spectrometry. We explore the potential of the infrared surface plasmon technique forbiological studies in aqueous solutions and compare it with the conventional surface plasmontechnique operating in the visible range. We demonstrate that the sensitivity of the SPR techniquein the infrared range is not lower and in fact is even higher. We show several examples of applyingFTIR-SPR for biological studies: �i� monitoring D-glucose concentration in solution and �ii�measuring D-glucose uptake by erythrocytes in suspension. We emphasize the advantages ofinfrared SPR for studying living cell cultures and show how this technique can be used forcharacterization of �i� cholesterol penetration into plasma membrane and �ii� transferrin-inducedclathrin-mediated endocytosis. © 2009 American Institute of Physics. �DOI: 10.1063/1.3116143�

I. GENERAL INTRODUCTION

Surface plasmon resonance �SPR� is the resonant excita-tion of the surface electromagnetic wave propagating at themetal-dielectric interface.1–3 This technique has become animportant research tool in biophysics. The interest in it hasbeen spurred by its potential for biosensing, the appearanceof commercial instruments, and the general interest inplasmonics.4 The SPR technique measures the refractive in-dex or optical absorption with high sensitivity and is particu-larly advantageous for biosensing since �i� it is a label-freemethod5 and �ii� it can monitor the kinetics of biologicalprocesses in real time. Several overviews summarize SPRapplications in biology and biochemistry.6–14 In particular,SPR in the visible range has been used to studymembranes15–22 and cell cultures.23–26 The SPR techniquehas been used in several modalities such as SPR-enhancedfluorescence,27 SPR imaging,3,28 and SPR spectroscopy.29–32

The majority of the SPR applications use laser sourcesoperating in the visible range. Recently, several SPR tech-niques operating in the near-infrared �NIR� rangeappeared,33–38 some of which use a Fourier transform IR�FTIR� spectrometer as a light source.39–42 In contrast withthe conventional SPR technique that operates with a lasersource and employs angular interrogation, the FTIR-SPRtechnique operates with a continuum wavelength source andemploys wavelength interrogation. So far, FTIR-SPR tech-niques utilized glass-based optics that limited its operation tothe NIR range. We extended the FTIR-SPR technique to themid-IR range using a ZnS prism.43,44 We found that newopportunities are offered by this technique.

• High sensitivity. Since conductive losses in the IRrange are lower than those in the visible range, the IR

SPR can be more sensitive than its visible range coun-terpart.

• Penetration depth of the surface plasmon �SP� in thevisible range is very short. This is beneficial for study-ing very thin layers, but detrimental for studying cellsand cell cultures. The IR SP penetrates much deeperand is more appropriate for studying cells.

• Fast multiwavelength measurements. The ability to de-tect SPR at varying wavelengths and/or varying anglesallows “tuning” the SPR to any desired spectral rangein order to achieve high sensitivity and appropriatepenetration depth.

• Spectroscopy. Since many biomolecules have specificabsorption bands in the IR range �“fingerprints”�, per-forming multiwavelength SPR measurements in thespectral range of fingerprints, in principle, allows thesebiomolecules to be identified selectively.

Next, we will analyze the SPR in the IR and identify thoseniches where it is especially useful for biosensing. There-after, we will discuss several FTIR-SPR applications devel-oped in our group.

II. THEORETICAL INTRODUCTION

A. Surface plasmon definition

SP is an electromagnetic wave Esp=E0e−ikxx−kzz thatpropagates along the metal-dielectric interface and exponen-tially decays in the perpendicular direction.1–3 The SP disper-sion relations in the dielectric and in the metal are corre-spondingly

kx2 − kzd

2 = �dk02, �1�

kx2 − kzm

2 = �mk02. �2�

Here, k0 is the wave vector in free space and �m and �d arethe complex dielectric permittivities of the metal and of thea�Electronic mail: golos@vms.huji.ac.il.

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dielectric, correspondingly, where �m� �0. When the SPpropagates along the interface between two unbounded me-dia �Fig. 1�, its wave vector is

kx� + ikx� = k0� �m�d

�m + �d�1/2

. �3�

SP is a TM wave. The ratio of the longitudinal and the trans-verse field components is �Ez /Ex�=��d / ��m� in the dielectricmedium and �Ez /Ex�=���m� /�d in the metal.

B. Surface plasmon excitation: Kretschmann’sgeometry

Because of the limited penetration of the SP wave intothe dielectric medium, it is widely used in monitoring therefractive index of thin dielectric layers. The most widelyused Kretschmann excitation geometry includes a high-refractive-index prism, a thin metal film, and a dielectric me-dium in contact with this film �Fig. 2�. The reflectivity as afunction of the incident angle �or wavelength� exhibits asharp minimum at a certain angle �sp corresponding to theSP excitation.6 By measuring the resonant angle/wavelength,one can determine the refractive index of the dielectric me-dium with high accuracy. The sensitivity of this technique islimited by the angular width of the SPR, ��sp. Next, we willdevelop compact expressions for the SPR angle and width,�sp, ��sp

The resonance condition is

kx = kp sin �sp, �4�

where np is the refractive index and kp=npk0 is the wavevector in the prism. Equations �3� and �4� yield the resonantangle

sin �sp =1

np� �m�d

�m + �d�1/2

. �5�

This angle always exceeds the critical angle

sin �cr =�d

1/2

np. �6�

To recast these expressions using an external angle �see Fig.2�, we apply Snell’s law and obtain

sin��ext − �p� = np sin��sp − �p� , �7�

where �sp is the angle at the base of the prism �Fig. 2�.Figure 3 compares the SPR in the visible ��=0.633 �m� andin the IR ��=1.4 �m� range. The SPR appears as a pro-nounced reflectivity dip. In the visible range, this dip is wideand appears well above the critical angle, whereas in the IRrange, this dip is very narrow and is very close to the criticalangle.

