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Optics Express

Optics Express

  • Editor: Andrew M. Weiner
  • Vol. 21, Iss. 9 — May. 6, 2013
  • pp: 10831–10840
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120 nm resolution and 55 nm structure size in STED-lithography

Richard Wollhofen, Julia Katzmann, Calin Hrelescu, Jaroslaw Jacak, and Thomas A. Klar  »View Author Affiliations


Optics Express, Vol. 21, Issue 9, pp. 10831-10840 (2013)
http://dx.doi.org/10.1364/OE.21.010831


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Abstract

Two–photon direct laser writing (DLW) lithography is limited in the achievable structure size as well as in structure resolution. Adding stimulated emission depletion (STED) to DLW allowed overcoming both restrictions. We now push both to new limits. Using visible light for two-photon DLW (780 nm) and STED (532 nm), we obtain lateral structure sizes of 55 nm, a Sparrow limit of around 100 nm and we present two clearly separated lines spaced only 120 nm apart. The photo-resist used in these experiments is a mixture of tri- and tetra-acrylates and 7-Diethylamino-3-thenoylcoumarin as a photo-starter which can be readily quenched via STED.

© 2013 OSA

1. Introduction

More than two decades ago, Denk, Strickler and Webb introduced two-photon fluorescence microscopy in order to establish an axial resolution without the need for confocalization [1

1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

]. Shortly after, the same group showed that two-photon excitation of a photopolymer allows for high density data storage [2

2. J. H. Strickler and W. W. Webb, “Three-dimensional optical data storage in refractive media by two-photon point excitation,” Opt. Lett. 16(22), 1780–1782 (1991). [CrossRef] [PubMed]

]. In 1997, Kawata and his group adapted the method for three dimensional two-photon-induced lithography [3

3. S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett. 22(2), 132–134 (1997). [CrossRef] [PubMed]

]. Nowadays, two-photon direct laser writing (DLW) facilitates writing features with lateral sizes of 90 [4

4. F. Burmeister, S. Steenhusen, R. Houbertz, U. D. Zeitner, S. Nolte, and A. Tünnermann, “Materials and technologies for fabrication of three-dimensional microstructures with sub-100 nm feature sizes by two-photon polymerization,” J. Laser Appl. 24(4), 042014 (2012). [CrossRef]

], 80 [5

5. J. F. Xing, X. Z. Dong, W. Q. Chen, X. M. Duan, N. Takeyasu, T. Tanaka, and S. Kawata, “Improving spatial resolution of two-photon microfabrication by using photoinitiator with high initiating efficiency,” Appl. Phys. Lett. 90(13), 131106 (2007). [CrossRef]

, 6

6. V. F. Paz, M. Emons, K. Obata, A. Ovsianikov, S. Peterhänsel, K. Frenner, C. Reinhardt, B. Chichkov, U. Morgner, and W. Osten, “Development of functional sub-100nm structures with 3D two-photon polymerisation technique and optical methods for characterization,” J. Laser Appl. 24(4), 042004 (2012). [CrossRef]

], and 65 nm [7

7. W. Haske, V. W. Chen, J. M. Hales, W. T. Dong, S. Barlow, S. R. Marder, and J. W. Perry, “65 nm feature sizes using visible wavelength 3-D multiphoton lithography,” Opt. Express 15(6), 3426–3436 (2007). [CrossRef] [PubMed]

] when pulsed lasers are used for two-photon excitation with wavelengths of 1030, 800, and 520 nm, respectively. A line width of 50 nm has been achieved by post deposition shrinking [8

8. X. Z. Dong, Z. S. Zhao, and X. M. Duan, “Improving spatial resolution and reducing aspect ratio in multiphoton polymerization nanofabrication,” Appl. Phys. Lett. 92(9), 091113 (2008). [CrossRef]

]. However, the dimensions of isolated structures must not be confused with the resolution of DLW. The latter is defined by the minimal spacing of two adjacent yet separated structures and is currently in the range of 200 nm [9

9. J. Fischer and M. Wegener, “Three-dimensional direct laser writing inspired by stimulated-emission-depletion microscopy,” Opt. Mater. Express 1(4), 614–624 (2011). [CrossRef]

].

