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Radial polarization induced surface plasmon virtual probe for two-photon fluorescence microscopy. K. J. Moh,1 X.-C. Yuan,2,* J. Bu,2 S. W. Zhu,3 and Bruce Z.
April 1, 2009 / Vol. 34, No. 7 / OPTICS LETTERS

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Radial polarization induced surface plasmon virtual probe for two-photon fluorescence microscopy K. J. Moh,1 X.-C. Yuan,2,* J. Bu,2 S. W. Zhu,3 and Bruce Z. Gao4 1

School of Electrical & Electronic Engineering, Nanyang Technological University, Nanyang Avenue, Singapore 639798, Singapore 2 Institute of Modern Optics, Key Laboratory of Optoelectronic Information Science and Technology, Ministry of Education of China, Nankai University, Tianjin, 300071, China 3 Tianjin Union Medicine Centre, Tianjin 300121, China 4 Department of Bioengineering, Clemson University, Clemson, South Carolina 29634, USA *Corresponding author: [email protected]

Received September 29, 2008; revised February 6, 2009; accepted February 7, 2009; posted February 23, 2009 (Doc. ID 102128); published March 20, 2009 Surface plasmons excited by a focused femtosecond radially polarized beam on a metal surface form a standing wave pattern with a sharp peak that can be used as a “virtual probe” for surface plasmon microscopy. The rotational symmetry of radially polarized light effectively provides the TM polarization required for coupling to the surface plasmons while the short pulse nature of the probe allows for nonlinear processes to be studied. © 2009 Optical Society of America OCIS codes: 180.4315, 180.2520, 240.6680, 260.5430.

Surface plasmons (SPs) are propagating electromagnetic surface waves existing on the interface of a dielectric and a metal layer. They are highly sensitive to perturbations in the environment, consequently making SPs useful for a host of optical sensing and imaging applications. However the long propagation length of the SP can be a limitation in applications requiring high lateral spatial resolution because their response to any surface artifacts is averaged over this distance [1]. To improve the lateral spatial resolution on a planar surface, a focused beam technique was proposed to confine the SPs in the region of the diffraction limited spot size of the incident light [2–5]. The coherent nature of the SPs enables the formation of an evanescent Bessel-like standing wave or fringelike interference patterns concentrated at the focal region [6,7]. In a recent work, we used the sharp central peak of the SP standing wave pattern as an immaterial point sensor (a virtual probe) and measured quantitatively in vitro the local refractive index distribution of biological cell-substrate contacts [8]. Here we extend the SP virtual probe technique to the nonlinear regime and demonstrate its application to two-photon excitation fluorescence microscopy. Our interest and motivation stem from the technique’s potential to increase nonlinear optical effects at interfaces [9], which may be relevant to studies in the visualization of fluorescent ligands clustered with numerous cytoskeletal and membrane signaling proteins at focal adhesions and the focal complexes with conformation changes caused by ligand binding. Directly imaging these molecular events is crucial to the understanding of cellular matrix mediated cell differentiation, growth, migration, or apoptosis. Figure 1 shows the basic schematic for the focused beam technique. The high index hemisphere lens is analogous to the prism in the Kretschmann and Raether (KR) coupling configuration and provides a 0146-9592/09/070971-3/$15.00

range of angles for SP excitation on the metal surface. The gap between the lens and the thin glass slide 共ng = 1.515兲 is filled with an index matching medium. For an incident radially polarized (RP) laser beam, the technique is in essence the KR optical configuration for SP excitation with complete rotational symmetry about the optical axis of the lens. This occurs because the axially symmetric polarization of RP light results in the entire beam being TM polarized with respect to the metal interface. Therefore SPs are simultaneously excited on the metal in all directions and interfere to form a Bessel-like standing wave pattern that has a maximum intensity confined to a small region on the metal layer [6]. Practically, this configuration can be realized by (i) focusing the RP light at the correct angle ␪in on a high index hemispherical lens or (ii) directly by a very high numerical aperture (NA) lens whose maximum angle is greater than the critical angle for the

Fig. 1. (Color online) Optical configuration for exciting SPs using a high index hemispherical lens. There is full rotational symmetry about the optic axis with RP light. The generated SP has a Bessel-like spatial distribution. © 2009 Optical Society of America

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Fig. 2. (Color online) Graphs of the calculated angle at which the SP is excited for various sample 共ns兲 refractive indices on a gold (Au) layer at 488, 532, 632.8, and 785 nm excitation.