The depth of the SPR in Kretschmann geometry is finetuned by the careful choice of the metal film thickness, d.Note that the SP propagating along a thin metal film is not atrue surface wave but rather a leaky wave that radiates aconsiderable part of its energy back into the prism.1,45 Forthe optimal metal film thickness, the reflectivity at the reso-nant angle achieves its minimum value and approaches zero�the so-called critical coupling regime�, whereas radiation

Dielectric,εd′ > 0

SP waveMetal,εm′ < 0

x

z

δzm

δzd Lx

FIG. 1. �Color online� Schematic of the SP propagating at the metal-dielectric interface. �zm and �zd are penetration depths into metal and dielec-tric media, correspondingly. Lx is the lateral propagation length that is de-termined by conductive, dielectric, and radiation losses.

θsp

PrismMetalDielectric

k p

k 0

kx

θextDetectorLight source

d

θp

FIG. 2. �Color online� Kretschmann geometry of the SP excitation in theattenuated total reflection regime using a high-refractive-index prism and athin metal film. k0 and kp are wave vectors in free space and in the prism. kx

is the SP wave vector. �sp is the angle of incidence at the prism/metal/dielectric interface that corresponds to the resonant excitation of the SP. �p

is the angle at the base of the prism and �ext is the external angle that isdetermined by �sp and by the prism material and shape. Upon proper choiceof the metal film thickness d, the resonance reflectivity approaches zero andthe off-resonance reflectivity approaches unity.

0

0.2

0.4

0.6

0.8

1

35 40

Ref

lect

ivit

y

Θsp

(deg.)

λ=1.4 µm λ=0.633 µm

Θcrit

FIG. 3. �Color online� SPR at the Au/air interface. The SPR in the visiblerange was excited using a He–Ne laser and a 60° SF-10 prism coated with a47-nm-thick Au film. The SPR in the IR range was excited using an FTIRspectrometer and an SF-11 right-angle prism coated with a 30.4-nm-thickAu film. Note the wide SPR in the visible range ��=0.633 �m� and thesharp resonance in the IR ��=1.4 �m�. The SPR in the visible range is welldisplaced with respect to the critical angle whereas the SPR in the IR rangeis very close to the critical angle.

102036-2 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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losses are equal to intrinsic losses: �m� ,�d�. The radiation af-fects the SP dispersion relation �Eq. �3�� as well. The reso-nant angle �Eq. �5�� is slightly shifted by 1°–1.5°, whereas kx�is changed more significantly; in the critical coupling regimeit increases by a factor of 2 with respect to that given by Eq.�3�.45

C. IR surface plasmon resonance propagating alonga thin metal film: Modeling

Since we focus on the biological applications of theSPR, we will next consider only water-based samples. Wewill assume that the metal film is composed of gold and thatits thickness is optimal, i.e., it corresponds to the criticalcoupling regime. Since the dielectric constant of gold at IRfrequencies is very high, �m� ��d1, conductor losses aresmall, �m� /�m� 1. If the dielectric losses are also small, then�d� /�d�1, then kx��kx�. Under these conditions, we obtainuseful expressions for the real and imaginary parts of the SPwave vector

kx� k0� �m� �d�

�m� + �d��1/2

�8�

kx� k0� �m� �d�

�m� + �d��3/2� �d�

�d�2 +

�m�

�m�2� �9�

kzm = k0� �m2

− �m − �d�1/2

�10�

kzd = k0� �d2

− �m − �d�1/2

�11�

�Note that �m� �0.� The SP penetration depth into the dielec-tric is

�zd =1

2kzd=

�0

4�− �m� − �d�

�d�2 �1/2

�12�

The SP propagation length along the metal-dielectric inter-face is1,45

Lx =1

2kx�=

�0

4� �m� + �d�

�m� �d��3/2� �d�

�d�2 +

�m�

�m�2�−1

�13�

Since the dielectric constant of the metal in the IR range isvery high, ��m� ��1, then �m� /�m�

21 �this is valid even if theloss tangent of the metal is high, �m� / ��m� �1�. Therefore, theconductor loss contribution to Lx is negligible and is deter-mined mainly by the dielectric loss.

To estimate the SPR width ��sp we noted that Eq. �4�yields �kx=cos �sp��sp. We replaced there �kx by kx� andfound

��sp = Akx�

kp cos �sp, �14�

where A is a numerical constant that depends on how theSPR width is defined �full width at half maximum or differ-ently�. Using Eqs. �7� and �14�, we obtain

��ext = Acos��p − �sp�

cos��p − �ext�cos �sp

kx�

k0. �15�

For realistic parameters of the prism and the sample �np

=2–2.3,�p=45° ,nd=1.25–1.45�, the angular dependenceof the prefactor, cos��p−�sp� /cos��p−�ext�cos �sp, is veryweak and can be replaced by a numerical constant. Using Eq.�9�, we recast Eq. �15� as follows:

��ext A�d3/2� �m�

�m�2 +

�d�

�d�2� , �16�

where A absorbs all numerical constants. A comparison ofthe exact numerical calculations for the ZnS prism and water

as a dielectric medium yields A=1.85. An approximate ex-pression for the SPR width is

�� = ���ext2 + ��div

2 , �17�

where ��div is the incident beam divergence. Although theSPR width can be calculated exactly using Fresnel reflectiv-ity formulae, Eqs. �16� and �17� are more amenable to analy-sis.

The SPR width is a crucial factor that determines thesensitivity of the SPR technique. The latter is usually6,45 de-fined as �R /�n, where R is the reflectivity and n=�d

1/2. Afterrecasting this expression using an angular variable, we obtainSbulk= ��R /��ext����ext /�n�. The maximum sensitivity isachieved at some angle that corresponds to the sloping edgeof the SPR, where reflectivity sharply drops from unity toalmost zero. Hence

Sbulk 1

��

��ext

�n. �18�

The first factor here is determined by the SPR width �Eqs.�15� and �17��, whereas the second factor ��ext /�n is asmooth function of the refractive index derived from Eqs. �5�and �7�.