In 1994, a major proposal was set out to ultimately break Abbe’s resolution limit [10

10. E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873). [CrossRef]

] in fluorescence microscopy by deliberately switching off the fluorophores in the outer rim of the point spread function (PSF) via stimulated emission depletion (STED) [11

11. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]

]. Meanwhile, STED-nanoscopy has proven to provide even sub-10 nm resolution [12

12. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009). [CrossRef]

] and has found many applications in biology [13

13. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006). [CrossRef] [PubMed]

16

16. P. A. Pellett, X. L. Sun, T. J. Gould, J. E. Rothman, M. Q. Xu, I. R. Corrêa Jr, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express 2(8), 2364–2371 (2011). [CrossRef] [PubMed]

] or materials science [17

17. K. Friedemann, A. Turshatov, K. Landfester, and D. Crespy, “Characterization via two-color STED microscopy of nanostructured materials synthesized by colloid electrospinning,” Langmuir 27(11), 7132–7139 (2011). [CrossRef] [PubMed]

]. Similar to the further development of two-photon microscopy into two-photon lithography [2

2. J. H. Strickler and W. W. Webb, “Three-dimensional optical data storage in refractive media by two-photon point excitation,” Opt. Lett. 16(22), 1780–1782 (1991). [CrossRef] [PubMed]

, 3

3. S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett. 22(2), 132–134 (1997). [CrossRef] [PubMed]

], it was pointed out already in the early reports of STED that the confined excitation volume can be applied to spatially control chemical reactions on the nanometer scale [18

18. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999). [CrossRef] [PubMed]

, 19

19. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]

]. STED-inspired diffraction-unlimited DLW has been realized recently [20

20. T. F. Scott, B. A. Kowalski, A. C. Sullivan, C. N. Bowman, and R. R. McLeod, “Two-color single-photon photoinitiation and photoinhibition for subdiffraction photolithography,” Science 324(5929), 913–917 (2009). [CrossRef] [PubMed]

22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

]. In the concept closest to STED [22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

], one laser pulse excites photo-initiators for radical polymerization and a second laser locally inhibits the ability of the photo-initiator to start the polymerization in the outer rim of the PSF. Thereby, polymerization is restricted to the inner part of the PSF thus creating a shrunken “effective PSF”. Such a STED based approach facilitates the generation of structures with feature sizes below the limits of two-photon polymerization. Theoretically, it allows for light induced nano-patterning of unlimited small feature sizes [22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

]. So far, a minimal lateral resolution of 175 nm was reported [9

9. J. Fischer and M. Wegener, “Three-dimensional direct laser writing inspired by stimulated-emission-depletion microscopy,” Opt. Mater. Express 1(4), 614–624 (2011). [CrossRef]

], which is considered as the state of the art in DLW resolution [23

23. J. Fischer and M. Wegener, “Three-dimensional optical laser lithography beyond the diffraction limit,” Laser Photon. Rev. 7(1), 22–44 (2013). [CrossRef]

]. The minimal lateral diameter for “0D” voxels reported so far is 40 nm [24

24. Y. S. Cao, Z. S. Gan, B. H. Jia, R. A. Evans, and M. Gu, “High-photosensitive resin for super-resolution direct-laser-writing based on photoinhibited polymerization,” Opt. Express 19(20), 19486–19494 (2011). [CrossRef] [PubMed]

] and the minimal width of 1D lines is 65 nm [22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

].

In this article, we address the question of minimal lateral (two dimensional) structure size and minimal lateral resolution in STED-DLW. We show that single lines can be written with a lateral width of only 55 nm and a full width of half maximum of 34 nm. This is a new hitherto unreached limit in lithography using low energetic visible light photons. We further find that the resolution in STED-DLW can be pushed down to 120 nm. At this distance, two adjacent, yet separated lines can be written. We deliberately focus ourselves on the lateral dimensions this time for two reasons: First, it has been shown that it is of utmost importance to find the right two-photon excitation intensity in DLW in order to achieve the smallest feature sizes and the best resolution. This intensity is decisively different for 3D lithography in the volume of a photoresist compared to 2D lithography close to a glass surface [25

25. D. Kunik, S. J. Ludueña, S. Costantino, and O. E. Martínez, “Fluorescent two-photon nanolithography,” J. Microsc. 229(3), 540–544 (2008). [CrossRef] [PubMed]

]. Second, in STED nanoscopy the de-excitation focus is either optimized for a 3D or for a 2D improvement of resolution. For best results in 3D, a “bottle shaped” STED-PSF is preferred [19

19. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]

], while for pure 2D improvement a 2D donut-shaped STED PSF is the best choice [13

13. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006). [CrossRef] [PubMed]

], and is therefore used in this work.

