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Total Internal Reflection Fluorescence Microscopy of Cell Adhesion on Patterned Self-Assembled Monolayers on Gold Diana K. Hoover, Eun-Ju Lee, and Muhammad N. Yousaf* Department of Chemistry, Carolina Center for Genome Science, UniVersity of North Carolina, Chapel Hill, North Carolina 27599-3290 ReceiVed NoVember 27, 2008. ReVised Manuscript ReceiVed January 26, 2009 We report the use of total internal reflection fluorescence microscopy (TIRFM) to study cell adhesion on patterned self-assembled monolayers (SAMs) on gold surfaces. Microcontact printing was used to pattern hydrophobic features to which the extracellular protein fibronectin was adsorbed, while dip-pen nanolithography was used to produce electroactive nanoarrays of hydroquinone-terminated alkanethiol on gold-coated quartz substrates. The hydroquinone was electrochemically oxidized to the corresponding quinone, and an oxyamine-tethered linear Arg-Gly-Asp (RGD) peptide was chemoselectively immobilized. A prism-based method of TIRFM was used to examine adhered cells on both the microscale and nanoscale features. We also demonstrate that, following imaging with TIRFM, the substrates can be visualized using conventional fluorescence microscopy.
Introduction Total internal reflection fluorescence microscopy (TIRFM) is a technique that has been used for the past two decades to study events at the cell plasma membrane.1 One early application of TIRFM in cell biology was to study the cell-substrate contact regions of adherent cells on glass coverslips.2 TIRFM is based on the principle of total internal reflection (TIR). In order to establish TIR, a light beam traveling through a medium with a refractive index n1 (i.e., quartz, n1 ) 1.46) must encounter an interface with a second medium with refractive index n2 (i.e., the cell cytoplasm, typically n2 ) 1.36-1.373), such that n2 < n1. TIR will occur at all incidence angles Θ that are greater than some critical angle Θc defined by the expression Θc ) arcsin(n2/ n1). The result of the total reflection of the light beam will be an evanescent electromagnetic field extending into the second medium.4 The evanescent field strength decays exponentially as a function of the distance from the interface. As a result, fluorophores within the evanescent field will be excited and will emit, thus allowing the study of structures near the interface. TIRFM has been used to study a variety of systems, including single molecule fluorescence5 as well as events and structures at the cell surface such as exocytosis6 and ion channels,7 and has also been integrated with other methods such as interference * To whom correspondence should be addressed. E-mail: mnyousaf@ email.unc.edu. (1) (a) Burmeister, J. S.; Olivier, L. A.; Reichert, W. M.; Truskey, G. A. Biomaterials 1998, 19, 307–325. (b) Steyer, J. A.; Almers, W. Nat. ReV. Mol. Cell Biol. 2001, 2, 268–275. (2) Axelrod, D. J. Cell Biol. 1981, 89, 141–145. (3) Mathur, A. B.; Truskey, G. A.; Reichert, W. M. Biophys. J. 2000, 78, 1725–1735. (4) (a) Sako, Y.; Uyemura, T. Cell Struct. Funct. 2002, 27, 357–365. (b) Schneckenburger, H. Curr. Opin. Biotechnol. 2005, 16, 13–18. (5) Hanasaki, I.; Takahashi, H.; Sazaki, G.; Nakajima, K.; Kawano, S. J. Phys. D: Appl. Phys. 2008, 41, 095301–095309. (6) (a) Ohara-Imaizumi, M.; Fuijiwara, T.; Nakamichi, Y.; Okamura, T.; Akimoto, Y.; Kawai, J.; Matsushima, S.; Kawakami, H.; Watanabe, T.; Akagawa, K.; Nagamatsu, S. J. Cell Biol. 2007, 177, 695–705. (b) Nofal, S.; Becherer, U.; Hof, D.; Matti, U.; Rettig, J. J. Neurosci. 2007, 27, 1386–1395. (c) Zhang, Z.; Chen, G.; Zhou, W.; Song, A.; Xu, T.; Luo, Q.; Wang, W.; Gu, X. S.; Duan, S. Nat. Cell Biol. 2007, 9(8), 945–957. (7) (a) Demuro, A.; Parker, I. Cell Calcium 2005, 40, 413–422. NechyporukZloy, V.; Dieterich, P.; Oberleithner, H.; Stock, C.; Schwab, A. Am. J. Physiol. 2008, 294, 1096–1102. (b) Staruschenko, A.; Adams, E.; Booth, R. E.; Stockand, J. D. Biophys. J. 2005, 88, 3966–3975.