To characterize thin dielectric layers with a thicknesssmaller than the SP penetration depth �zd, Eq. �12�, the SPRsensitivity is defined as follows:45

Slayer =Sbulk

�zd. �19�

D. IR surface plasmon resonance at the ZnS/Au/waterinterface: Practical aspects

We will now consider practical aspects of the IR SPR inthe water-based samples such as biological specimens. Al-though SPR at IR frequencies can be excited in manymetals,46 the requirement of biocompatibility leaves gold asthe only choice. For the near IR, one can use BK-7 or SF-11glass prisms, whereas for mid-IR, one can use ZnS or ZnSeprisms.

The range of wavelengths/angles where SPR can be ex-cited is determined by the dielectric losses in water and bythe conductive losses in the gold film. Figure 4 shows dielec-tric losses �upper panel� and the refractive index of water�lower panel�. It is clear that SPR at the Au/water interface

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can be excited only in several “allowed” spectral windowswhere water absorption is sufficiently low. Figure 4 showsthe SPR angle versus the wavelength, as predicted by Eqs.�5� and �7� �lower panel, right y-axis�. This dependence mim-ics the refractive index of water. Note that in the angularinterval, �ext=20° –25°, for each angle there are two SPRsat different wavelengths.

Figure 5 shows SP propagation length Lx as predicted byEq. �13�. This length should be compared to the SP wave-length, �sp=2 /kx�. In the “allowed” spectral ranges, the SPattenuation is low and SP propagates to long distances,Lx /�sp�1. In the “forbidden” spectral ranges, the SPR isoverdamped and the SP wave quickly attenuates, yieldingLx /�sp�1.

In the context of sensing biological cell cultures, it isuseful to compare the SP propagation length Lx to the lateralsize of the cell, dcell10–20 �m. In the spectral ranges ��1.2 �m and ��4.5 �m, the SP propagation length issmall, Lx�dcell, and reflectivity in the SPR regime is the sumof the contributions of individual cells. In the spectral ranges�sp=1.2–2.6 �m and �sp4 �m, the SP propagationlength is long, Lx�dcell; hence, the SP wave senses an “ef-fective medium” consisting of cells and gaps between thecells.

Figure 6 shows the SP penetration depth into a water-based dielectric medium. In the visible and NIR ranges, it isexceedingly small, �z�0.24 �m. In the context of cell cul-

tures grown on gold, such a small penetration depth is ad-vantageous for studying the cell membrane and cell adhe-sion. However, a small penetration depth precludes studyingthe cell interior. In the spectral range �=2–2.5 �m, the SP

10-5

10-4

10-3

10-2

10-1

(ε"/ε') w

ater

10 5 4 3 2 1Wavelength (µm)

1

1

1.1

1.2

1.3

1.4

0

10

20

30

0 2000 4000 6000 8000 1 104

nw

ater

Θex

t(deg

.)

Wavenumber (cm-1)

FIG. 4. �Color online� Upper panel: loss tangent of water. The colored areasindicate spectral windows where water absorption is sufficiently low and theSP can be excited. Lower panel: the continuous blue line shows the refrac-tive index of water. Purple crosses denote the SPR angle �ext for the ZnS/Au/water interface, as given by Eqs. �5� and �7� �right y-axis�. Red circlesindicate our experimentally found angles for a 18-nm-thick Au film. Thewavelength dependence of the SPR angle mimics the water refractive index.

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

Lx

Pro

pag

atio

nle

ng

th,L

x(µ

m)

lateral cell size

ΙΙ

λsp

Wavelength (µm)

Ι

sing

lece

llsce

llla

yer

Au/water

FIG. 5. �Color online� The open circles denote SP propagation length Lx atthe ZnS/Au/water interface, as given by Eq. �13� for the optimal Au filmthickness. The dash-dotted blue line denotes SP wavelength �sp. The coloredareas indicate spectral windows where SPR can be excited �see Fig. 4�. Inthe “allowed” spectral windows �0.5–3 �m and 3.3–6 �m� Lx��sp,whereas in the “forbidden” windows �3–3.3 �m and above 6 �m�, Lx

��sp. The dashed line denotes lateral cell size. When SP propagates throughthe cell monolayer grown on gold, it senses either single cells �spectralwindows of 0.5–1 �m and 4.5–6 �m, see Fig. 10�e�� or some effectivemedium consisting of cells and growth solution �spectral windows of1–2.7 �m and 3.5–4.5 �m�.

0.01

0.1

1

10

100

1 10

100010000

Wavelength (µm)

Pen

etra

tio

nd

epth

,δzd

(µm

)

membrane thickness

cell height

Wavenumber (cm-1)

Au/water

FIG. 6. �Color online� SP penetration depth into biolayer �aqueous solution�as defined by Eq. �12�. The colored areas show spectral windows where SPRat the Au-water interface can be excited. In studying cell monolayers, it isimportant to compare penetration depth �open circles�, cell height, andmembrane thickness �dashed lines�. Penetration of the short-wavelength SPinto the cells is strongly limited; hence, it is mostly sensitive to eventsrelated to the cell membrane. The long-wavelength SP penetrates deeper intocells and senses the whole cell volume.

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penetration depth is �zd=1–2 �m. Hence, the SP wave pen-etrates deep enough to study events in the cell cytoplasm butnot in the nucleus. For the wavelength range �=4–6 �m,the penetration depth is comparable to the cell size; hence,the whole cell volume is accessible for the SP.