2. Experimental

The composition of the photo-resist is a 40:60 mixture (weight ratio) of pentaerythritol tetraacrylate (PETTA) and pentaerythritol triacrylate (PETA) including 300-400 ppm monomethyl ether hydroquinone (Sigma Aldrich). We added 0.25 wt. % photosensitizer DETC (7-Diethylamino-3-thenoylcoumarin, Acros Organics). The photo-resist was stirred for 20 min to thoroughly mix all the components. The DETC dissolved in PETA shows a one-photon absorption peak with a maximum at 420 nm wavelength and a fluorescence emission with a maximum at 480 nm, dropping off to 20% at 532 nm (Fig. 2(b)
Fig. 2 (a) Setup for STED-lithography. Two photon excitation at 780 nm and depletion at 532 nm. PH: pinholes for mode purification, PP: 2π spiral phase plate to create donut beam, objective lens: 100x NA = 1.46, APD: avalanche photodiode. (b) Spectra of photoinitiator DETC in PETA. (c) Measured excitation PSF and (d) depletion PSF, lateral (x,y)-cross sections in focal plane (measured via back-reflection from a gold nanoparticle, diameter 50 nm). (e,f) SEM images of solitary polymerized lines written with (e) ordinary two photon DLW and (f) STED-DLW.
). The setup is sketched in Fig. 2(a). To initiate the polymerization, DETC is excited by laser pulses of 780 nm (82 MHz repetition rate, 110 fs, FFS-tSHG, Toptica, Gräfelfing, Germany). STED is performed via a continuous wave (CW) 532 nm laser (Verdi-V5, Coherent, Santa Clara, CA, USA).

An alpha-Plan Apochromat, 100x, NA = 1.46 oil immersion lens (Zeiss, Oberkochen, Germany) is used. Stage scanning is performed via a capacitively coupled three axes piezo stage (P-611.3S NanoCube, Physik Instrumente PI, Karlsruhe, Germany) mounted on top of a mechanical three axes micrometer driven stage (Thorlabs, Newton, NJ, USA) which exhibits a thermal drift of less than 10 nm/min. A 2π phase spiral (RPC Photonics, Rochester, NY, USA) is used to create the donut mode of the STED beam. The STED laser is circularly polarized via a λ/4 plate before it transmits the phase spiral in order to match its chirality. An avalanche photo diode (APD, SPCM-AQRH, Perkin Elmer, Waltham, MA, USA) is used to monitor the (weak) back-reflected beams transmitting the two last mirrors before the objective lens in order to adjust and properly overlap the two beams on the cover slip. The excitation and the STED PSFs were measured using the back-reflected light from a gold nanoparticle (50 nm diameter) and are shown in Figs. 2(c) and 2(d), respectively. The setup is controlled via a LabView© routine.

The photo-resist was directly pipetted on a cover slip of ~170 µm thickness. The center of the focal volume was set to z = 50 ± 15 nm above the surface of the cover slip (using the faint back reflection from the glass/resist interface as a reference). The lines were written with a speed of 90 µm/s. After exposure, the structures were gently rinsed with 98.4% xylol. Structure characterization was performed with either scanning electron microscopy (SEM) or atomic force microscopy (AFM). Before SEM imaging, the samples were evaporated with approx. 10 nm of platinum to render them conductive. For AFM analysis, the samples were scanned in non-contact mode.

3. Results and discussion

First, we wrote single lines on silane coated cover slips and optimized the intensities of the two-photon excitation and the STED beam. Minimal line widths are found for 3.2 mW and 5.8 mW of excitation and STED power in the focus, respectively. This allows writing lines with a width of 87 nm if only two-photon DLW but no STED beam is applied, see the scanning electron micrograph (SEM) in Fig. 2(e). With the addition of the STED beam, a line width of 54 nm is achieved (Fig. 2(f)). Hence, the addition of the STED beam allows for a substantial reduction of line width as it is expected from the narrowed effective PSF in the case of STED-DLW as compared to two-photon DLW (compare: Figs. 1(d, g)). In order to quantify the line widths in more detail, AFM profiles are taken. One such profile is shown in Fig. 3
Fig. 3 AFM height profile of a line polymerized by STED-DLW showing a width of 57 nm at the base of the line and a FWHM of 34 nm.
, whereby 10 adjacent line scans are averaged in order to reduce noise. We deduce a line width of 57 nm indicated by two vertical blue lines in Fig. 3.