system. We explored both approaches in this Letter, where a total internal reflection fluorescence (TIRF) microscope objective was used in the later case (ii) and a considerably less expensive “off-the-shelf” sapphire hemisphere lens with another objective lens of a lower NA (compared to the TIRF) for the former (i). In either case, the angle ␪sp that corresponds to the SP excitation/resonance condition lies in between the maximum angle of the lens ␪NA and the critical angle ␪crit for total internal reflection as seen in Fig. 1. The angle ␪sp is influenced by the metal layer, the refractive index of the sample ns, the incident wavelength ␭ of the light, and the refractive index of the incident medium. It is found by relating the wave vector kx of the incident beam to the SP ksp,

␪sp = sin−1



␧ 2␧ 3 ␧1共␧2 + ␧3兲

,

共1兲

where ␧1, ␧2, and ␧3 are the permittivity of the incident medium, metal layer, and sample, respectively. Figure 2 shows the theoretical plots of ␪sp at four common laser wavelengths (␭ = 488, 532, 632.8, and 785 nm) for a range of sample refractive indices on a thin layer of gold (Au). The two horizontal arrows indicate the finite angular limit imposed by the objective lens NA. To the right of the vertical arrow in Fig. 2 denotes the region where the sample has the same refractive index as water 共⬃1.327兲 or is located in an

aqueous medium, the in vitro condition typical for most biological samples. The graphs in Fig. 2 show that the point required for SP coupling occurs at lower angles for longer wavelengths and over a longer range of refractive indices for wavelengths in the near infrared (NIR) compared to the lower portions of the spectrum. Both Fig. 2 and Table 1 indicate that the NIR SP virtual probe has a working range extending into the aqueous medium region. However the photon energy of the NIR wavelength is too low to excite fluorescent markers. In contrast, the shorter wavelengths are preferred in fluorescence microscopy (e.g., 488 nm for Alexa Flour dyes and 532 nm for Rhodamine dyes) but are not able to function as the virtual probe wavelengths in the useful aqueous medium region. Although there are specialized dyes for the 632 nm wavelength, the working range extends only slightly into the aqueous medium region at a high NA. The pulsed NIR laser fulfills these opposing requirements by both enabling nonlinear mutliphoton absorption in fluorescence markers and is also able to form the SP virtual probe in the aqueous medium region. An additional advantage in using NIR lasers is that living organisms and cells are generally transparent in this wavelength region and are therefore less susceptible to photo damage. A Ti:sapphire femtosecond laser with an ⬃200 fs pulse duration at a 76 MHz repetition rate was focused onto a 40 nm thick gold layer in our experiment. The beam was originally linearly polarized and has a full width at half-maximum (FWHM) bandwidth of ⬃5.27 nm centered at 785 nm. We applied a relatively simple polarization conversion technique composed of a microfabricated spiral phase plate and an azimuthal-type linear analyzer to generate RP light; interested readers are directed to [10] for details of the method. Figure 3 shows the emitted fluorescence observed through a second objective lens when Rhodamine B dye was excited by the SP virtual probe in our setup using the TIRF lens approach. A 575– 625 nm bandpass filter was used to ensure that only the emitted fluorescence signal (⬃572 nm peak) reached the detector. The strength of the detected signal was observed to be highly polarization dependent and can be attributed to the vector properties (i.e., focusing geometry) and focus profiles of linear, azimuthal, or RP light [11]. In the case of [Fig. 3(a)] azimuth polarization, the incident light is completely TE polarized with respect to the metal surface and cannot couple into the SP modes; therefore very little or no fluores-

Table 1. Refractive Index „ns… Range for SP Excitation Wavelength (nm) Objective Lens

488

NA 1.49 NA 1.4

1–1.081 1–1.045

Gold ␧2

−2.4645+ i3.2151

532 1–1.218 1–1.168 Relative Permittivity −4.4764+ i2.5318

632.8

785

1–1.345 1–1.278

1–1.411 1–1.334

−9.7997+ i1.9649

−19.248+ i2.0315

April 1, 2009 / Vol. 34, No. 7 / OPTICS LETTERS

Fig. 3. (Color online) Emitted fluorescence for (a) azimuth (TE), (b) linear, (c) circular, and (d) radial (TM) polarization. The values indicate the mean detected flouresence intensity for each polarization compared to the RP beam.