Figure 7 shows the SPR width versus wavelength, asgiven by Eqs. �16� and �17�. The exact calculation, using theFresnel reflectivity formulae, yields almost the same results.Note that in the NIR range ���1.3 �m�, where water opti-cal absorption is negligible, the SPR width is determined byconductor losses. On the other hand, at ��2.5 �m, conduc-tor losses are negligible and the SPR width is determined bywater absorption. In the intermediate range, �=1.3–2.5 �m, the SPR width achieves its minimum valueof 0.3°, whereas the contributions of water absorption andconductor losses are comparable. However, to take full ad-vantage of such a narrow resonance, the incident beamshould be very well collimated, otherwise the SPR width inthis spectral range would be limited by beam divergence.

The inset in Fig. 7 shows the optimal gold film thicknessd required to achieve critical coupling. d decreases with in-creasing wavelength, in such a way that the operation inmid-IR requires 10–12-nm-thick gold films; this approachesthe practical limit at which continuous gold films can befabricated. When the film thickness deviates from its optimalvalue, the SPR is excited, although the SPR minimum is not

so deep.1 The symbols in Fig. 7 show our experimental re-sults achieved in two experiments with a 12-nm-thick goldfilm that was optimized for the operation at �=5 �m. At��3 �m, we observe good correspondence between the ap-proximate model and experimental SPR values, whereas at��2.5 �m, the experimental SPR is considerably broad-ened due to beam divergence and deviation of the film thick-ness from the optimal value corresponding to this wave-length.

Figure 8 shows the bulk sensitivity of the SPR techniqueestimated according to Eq. �18�. The bulk sensitivity in theIR range is considerably higher than that in the visible range.In the range �=0.6–2.5 �m, the sensitivity is more or lessconstant, whereas in the long-wavelength window, �=3.3–5 �m, it is lower but is still comparable to that in thevisible range. This means that both these spectral windowsmay be used for SPR spectroscopy.

Figure 9 shows the thin layer sensitivity. The maximumsensitivity is achieved in NIR at �=800 nm, whereas thesensitivity in the mid-IR is lower. Hence, the NIR range isespecially advantageous for studying thin dielectric films ongold substrate.

E. Surface plasmon propagation in biologicalsamples

We consider several regimes in which the FTIR-SPRtechnique can be used to study biological objects. These ob-jects �cell membrane, single cell, cell culture, etc.� have dif-ferent sizes and the SP propagation there is also different.

0

1

2

3

4

5

6

7

0 5000 1 104 1.5 104

∆Θ

ext(d

eg.)

Wavenumber (cm-1)

∆Θdiv

=1o

∆Θdiv

=0

Au Au/water

10 5 4 3 2 1 0.70.9 0.8Wavelength (µm)

0

10

20

30

40

50

60

0 5000 1 104 1.5 104

do

pti

mal

(nm

)

ν (cm-1)

Au/water

FIG. 7. �Color online� SPR width for the ZnS/Au/water interface. The linesshow SPR width, as determined by the absorption in water and in Au and bythe coupling �radiation� losses, as predicted by Eq. �16�. The continuous redline denotes zero beam divergence and the dash-dotted blue line denotes abeam divergence of 1°. The dashed red line indicates the prediction of Eq.�14�, assuming negligible losses in water. Blue circles indicate our experi-mental results obtained with a 14-nm-thick Au film. The beam divergencewas ��div=0.8°. In all calculations, we assumed that the Au film thicknessis optimal, i.e., it satisfies the critical coupling condition at a given wave-length. The inset shows the optimal Au film thickness that was determinedfrom numerical simulations based on Fresnel reflectivity formulae.

0

50

100

150

200

250

0 5000 1 104 1.5 104

Sb

ulk

(RIU

-1)

∆Θdiv

=0o

∆Θdiv

=1o

∆Θdiv

=0.5o

Au/water

10 5 4 3 2 1 0.70.9 0.8

Wavelength (µm)

Wavenumber (cm-1)

FIG. 8. �Color online� The open circles denote the calculated sensitivity ofthe SPR technique, Sbulk=�R /�n, for different beam divergences, as definedby Eq. �18� and Fig. 4. The lines are the guides to the eye. �Our calculationdoes not extend into spectral region between 7670 and 9670 cm−1 owing touncertainty in the optical parameters of Au arising from the mismatch be-tween two data sets �Ref. 50�.� Sensitivity in mid-IR is higher than that inthe visible range.

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Indeed, the SP propagation depends on the relation betweenthe spatial extent of the SP wave and the characteristic sizeof the sample. Hence, by selecting the proper wavelength,the SPR can be made especially sensitive to the processesoccurring at a targeted spatial scale. Figure 10 illustrates sev-eral regimes of SP sensing.

• �Figs. 10�a� and 10�b�� Thick and macroscopically uni-form biolayers. Here, SP probes the bulk refractiveindex of the biolayer. This can be used for monitoringbiologically important molecules in solutions, for ex-ample, glucose. An interesting modification is a sus-pension of floating cells, for example, erythrocytes.

• �Fig. 10�c�� A very thin biolayer attached to the goldfilm, for example, a membrane, or biomolecular rec-ognition layer with a thickness of only 1–5 nm. Al-though the SP penetration depth usually exceeds thisthickness, the short-wavelength SP is sensitive enoughto characterize them.

• �Fig. 10�d�� A moderately thick biolayer with thicknesscomparable to the wavelength. Here, in addition to theSP wave, additional waveguide modes can appear.These can be useful for depth-resolved biosensing andfor probing cell adhesion and cell-cell attachment.

• �Figs. 10�e� and 10�f�� Cell cultures grown directly onmetal. Here, depending on the propagation length, theSP wave can probe either individual cells or the celllayer as a whole.