reflection microscopy8 and fluorescence correlation spectroscopy.9 Due to the flexibility of surface chemistry and conductivity, gold has been widely used for biointerfacial studies and as a platform for many biotechnologies. However, due to gold’s efficient quenching of fluorescence, limited optical transparency, and lack of long-term stability due to monolayer desorption, it has not found wide use in practical biosensor applications. Until recently, it was thought that gold surfaces precluded the use of live-cell high resolution fluorescence microscopy to study internal cell structure dynamics. Fluorescence microscopy is a fundamental tool in cell biology research to study cell behavior during adhesion, polarization, and migration.10 Moreover, the use of gold surfaces in cell biology as model substrates allows for the development of sophisticated immobilization strategies to install a variety of biologically relevant ligands on the surface. There have been conflicting reports on whether the presence of gold impedes the use of TIRFM in studying cell adhesion to gold substrates. Borisy and co-workers have stated that gold surfaces prevent the use of TIRFM,11 while Ariwa and Iwata have reported studying the adhesion of cells with fluorescently labeled plasma membranes on self-assembled monolayers (SAMs) of varying properties using TIRFM.12 However, the latter did not examine the internal structures of the cell, nor were the surfaces tailored with biologically relevant ligands. In this Letter, we report the combination of microcontact printing or an electroactive chemoselective immobilization strategy, dip-pen nanolithography, and TIRFM to visualize cell adhesion on tailored and patterned gold surfaces. This report is the first to show that TIRFM can be used to visualize internal features of a cell on chemoselectively tailored gold SAM surfaces and will open (8) Llobet, A.; Beaumont, V.; Lagnado, L. Neuron 2003, 40, 1075–1086. (9) Thompson, N. L.; Steele, B. L. Nat. Protoc. 2007, 2, 878–890. (10) (a) Hodgson, L.; Chan, E. W. L.; Hahn, K. M.; Yousaf, M. N. J. Am. Chem. Soc. 2007, 129, 9264–9265. (b) Hoover, D. K.; Chan, E. W. L.; Yousaf, M. N. J. Am. Chem. Soc. 2008, 130, 3280–3281. (c) Lamb, B. M.; Westcott, N. P.; Yousaf, M. N. ChemBioChem 2008, 9, 2220–2224. (d) Lamb, B. M.; Westcott, N. P.; Yousaf, M. N. ChemBioChem 2008, 9, 2628–2632. (e) Chan, E. W. L.; Yousaf, M. N. Mol. BioSyst. 2008, 4, 746–753. (11) Kandere-Grzybowska, K.; Campbell, C.; Komarova, Y.; Grzybowski, B. A.; Borisy, G. G. Nat. Methods 2005, 2(10), 739–741. (12) Ariwa, Y.; Iwata, H. J. Mater. Chem. 2007, 17, 4079–4087.
10.1021/la803927k CCC: $40.75 2009 American Chemical Society Published on Web 01/30/2009
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Figure 1. Schematic depiction of TIRFM experiment. The sample was placed between a water immersion 60× objective and a quartz prism as shown (using water and immersion oil, respectively, for similar refractive indices). Fluorescence excited by TIR was collected through the objective, filtered, and captured on a CCD camera. (Complete optics path has been omitted for clarity.)
many avenues for integrating TIRFM with material science and cell signaling dynamics to study cell behavior.
Results and Discussion A prism-based method of TIRFM was used to examine cells adhered on patterned SAMs on gold-coated quartz surfaces. As shown in Figure 1, the prepared slide was placed in contact between a quartz prism and a 60× water immersion objective, using immersion oil and water, respectively, to maintain a similar refractive index between each interface. TIR was established by adjusting the quartz prism, thus changing the angle of incidence (Θ) of a 532 nm green diode laser beam. The emitted fluorescence of fluorophores excited by the evanescent wave created was filtered and captured by a CCD camera. SAMs of hexadecanethiol on gold-coated quartz slides were patterned using microcontact printing (Figure 2A). Scanning electron microscopy (SEM) was used to characterize the microcontact printed features, as shown in Figure 2B. Following backfilling with tetra(ethylene)glycol alkanethiol (EG4C11SH), the extracellular matrix protein fibronectin was adsorbed to the hydrophobic regions in order to support cell adhesion. 3T3 Swiss Albino mouse fibroblasts were allowed to adhere to the patterns, fixed, and stained with an antibody targeting paxillin, a protein found in focal adhesions,15 followed by a fluorescently labeled secondary antibody. As can be seen in the TIRFM micrograph in Figure 2C, paxillin is distributed throughout the cells. An added level of complexity was introduced by integrating the previously described electroactive chemoselective ligand immobilization strategy and dip-pen nanolithography (DPN) in order to produce electroactive nanoarrays on gold-coated quartz substrates.16 An oxyamine-tethered ligand, linear Arg-Gly-Asp (RGD-ONH2), was reacted following electrochemical activation of the nanopatterned surface (Figure 3). RGD is a well-known peptide found in the main binding domain of fibronectin.17 3T3 (15) Turner, C. E.; Glenney, J. R.; Burridge, K. J. Cell Biol. 1990, 111, 1059– 1068. (16) (a) Chan, E. W. L.; Park, S.; Yousaf, M. N. Angew. Chem., Int. Ed. 2008, 47, 6267–6271. (b) Chan, E. W. L.; Yousaf, M. N. ChemPhysChem 2007, 8, 1469–1472. (c) Barrett, D. G.; Yousaf, M. N. Angew. Chem., Int. Ed. 2007, 46, 7437–7439. (d) Westcott, N. P.; Yousaf, M. N. Langmuir 2008, 24, 2261–2265. (e) Luo, W.; Westcott, N. P.; Pulsipher, A.; Yousaf, M. N. Langmuir 2008, 24, 13096–13101. (17) Pierschbacher, M. D.; Ruoslahti, E. Nature 1984, 309, 30–33.