The blue lines indicate the full width of the region where the AFM height profile is elevated above background. Taking this full width is the most conservative measure we can give for the line width. The width of 57 nm resulting from the AFM scan is in good agreement to the 54 nm width determined by inspecting the SEM image (Fig. 2(f)). In fluorescence microscopy, PSFs are usually quantified by the full width at half maximum (FWHM). For comparison, we can also deduce the FWHM of a polymerized line from the AFM profile. This is indicated by the green lines in Fig. 3 and measures 34 nm only. Remember that this line was written with 780 nm light and 532 nm light was used for STED. Both SEM and AFM might overestimate the polymer line width: the former because the SEM images are taken after the evaporation of a few nanometers of platinum onto the polymer structure, the latter because an AFM image is always a convolution of the structure itself and the AFM tip geometry. It is reasonable to assume that vertical evaporation of platinum does not compromise the steepness of the edges of the polymerized lines as much as the convolution with the AFM tip does. This explains why the AFM scans give slightly broader line widths than the SEM images. In any case, it is likely that the true width of the polymerized line is even below 55 nm.

As can be seen in Fig. 3, the lines are only 20 nm in height. This is a well-known feature of 2D DLW on glass surfaces when the intensity is optimized to achieve the smallest lateral features and has been discussed in detail by Kunik et al. [25

25. D. Kunik, S. J. Ludueña, S. Costantino, and O. E. Martínez, “Fluorescent two-photon nanolithography,” J. Microsc. 229(3), 540–544 (2008). [CrossRef] [PubMed]

]. A possible explanation is that the surface provides polymerization nuclei enabling polymerization only at the surface if low power excitation is applied.

With the addition of a 532 nm STED laser, a minimal line width of 65 nm has been reported by Fischer and Wegener [22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

], while we achieve 55 nm line width. One difference to our work is that they used pure PETA, while a mix of PETA and PETTA is used in this study as precursor for polymerization. A second possible reason for narrower structuring could be that a “true” STED dye (DETC) is used instead of isopropyl thioxanthone (ITX) as used in Ref [22

22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

]. It has been found for DETC that the 532 nm STED-beam induces predominantly a fast de-excitation of the first singlet state and hence effectively prevents inter-system crossing to the triplet state, from where radical-polymerization is initiated [32

32. T. J. A. Wolf, J. Fischer, M. Wegener, and A. N. Unterreiner, “Pump-probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography,” Opt. Lett. 36(16), 3188–3190 (2011). [CrossRef] [PubMed]

, 33

33. J. Fischer and M. Wegener, “Ultrafast polymerization inhibition by stimulated emission depletion for three-dimensional nanolithography,” Adv. Mater. 24(10), OP65–OP69 (2012). [CrossRef] [PubMed]

]. In contrast, ITX may cross to the triplet state and the initiation of radical polymerization is hindered only subsequently by excitation within the triplet system [32

32. T. J. A. Wolf, J. Fischer, M. Wegener, and A. N. Unterreiner, “Pump-probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography,” Opt. Lett. 36(16), 3188–3190 (2011). [CrossRef] [PubMed]

, 34

34. B. Harke, P. Bianchini, F. Brandi, and A. Diaspro, “Photopolymerization inhibition dynamics for sub-diffraction direct laser writing lithography,” ChemPhysChem 13(6), 1429–1434 (2012). [CrossRef] [PubMed]

]. It is thinkable that the latter is less effective to prevent polymerization because the triplet state of the ITX molecules is populated at least for some time. In the case of DETC, inter-system crossing is prevented from the first by true STED. To conclude, to the best of our knowledge 55 nm, achieved in this study on lateral feature size, is the narrowest width of polymer lines written with STED-DLW so far.