III. EXPERIMENTAL

Next, we will present several biological applications ofthe FTIR-SPR technique. Figure 11 shows our experimental

setup based on the Bruker Equinox 55 FTIR spectrometer.The light source is a tungsten lamp equipped with the KBrbeam splitter. The IR beam is emitted from the external portof the spectrometer and passes through a collimator consist-ing of a pinhole mounted at the common focal plane of twoAu-coated off-axis parabolic mirrors with a focal length of76.2 and 25.4 mm, correspondingly. For the 1-mm-diameterpinhole, the beam diameter is 3–4 mm and the beam diver-gence is ��div=0.8°. The collimated beam passes throughthe grid polarizer �Specac, Ltd.� and is reflected from theright-angle ZnS prism �ISP Optics, Inc.� mounted on a �-2�rotating table. Another parabolic mirror focuses the reflectedbeam on the liquid-nitrogen-cooled HgCdTe �MCT� detector.A temperature-stabilized flow chamber �with a volume of 0.5ml� is in contact with the gold-coated base of the prism. Thegold film thickness is chosen according to the targeted wave-length �Fig. 7, inset�.

To operate this setup, we first choose the desired incidentangle. Then we mount the sample and measure the reflectiv-ity spectrum for the s-polarized beam. This spectrum is usedas a background for further measurements. Then we measurethe reflectivity spectrum for the p-polarized beam. Normally,this is done with 4 cm−1 resolution and 16 scan averaging.

0

200

400

600

800

1000

0 5000 1 104 1.5 104

sensitivity-layer-intrinsic

sens-layer-div0.5

sens-layer-div1

Sla

yer(µ

m-1)

∆Θdiv

= 0.5o

∆Θdiv

= 1o

∆Θdiv

= 0o

Au/water

Wavenumber (cm-1)

10 5 4 3 2 1 0.70.9 0.8

Wavelength (µm)

FIG. 9. �Color online� Sensitivity of the SPR technique to a thin layer withthickness much smaller than the SP penetration depth, as defined by Eq.�19�. The number at each curve indicates beam divergence. The maximumsensitivity is achieved for �0.8 �m.

SPbiolayer

metal

solution

SPbiolayer

metal

solution

SP

metal

erythrocytesuspension

(a) (b)

(d)(c)

(e) (f)

Lx

metal

cell monolayerSP

solution

Lx

metal

SP

solution

cell monolayer

SP

solution

TE0TE2

biolayer

metal

FIG. 10. �Color online� SP propagation in biological samples. �a� Thickbiolayer �solution or bulk sample�. SP decays within this layer and is notsensitive to anything beyond it. �b� Cell suspension, for example, erythro-cytes. SP partially or fully penetrates into cells that are close to the metal. SPcan be used to study these cells provided the SP penetration depth is com-parable to the cell size. �c� A very thin biolayer in contact with metal �mem-brane, adsorbed molecules�. To study such thin layers, the SP penetrationdepth should be short enough. �d� A biolayer of intermediate thickness incontact with metal. Here, in addition to the exponentially decaying SP wave,the guided modes can be excited as well. These waveguide modes havesinusoidal field distribution inside the layer and may be advantageous forlayer characterization. �e� Cell culture grown on metal. The SP propagationlength is smaller than the lateral cell size. The reflectivity in the SPR regimerepresents a sum of the contributions of individual cells. �f� SP propagationlength exceeds lateral cell size. The SP probes an “effective medium” con-sisting of cells and extracellular space.

102036-6 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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A. Solutions

1. Glucose concentration in water

We used the FTIR-SPR technique for precisely measur-ing the refractive index of solutions. Our motivation was tomonitor physiologically important glucose concentrations inwater and in human plasma.43 Similar studies have been per-formed earlier using SPR in the visible range.47–49 Figure 12shows the reflectivity spectra of pure water and of 1%D-glucose solution measured using our setup. The spectralrange around 4000 cm−1 corresponds to the short-wavelength SPR and the small peak at 2000 cm−1 corre-sponds to the long-wavelength SPR. Adding glucose affectsthe whole reflectivity spectrum, in such a way that the SPRresonance is redshifted. The maximal reflectivity change oc-curs at 4600 cm−1. The inset shows that the reflectivity at4600 cm−1 linearly increases with increasing glucose con-centration. The slope of this linear dependence yields sensi-tivity, Sbulk=�R /�n=75 RIU−1. Taking into account the ac-tual beam divergence of 0.8°, the experimental sensitivity isconsistent with the approximate expression given by Eq. �18��see also Fig. 8�.

2. Glucose uptake by erythrocytes

Here we studied glucose uptake by erythrocyte suspen-sion in the PBS medium.51 Briefly, fresh human red bloodcells �RBCs� were washed four times in PBS by centrifuga-tion. The supernatant and buffy coat cells were discarded andRBCs were resuspended in glucose-free PBS to yield ce

=5% v /v. For complete glucose depletion, cells were incu-bated for 60 min at 22 °C in PBS. Then, RBCs were centri-fuged and resuspended in fresh PBS containing the requiredconcentrations of D-glucose supplemented with L-glucose to

keep the total glucose concentration �osmolarity� equal to 20mM. The SPR measurements were performed at 22 °C toslow down cellular glycolysis.

Our measurements were performed using a ZnS prismcoated with a 12 nm thick Au film. We chose the wavelength�=4 �m at which the SP penetration depth, �zd=4.5 �m�Fig. 6�, is comparable to the typical erythrocyte diameter.Although the sensitivity at this wavelength is low �Fig. 8�,the use of such a long wavelength here is mandatory in orderto sense the processes inside erythrocytes. The average dis-tance between the erythrocytes is Dece

1/3=17 �m. The SPpropagation length, Lx=30 �m �Fig. 5�, exceeds the dis-tance between erythrocytes. Hence, in the “coherence area”of the SP wave, ��zdLx�, there are one to two erythrocytes.

The erythrocytes intensively absorb D-glucose, whichaffects their refraction index and probably their shape. Theoptical reflectivity from the erythrocyte suspension changescorrespondingly. Indeed, Fig. 13 shows that, upon addition ofD-glucose, reflectivity in the SPR regime rapidly increaseswith time until saturation occurs. The rate of this processincreases when cells are exposed to higher glucose concen-trations.