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Figure 2. (A) Microcontact printing was used to pattern 50 µm features of C16-SH on gold substrates. The remaining bare gold regions were backfilled with EG4-SH, and fibronectin (Fn) was adsorbed to the features. Cells were then seeded on the surfaces. (B) SEM micrograph of microcontact printed C16-SH on gold. (C) Representative micrograph of a fibroblast adhered to a 50 µm microcontact printed feature. The cells were stained with anti-paxillin and a TRITC secondary antibody to visualize the focal adhesions in the cell. TIRFM signal from TRITC labeled secondary antibody is pseudocolored red.
Swiss Albino mouse fibroblasts were then seeded to the substrates, fixed, and stained for nuclei, actin, and paxillin. As shown in Figure 4A, the paxillin signal found in TIRFM is localized primarily to the perimeter of the cell. We then imaged the same substrates using conventional fluorescence microscopy. The representative fluorescent micrograph in Figure 4B was taken of a cell on a nanoarray of immobilized linear RGD using standard fluorescent microscopy following TIRFM imaging. Interestingly, there is not a significant amount of photobleaching of the tetramethylrhodamine B isothiocyanate (TRITC) secondary antibody following extended imaging using TIRFM.
Conclusion In this report, we have demonstrated that TIRFM can be used to visualize the internal structures of cells adhered to SAMs on gold surfaces. Second, we have shown that this TIRFM imaging can be used in conjunction with more traditional methods of imaging, such as fluorescence microscopy to study cells. Finally, we show the combination of TIRFM with surface patterning techniques (microcontact printing, dip-pen nanolithography) and an electroactive immobilization strategy to tailor surfaces with ligands to study cell behavior. In the future, TIRFM could be used to explore the intricacies of the cell nanoarchitecture on surfaces presenting various patterns of biospecific ligands. In particular, this technique could be used to study signaling dynamics in cell adhesion, polarization, and migration on welldefined surfaces presenting more complex surface chemistries such as gradients or by integration with patterned dynamic surfaces.
Experimental Section Preparation of Gold-Coated Quartz Substrates. Quartz microscope slides were cleaned using Piranha solution (use with caution, 1:1 (v/v) 30% H2O2/concentrated H2SO4). An adhesion layer of titanium (6 nm), followed by a layer of gold (24 nm) was thermally evaporated onto the quartz slides. The surfaces were cleaned with absolute ethanol and dried with a stream of air before use.
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Figure 3. (A) DPN was used to produce electroactive nanoarrays of hydroquinone-terminated alkanethiol (H2Q-C11-SH). Following backfilling with EG4-SH, the nanoarrays were electrochemically oxidized and an oxyamine-tethered ligand (linear RGD-ONH2) was chemoselectively immobilized. (B) Expanded lateral force microscopy image of an electroactive nanoarray on gold.
Figure 4. (A) Representative TIRFM micrograph of an adherent Swiss Albino mouse fibroblast on a nanoarray of immobilized linear RGD peptide. The cell was stained with DAPI, FITC-phalloidin, and anti-paxillin with a TRITC secondary antibody. TIRFM signal from TRITC labeled secondary antibody is pseudocolored red. (B) Representative micrograph of a 3T3 Swiss Albino fibroblast taken with fluorescence microscopy following imaging using TIRFM. This cell was stained with DAPI (nuclei, blue), FITC-phalloidin (actin, green), and anti-paxillin followed by a TRITC secondary antibody (focal adhesions, red).