cence is excited in this case. The low residual signal detected for Fig. 3(a) was due to unwanted s- and p-polarization phase shifts from reflecting optics in our experiment, which resulted in an impure azimuthally polarized beam. For [Fig. 3(b)] linear polarization, only the incident rays in the same plane of Fig. 2 are completely TM polarized while the vertical out-of-plane rays are TE polarized. The circular case [Fig. 3(c)] is a time averaged result of Fig. 3(b); thus there is partial coupling into the SP modes for these two cases. The strongest signal was obtained for RP [Fig. 3(d)], which is fully TM polarized with respect to the metal layer; thus the rotationally symmetric RP beam provides the most efficient means of inducing the SP virtual probe. Consequently for a proof-of-concept demonstration of SP excited two-photon microscopy, thin slices of leaf petiole/stem were soaked in Rhodamine B and placed on the gold substrate. The high index hemispherical lens approach was used in this case, where the image was acquired by scanning the virtual probe through the sample and collecting the fluorescence signal with a photomultiplier tube. The obtained twophoton excited fluorescence image in Fig. 4(a) shows the vascular transport structures in the transverse

Fig. 4. (Color online) Two-photon excitation fluorescence image of a leaf petiole obtained by (a) scanning the SP virtual probe in a 1 ⫻ 1 mm region and (b) nonSP excited fluorescence.

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section of the leaf petiole sample. For comparison with a nonSP excited two-photon image, a second experiment [shown in Fig. 4(b)] was conducted for a similar sample placed on clear glass without the metal layer. An opaque disc was placed in the path of the incident beam and directly in front of the objective to block angles lower than the critical angle for reflection. Therefore only the peripheral light contributed to the TIRF signal. Similar to [9], which reported SP intensified multiphoton excited fluorescence, we observed in Fig. 4(a) higher contrast and better signal-to-noise ratios for SP excited twophoton virtual probe microscopy. In summary we demonstrate an SP virtual probe excited by RP light in the femtosecond domain. This immaterial tip may be of interest in scanning microscopy, where it enables direct access to a biological specimen surrounded by a fluid medium sealed in a controlled environment. The application to twophoton excitation fluorescence imaging shown here can provide concurrent information to the quantitative refractive index measurements obtained from a specimen via the virtual probe [8]. Work is in progress to investigate several other nonlinear optical effects at interfaces using the technique, e.g., surface enhanced Raman spectroscopy and SP coupled emission. This work was partially supported by National Research Foundation of Singapore under grant NRF-GCRP 2007-01. Support also derives from Ministry of Education under ARC 3/06, RGM6/05, and RGM37/06 for manpower support. X.-C. Yuan, J. Bu, and S. W. Zhu acknowledge National Natural Science Foundation of China (NSFC) for grant 60778045. B. Z. Gao acknowledges partial financial support from the National Institutes of Health (NIH) [South Carolina Idea Networks of Biomedical Research (INBRE)] grant 2p20RR16461-05 and K25 award 1K25HL088262-010. References 1. B. Rothenhausler and W. Knoll, Nature 332, 615 (1988). 2. H. Kano, S. Mizuguchi, and S. Kawata, J. Opt. Soc. Am. B 15, 1381 (1998). 3. H. Kano and W. Knoll, Opt. Commun. 153, 235 (1998). 4. H. Kano and W. Knoll, Opt. Commun. 182, 11 (2000). 5. M. G. Somekh, S. G. Liu, T. S. Velinov, and C. W. See, Opt. Lett. 25, 823 (2000). 6. Q. Zhan, Opt. Lett. 31, 1726 (2006). 7. A. Bouhelier, F. Ignatovich, A. Bruyant, C. Huang, G. Colas des Francs, J. C. Weeber, A. Dereux, G. P. Wiederrecht, and L. Novotny, Opt. Lett. 32, 2535 (2007). 8. K. J. Moh, X. C. Yuan, J. Bu, S. W. Zhu, and B. Z. Gao, Opt. Express 16, 20734 (2008). 9. H. Kano and S. Kawata, Opt. Lett. 21, 1848 (1996). 10. K. J. Moh, X. C. Yuan, J. Bu, R. E. Burge, and B. Z. Gao, Appl. Opt. 46, 7544 (2007). 11. S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, Appl. Phys. B 72, 109 (2001).