In another experiment, cells were exposed to a solutioncontaining 20 mM D-glucose supplemented with 20 �M ofthe GLUT-1 inhibitor, cytochalasin B �CB�. A negligible re-flectivity change was observed in the presence of the inhibi-

ZnS prism

M3

iris

growth medium

flow chamberoutle

t

inlet

cellmonolayer

Au film

grid polarizer

M2M1

pinhole

FTIRsource

MCT

FIG. 11. �Color online� The FTIR-SPR setup. The IR beam from the FTIRspectrometer is collimated by two parabolic mirrors, M1 and M2, and apinhole. The collimated beam is polarized by the grid polarizer, it passesthrough the ZnS prism, and is reflected from its base. The reflected beam isfocused by the parabolic mirror, M3, on the MCT detector. The base of theprism is Au-coated and it is in contact with the flow chamber. The samplescan be of two kinds: �i� a biosolution in the flow chamber or �ii� a biologicalmembrane or cell monolayer grown directly on the base of the Au-coatedprism. In the latter case, the flow chamber is filled with growth medium.

0

0.5

1

1.5

0 2000 4000 6000 8000 1 104 1.2 104

Ref

lect

ivit

y(a

rb.u

nit

s)

Wavenumber (cm-1)

10 5 4 3 2 11.5 0.9

Wavelength (µm)

waterwater +1wt.% D-glucose

0.001

0.01

0.1

0.01 0.1 1

∆R

cglucose

(wt.%)

0.070

0.075

4300 4350ν (cm-1)

∆

R

FIG. 12. �Color online� Reflectivity from the ZnS/Au/water interface �blackdots�. The gold film thickness is 13 nm and the incident angle is �ext=21°.The red dots denote the corresponding results for 1 wt % of D-glucosewater solution. The SPR for the Au/water interface is manifested by a pro-nounced dip at 4334 cm−1. Addition of 1 wt % of D-glucose shifts this dipto 4310 cm−1. A secondary dip at 1780 cm−1 is a long-wavelength SP; thefeatures at 5145 cm−1 and at 6900 cm−1 indicate the water absorptionpeaks. The inset shows that �R at 4600 cm−1 �this wave number corre-sponds to the slope of the SPR and the reflectivity here is extremely sensi-tive to slight variations in the refractive index� linearly depends on theD-glucose concentration. The slope of this linear dependence yields a bulksensitivity, Sbulk=dR /dn=75 RIU−1.

102036-7 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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tor. Similarly, minor changes in the reflectivity were detectedwhen cells were exposed to a plain buffer solution. We inter-pret these results as follows: the erythrocytes absorbD-glucose �but not L-glucose� and this affects their refractiveindex. Although the exact mechanism by which the SP waveinteracts with the erythrocytes is not yet clear, we believethat the observed SPR variation is associated with D-glucoseuptake by erythrocytes because �i� the rate of SPR change isdetermined by the glucose concentration and �ii� the SPRchange is inhibited by CB, as expected. Therefore, we canconclude that FTIR-SPR measurements specifically sense aglucose-dependent transport process. This conjecture is cor-roborated by the dielectric spectroscopy measurements onsimilar suspensions.52

B. Cell culture studied by the SPR: The physicsinvolved

We succeeded in growing cell cultures directly on theAu-coated prism.42,44 These include human melanoma �MEL1106�, MDCK, and HeLa cells that form monolayers with atypical confluence of 60%–80%. In a typical experiment, theprism with a cell culture was removed from the incubator,

attached to a flow chamber, mounted in the FTIR-SPR setup,and then exposed to HEPES buffered medium �MEM� at37 °C. The temperature in the flow chamber was controlledwithin 0.1 °C while the buffer solution was constantlypushed through the chamber at a flow rate of 5 �l /min us-ing a motorized bee syringe pump. After 5–7 min of equili-bration, we measured the reflectivity in the SPR regime. Themeasurements lasted up to 6 h, after which, the cells usuallydie, most probably from the lack of CO2.

1. Surface plasmon propagation in the cell culture

Understanding SP propagation in the cell monolayer ne-cessitates further discussion. Indeed, the cells have irregularshapes with a lateral size of 10 20 �m2 and a height of 1to 6 �m. Since the cells contain up to 25% organic mol-ecules, their average refractive index exceeds that of water.This results in an angular shift of the SPR resonance �Eq.�5�� for the ZnS/Au/cell interface, compared to the ZnS/Au/water interface. However, even at high cell confluence, someparts of the Au-coated prism can be uncovered by cells.These Au patches may exhibit an unshifted SPR, providedtheir size exceeds the SP propagation length, lpatch�Lx.

Figure 14 summarizes our studies of the HeLa cellmonolayers grown on an SF-11 gold-coated glass prism.Here we used a relatively short wavelength �=1.6 �m. Thecorresponding penetration depth is also small, �zd=0.7 �m�Fig. 6� but sufficient to probe the cell’s interior. The propa-gation length, Lx=46 �m �Fig. 5�, exceeds the average cellsize. Figure 14 shows angular-dependent reflectivity for dif-

0

0.05

0.1

0 1 2 3 4 5

15 mM10 mM5 mM0 mM20 mM

Time (min.)

∆R

0.26

0.27

0.28

0.29

2500 2550 2600

t=1 min.

t=5 min.