Figure 5(a)
Fig. 5 (a) AFM profiles of twin lines at nominal distances δ = 100, 120, 140, 160, 180, and 200 nm. 20 adjacent line scans were averaged for each profile. (b) AFM images for distances of 100, 120, 160, and 200 nm. Adjacent lines with δ ≥ 120 nm line distance are clearly resolved in (a) and (b).
shows AFM profiles of twin lines with nominal distances from 100 to 200 nm in steps of 20 nm. For each AFM profile, 20 nearby AFM scan lines across a specific twin line were averaged. Figure 5(b) shows four examples of AFM images of twin lines with nominal distances of 100, 120, 160, and 200 nm. From both the images in Fig. 5(b) and the profiles in Fig. 5(a), it is obvious that the limit of writing two clearly separated lines is given by a mutual distance of 120 nm. Hence, at a distance of 120 nm, a two-photon writing power of 3.2 mW and a STED power of 5.8 mW, the polymerization threshold is located in between the two maxima of the effective PSF and the central minimum (c.f. Fig. 1(i)). Most important, it is not necessary for writing two separated lines that the intermediate minimum of the effective PSF reaches zero intensity. The only requirement is a minimum below the polymerization threshold. This holds already for ordinary two-photon DLW (Fig. 1(f)). However, resolution is further enhanced by the narrower effective STED PSF (Fig. 1(i)), where both the chemical nonlinearity and the nonlinearity of polymerization suppression by STED add up to increase resolution. Compared to the best lateral resolution reported so far in ordinary DLW without STED, which is 200 nm, and for STED-DLW, which is 175 nm, (both reported in Ref [9

9. J. Fischer and M. Wegener, “Three-dimensional direct laser writing inspired by stimulated-emission-depletion microscopy,” Opt. Mater. Express 1(4), 614–624 (2011). [CrossRef]

].) our current finding of a resolution of 120 nm is a substantial improvement.

At a nominal distance of δ = 100 nm, the twin lines are not resolvable any more (Fig. 5(a) and (b)). At this distance, the threshold of polymerization is apparently below a local minimum in between the two effective PSFs of STED-DLW. As we have optimized the powers of two-photon excitation and STED (i.e. in order to get thinnest single lines and best resolution, but yet still connected, unbroken lines at the applied scanning speed of 90 µm/s), it is reasonable to assume that the polymerization threshold is quite close to the peak of the effective PSFs. Hence, we conclude that a spacing of 100 nm is close to the situation sketched in Fig. 1(h), where the Sparrow limit is reached. Since the Sparrow limit is defined as the distance, where the minimum in the center of the summed PSF just vanishes, a broad polymerized line is expected as depicted in Fig. 1(h) and indeed measured as shown in Fig. 5 (δ = 100 nm distance).

We now discuss the “memory” of the photo-resist. The situation depicted in Figs. 1(d) to 1(i) holds for “perfect non-forgetting” photo-resists [23

23. J. Fischer and M. Wegener, “Three-dimensional optical laser lithography beyond the diffraction limit,” Laser Photon. Rev. 7(1), 22–44 (2013). [CrossRef]

]. This means that the below-threshold exposure in the tails of the PSF of the line which is written first and the exposure of the subsequently written line fully add up (for example in the case of the Sparrow-criterion, Figs. 1(e, h)). In contrast, a “perfect forgetting” resist would polymerize only within the narrow region where the exposure exceeds the threshold (Figs. 1(d, g)) and the below threshold exposed tails would not polymerize and hence “forget” their exposure completely by diffusion exchange before the second line is written beneath the first one. In this case, the limit for the structure size and the resolution limit would coincide [23

23. J. Fischer and M. Wegener, “Three-dimensional optical laser lithography beyond the diffraction limit,” Laser Photon. Rev. 7(1), 22–44 (2013). [CrossRef]

]. This seems not to be the case for the currently used mixture of PETA, PETTA and DETC because our current limit for line size amounts to 55 nm while the resolution is 120 nm. Hence, we conclude that the applied mixture of photo-resist is at least partially non-forgetting.