Substrate Patterning by Microcontact Printing. A gold-coated quartz slide was patterned by microcontact printing as previously described.13 Briefly, a poly(dimethylsiloxane) (PDMS) stamp with 50 µm features of various shapes was used to microcontact print hexadecanethiol (C16SH, 1 mM in ethanol) on gold, as shown in Figure 2A. The remaining bare gold region was then backfilled using 11-mercaptoundecyl-tetra(ethylene glycol) (EG4C11SH, 1 mM in ethanol, 12 h). Bovine fibronectin (0.1 mg/mL in H2O, Fisher) was adsorbed for 1 h. Substrate Patterning by Dip-Pen Nanolithography. All dippen nanolithography (DPN) experiments were performed as previously described14 using a MFP-3D Stand Alone atomic force microscope (Asylum Research, Santa Barbara, CA). Silicon AFM tips (MikroMasch USA, Wilsonville, OR) were immersed in a solution of hydroquinone-terminated alkanethiol (H2QC11SH, 5 mM in acetonitrile) and then gently dried with a stream of air. Nanoarrays of dots were then produced (20 × 20, 3 µm pitch, 30 s dwell time, ∼500 nm diameter). The remaining exposed areas of gold were passivated by soaking the substrate in EG4C11SH (1 mM in ethanol, 12 h). The electroactive nanoarrays of H2QC11SH were electrochemically oxidized to the resulting quinone (800 mV vs Ag/AgCl for 15 s in 1.0 M HClO4). The substrate was then reacted with the oxyamine-tethered linear RGD peptide (10 mM in H2O) for 2 h. Cell Seeding and Staining Procedures. 3T3 Swiss Albino mouse fibroblasts (Tissue Culture Facility, UNC) were seeded on the surface, (13) Xia, Y. N.; Whitesides, G. W. Angew. Chem., Int. Ed. 1998, 37, 550–575. (14) Hoover, D. K.; Lee, E. J.; Chan, E. W. L.; Yousaf, M. N. ChemBioChem 2007, 8, 1920–1923.
incubated overnight (37 °C, 5% CO2) in serum-containing media (Dulbecco’s Modified Eagle’s Medium (Sigma) with 10% calf bovine serum and 1% penicillin/streptomycin), and then fixed with 3.2% formaldehyde in PBS-T (Dulbecco’s PBS buffer (Sigma) containing 0.1% Triton X-100). For studies using microcontact printed surfaces, the cells were stained using anti-paxillin (1:400 dilution, BD Bioscience, San Jose, CA) followed by a tetramethylrhodamine B isothiocyanate (TRITC) conjugated goat anti-mouse IgG secondary fluorescent antibody (1:400 dilution, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and visualized with TIRFM. For the DPN patterned surfaces, the cells were stained with three dyes 4′,6-diamidino-2-phenylindole hydrochloride (DAPI, 1:500 dilution, Sigma), fluorescein isothiocyanate (FITC) labeled phalloidin (1:50 dilution, Sigma), and anti-paxillin (1:400 dilution), followed by TRITC-conjugated goat anti-mouse IgG (1:400 dilution), and then imaged using TIRFM followed by fluorescence microscopy. Cell Visualization Using TIRF Microscopy and Fluorescence Microscopy. Following the cell staining procedure, patterned substrates were prepared for TIRFM analysis. A glass coverslip (Fisher) was flamed and placed over the area of patterned cells, trapping a layer of PBS between the glass and quartz. The edges of the coverslip were sealed using 90 s curing epoxy (Araldite 2043, McMaster-Carr, Princeton, NJ). All TIRFM measurements were made using a prism-based method with an Olympus IX51 inverted microscope and Olympus UPLSAPO 60× water immersion objective (NA ) 1.2, Olympus America, Inc., Center Valley, PA), as shown in Figure 1. The excitation radiation was supplied by a green diode laser module (532 nm, 30 mW, GDLM-5030 L, Photop Technologies,
2566 Langmuir, Vol. 25, No. 5, 2009 Chatsworth, CA). The emitted radiation was sent through a green emission filter (585 nm/70 nm band pass, Chroma HQ585/70, Chroma Technology, Rockingham, VT) and captured by a CCD camera (Cascade II 512B, Photometrics, Tucson, AZ). Final image analysis was performed using MatLab R2007B (The Mathworks, Inc., Natick, MA). Fluorescence microscopy images were taken using an inverted Nikon Eclipse TE2000-E microscope (Nikon USA, Melville, NY).
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Acknowledgment. This work was supported by the Carolina Center for Cancer Nanotechnology Excellence and the Burroughs Wellcome Foundation (Interface Career Award). We thank Vanessa DeRocco and Erika Pearson of Dr. Dorothy Erie’s research group (UNC) for helpful discussions. LA803927K