∆R

ν(cm-1)

cgl=10 mM

FIG. 13. �Color online� Glucose uptake by erythrocyte suspension. We usedhere a long-wavelength SP at �4 �m and a 12-nm-thick Au film in orderto achieve a sufficiently high penetration depth, �zd=4.5 �m, which is com-parable to the typical erythrocyte diameter. The left-hand inset schematicallyshows SP penetration into erythrocytes. The right-hand inset shows the SPRreflectivity from the erythrocyte suspension before and after exposure to 10mM of D-glucose. The SPR minimum shifts with time to longer wave-lengths �redshift�, indicating glucose uptake by erythrocytes. The open sym-bols in the main panel indicate SPR reflectivity change �at a fixed wave-length� for erythrocyte suspension in the PBS buffer with differentconcentrations of D-glucose. Upon addition of D-glucose, �R increases andachieves saturation. Continuous lines indicate exponential fit �R� �1−e−�t�, whereas the rate � increases with glucose concentration. The orangecrosses indicate the results of another experiment where cells were exposedto solution containing 20 mM D-glucose supplemented with 20 �M of theGLUT-1 inhibitor, CB. As expected, a negligible reflectivity variation wasobserved in the presence of the inhibitor.

0

0.2

0.4

0.6

0.8

1

50 52 54 56 58 60

80%

60%

40%

0%

Ref

lect

ivit

y

Θext

(deg.)

λ=1.6 µm

Au

cells

ncell

-nwater

= 0.03

FIG. 14. �Color online� Angular dependence of the SPR from the HeLa cellmonolayers with different cell coverage �confluence�. We used here anSF-11 right-angle glass prism coated with a 35-nm-thick Au film. In theabsence of cells, there is a single SPR minimum at �ext=53.4°, which cor-responds to the reflectivity from the ZnS/Au/water interface. In the presenceof cells, an additional SPR dip appears at 55.8°, which corresponds to thereflectivity from the ZnS/Au/cells interface. The angular shift between thesetwo minima yields the refractive index difference, ncell−nwater=0.03. At highcell confluence �80%�, the SPR signal from the cells is dominant.

102036-8 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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ferent cell coverages �confluences�. In the absence of cells,there is a single dip at �sp=53.4° that corresponds to theSPR at the ZnS/Au/water interface. In the presence of HeLacells, an additional dip appears at �sp=55.8°, which corre-sponds to the ZnS/Au/HeLa cell interface. The SPR angularshift corresponds to ncell-nwater=0.03. For high cell conflu-ence, e.g., 80%, the SPR from uncovered gold patches isbarely seen, as expected.

2. Guided modes

Since the refractive index of cells exceeds that of thesurrounding aqueous medium �ncell�nwater� and the cellmonolayer thickness is comparable to the mid-IR wavelength�dcell��, the cell monolayer on gold could behave as ametal-clad optical waveguide.53 An indication of such behav-ior is seen in Fig. 15, which shows an SPR spectra �i� for ahuman MEL 1106 cell monolayer grown on an Au-coatedZnS prism at high confluence and �ii� for the similar Au-coated prism without cells. In both cases, there is a strongSPR at 3920 cm−1 ��=2.55 �m�. For the prism with cellculture, a short-wavelength satellite appears at 4385 cm−1

��=2.28 �m�. We associate this feature with the waveguidemode propagating in the cell monolayer �Fig. 10�d��.

Indeed, the cutoff condition for the TE and TM modes inthe planar metal-clad dielectric waveguide is3,53

k0dcell�ncell

2 − nwater2 = �m + 1

2� , �20�

where m=0,1 ,2¯. We substitute here nwater=1.25, ncell

−nwater=0.03, dcell=2 �m; for the lowest TE0 mode wefound that �0=2.3 �m ��=4348 cm−1�. This estimate cor-responds fairly well to our observation shown in Fig. 15.

Note that the SP propagation length at this wavelength isvery long, Lx=73 �m �Fig. 5�, as expected.

C. Cell cultures studied by the SPR technique: Thebiology involved

1. Cholesterol in the cell membrane

We studied cholesterol penetration into the plasma mem-brane of the HeLa cells42 using our FTIR-SPR technique�similar studies using SPR in the visible range were per-formed in Ref. 54�. It is well-known that cholesterol from theextracellular solution enters mostly into the plasma mem-brane rather than into cytoplasm. Therefore, to achieve highsensitivity to membrane-related events, we chose the NIRwavelength range, �sp=1–1.08 �m, characterized by a rela-tively low SPR penetration depth.

The HeLa cells were grown on an SF-11 glass prismcoated with a 35-nm-thick gold film. The prism with cellswas mounted into a flow chamber and was equilibrated ingrowth medium for 5–7 min at 37 °C. Then, we added 10mM of m�CD-chol �methyl-beta-cyclodextrin loaded withcholesterol�, which is known to enrich the cells with choles-terol.

We measured the FTIR-SPR spectra before and afteradding the drugs. Figure 16 shows that after exposure tom�CD-chol the SPR is redshifted. This corresponds to a re-fractive index increase because cholesterol has a higher re-fractive index than water. In contrast, when we add to agrowth solution a similar chemical, m�CD, which is notloaded with cholesterol, the SPR is blueshifted, which corre-

4000 6000 8000 1 1040

0.5

1

Ref

lect

ivit

y

Wavenumber (cm-1)

no cells

cell layer

SP

SP

guided mode

FIG. 15. �Color online� Reflectivity in the SPR regime for a melanoma1106-cell culture in MEM growth solution �upper curve, �=19.5°� andgrowth solution without cells �lower curve, �=18.6°�. The upper curve isdisplaced upwards for clarity. Note the main SPR dip at 3920 cm−1 in bothcurves and a satellite dip at 4385 cm−1 in the upper curve. We associate thisdip with the TE0 waveguide mode propagating in the cell monolayer.

-1000

-500

0

5000 10 20 30 40 50

∆ν

(cm

-1)

Time (min)

cholesteroldepletion

initial state

cholesterolenrichment

20.4 mM 24.9 mM 9.9 mM

λ=1.08 µm

red

shift

blue

shift

washmβCD-Chol mβCD

FIG. 16. �Color online� SPR shift upon cholesterol enrichment/depletion ofHeLa cell membranes. The drawings show cholesterol molecules in the cellmembrane �gray ovals�; the numbers below the drawings indicate choles-terol concentration in the membrane, as was determined biochemically onanalogous cell cultures. In order to be sensitive to membrane-related pro-cesses, the SPR measurements were performed at a short wavelength, �=1.08 �m, to ensure short penetration depth, �zd=0.36 �m �see Fig. 6�. Att=0 we injected 10 �M of m�CD-chol solution. The SPR becomes red-shifted, as expected, since the accumulation of cholesterol molecules with ahigh refractive index in the cell membrane increases the average refractiveindex. At t=15 min, the cells were washed with plain medium for 5 minand then 3 �M m�CD solution was added. The SPR became blueshiftedsince this drug depletes cell membranes from cholesterol in such a way thatthe average refractive index in the SPR-sensed region decreases.

102036-9 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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sponds to a refractive index decrease. This is expected sincem�CD depletes plasma membranes from cholesterol. Con-sistent with this explanation, the SPR reflectivity in thecholesterol-depleted state is lower than the initial SPR reflec-tivity.

2. Ferrotransferrin uptake

We applied our FTIR-SPR technique to studytransferrin-induced clathrin-mediated endocytic processesthat introduce Fe ions into the cell. Figure 17 shows a fluo-rescence image of a melanoma 1106 cell obtained by confo-cal microscopy.55 The inset shows the cell interior in black,the red areas indicate Fe penetration, and the green areasindicate extracellular space. It is clearly seen that the fluo-rescence intensity increases and then achieves saturation,whereas its kinetics in the peripheral part of the cell is fasterthan that in the whole cell.

We studied the same process using a long-wavelengthSP at �=2.54 �m. Its penetration depth, �zd=1.2 �m �Fig.6�, is long enough to penetrate the cells, although it sensesmostly the cell periphery. Figure 18 shows SPR reflectivityvariation upon introduction of Tfn into the solution. The ki-netics of the SPR reflectivity closely follows the fluorescencekinetics in the cell periphery �Fig. 17�, as expected. Thisexample demonstrates that the SPR results are consistentwith the confocal microscopy observations using fluorescenttags. The obvious advantage of the SPR technique is that it islabel-free.

Interestingly, the SPR here is blueshifted, indicating thatthe average refractive index in the measured volume de-creases �Fig. 18, inset�. This rules out the possibility that theSPR shift results from accumulation of organic molecules in

the cell �this would increase the refractive index�. The blue-shift indicates that the cells become “diluted,” as if thegrowth solution penetrates into cells. This could be explainedby formation of endocytic vesicles.

IV. DISCUSSION AND CONCLUSIONS

The conclusions are as follows:

• In the context of biosensing, the lack of convenientlaser sources and the enhanced water absorption wereconsidered as major impediments for extending theSPR technique into the mid-IR range. We demon-strated here that there are several spectral windows,where by a clever choice of wavelength, beam colli-mation, angle of incidence, and metal film thickness,the SPR technique can achieve sensitivity that is com-parable and even higher than that in the visible range.

• SPR in mid-IR can be used to study cells and cellcultures. This technique is well matched for the sens-ing of cells since the SP penetration depth in the IRrange is just on the order of the cell size. This is incontrast with the SP in the visible range, whose pen-etration depth is too short to probe the processes insidethe cell cytoplasm. However, to develop the FTIR-SPR technique into a useful tool for studying cells, onemust theoretically analyze SP propagation and scatter-ing in a cell monolayer.

• The waveguide modes in cell culture may develop intoa useful tool to study cell-cell attachment and cell ad-hesion to substrates.

• The most challenging task in the field of FTIR-SPR is

0

0.2

0.4

0.6

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1

1.2

0 5 10 15

Flu

ore

scen

cein

ten

sity

(arb

.un

its)

Time (min.)

whole cell

cell periphery

FIG. 17. �Color� Cell fluorescence resulting from the rhodamine-taggedferrotransferrin �Tfn� penetration into cells. The red circles represent theperipheral region of the cell, whereas the green triangles denote the fluores-cence from the whole cell. Note the fast fluorescence kinetics in the periph-eral regions of the cell, as compared with the slow kinetics in the whole cell.The inset shows the confocal microscope image of a single cell under partialpenetration of the ferrotransferrin.

0

0.01

0.02

0.03

0 5 10 15

∆R

Time (min.)

no cells

cell monolayer

ν(cm-1)

holo-Ft

R

0.25

0.3

4300 4350 4400

t=0t=15 min.

FIG. 18. �Color online� Reflectivity variation in the SPR regime followingthe introduction of holo-ferrotransferrin. The open circles indicate the resultsfor the ZnS/Au/growth solution interface when there is no cell culture. Here,introduction of the holo-Ft does not result in significant changes in reflec-tivity, indicating negligible holo-Ft adsorption on the Au surface. The filledcircles indicate a holo-Ft-induced reflectivity change from the ZnS/Au/Mel1006/water interface. There is pronounced SPR reflectivity change thatclosely follows the kinetics of fluorescence from the peripheral parts of thecells. The inset shows that the SPR in this case is blueshifted.

102036-10 Golosovsky et al. J. Appl. Phys. 105, 102036 �2009�

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to convert it into a spectroscopic tool that allows mea-suring the FTIR absorption/dispersion spectra in theregime of the SPR. The sensitivity of this techniqueshould surpass that of FTIR-ATR �Ref. 56� and allowdetection of biomolecules according to theirfingerprints.

ACKNOWLEDGMENTS

We are grateful to S. Shimron for preparing the cell cul-tures and for significantly contributing to the SPR experi-ments; to L. Livshitz for his help in erythrocyte studies, aswell as to N. Melamed-Book and A. M. Weiss for confocalmicroscopic images and image processing. This study wassupported in part by the Israel Science Foundation �BikuraGrant No. 1337/05�, by Johnson & Johnson, Inc., and byNofar Program of the Israeli Chief Scientist Office.

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