Sakellari and associates designed a forgetting photo-resist by adding highly mobile quenching molecules into the photo-resist formula [35

35. I. Sakellari, E. Kabouraki, D. Gray, V. Purlys, C. Fotakis, A. Pikulin, N. Bityurin, M. Vamvakaki, and M. Farsari, “Diffusion-assisted high-resolution direct femtosecond laser writing,” ACS Nano 6(3), 2302–2311 (2012). [CrossRef] [PubMed]

]. The quenchers are meant to erase the partial polymerization in the below threshold exposed area of the PSF. With this technique, they achieved lateral structure sizes down to 60 nm but the minimum lateral resolution wasnot quantified explicitly. A drawback of the diffusion-assisted perfect forgetting photo-resist is that the time between two laser scans must be longer than the diffusion time of quenchers back into the focal area. This renders the technique a factor of 5 slower compared to STED-DLW [35

35. I. Sakellari, E. Kabouraki, D. Gray, V. Purlys, C. Fotakis, A. Pikulin, N. Bityurin, M. Vamvakaki, and M. Farsari, “Diffusion-assisted high-resolution direct femtosecond laser writing,” ACS Nano 6(3), 2302–2311 (2012). [CrossRef] [PubMed]

].

Figure 6
Fig. 6 Combined plot of all achieved line-to-line distances obtained from AFM images. The “nominal distance” is the distance preset by the piezo scanning stage. The “achieved distance” is the distance determined via AFM measurements of the polymerized lines. No distances could be achieved for 100 nm as the lines are merged. For nominal line spacings at and above 140 nm, the deviation from the nominal distance is + 5 nm and −10 nm (1 sigma). For a nominal spacing of 120 nm, achieved distances substantially shorter than 120 nm (even down to 93 nm) are obtained in some cases.
shows a combined plot of all line-to-line distances determined from AFM line scans. The “nominal distance” is the distance preset by the piezo scanning stage with a positioning error of 1 nm. The thermal drift of the mechanical three axis stage which supports the piezo stage provides a negligible additional error of 0.1 nm within the time it takes to write a twin pair. Hence, the “nominal distance” can be considered as an absolute value because the error is in the range of 1%. The “achieved distance” is the distance determined via AFM measurements of the polymerized lines. No data is provided for a nominal distance of 100 nm because the lines are merged for 100 nm spacing (see Fig. 5). For a nominal line spacing of 140 nm or above, the deviation of the achieved from the nominal distance is + 5 nm and −10 nm (1 sigma). For a nominal spacing of 120 nm, achieved distances substantially shorter than 120 nm (even down to 93 nm) are obtained in some cases. The lines tend to adhere or to partially stick together in these cases. Capillary forces during the washing process might be a possible explanation. Post processing shrinkage in those cases where some polymerization occurred between the two lines could as well pull both lines together by shrinkage. Moreover several additional criteria might have an influence on the variation of the line to line distances, such as diffusion effects, statistics of involved chemical species etc [36

36. D. Van Steenwinckel, R. Gronheid, F. Van Roey, P. Willems, and J. H. Lammers, “Novel method for characterizing resist performance,” J. Micro-Nanolith. Mem. 7, 023002 (2008).

].

4. Conclusion

Acknowledgments

We would like to cordially thank Heidi Piglmayer-Brezina for taking the SEM images and Alois Mühlbachler and Alfred Nimmervoll for technical support. We would like to thank micro resist technology GmbH, Berlin, for providing the surface adhesive OrmoPrime08© free of charge, which we used in some cases as noted. The work was supported by Deutsche Forschungsgemeinschaft (DFG) via grant KL1432/5-1 within the priority program SPP 1327.

References and links

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W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990). [CrossRef] [PubMed]

2.

J. H. Strickler and W. W. Webb, “Three-dimensional optical data storage in refractive media by two-photon point excitation,” Opt. Lett. 16(22), 1780–1782 (1991). [CrossRef] [PubMed]

3.

S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett. 22(2), 132–134 (1997). [CrossRef] [PubMed]

4.

F. Burmeister, S. Steenhusen, R. Houbertz, U. D. Zeitner, S. Nolte, and A. Tünnermann, “Materials and technologies for fabrication of three-dimensional microstructures with sub-100 nm feature sizes by two-photon polymerization,” J. Laser Appl. 24(4), 042014 (2012). [CrossRef]

5.

J. F. Xing, X. Z. Dong, W. Q. Chen, X. M. Duan, N. Takeyasu, T. Tanaka, and S. Kawata, “Improving spatial resolution of two-photon microfabrication by using photoinitiator with high initiating efficiency,” Appl. Phys. Lett. 90(13), 131106 (2007). [CrossRef]

6.

V. F. Paz, M. Emons, K. Obata, A. Ovsianikov, S. Peterhänsel, K. Frenner, C. Reinhardt, B. Chichkov, U. Morgner, and W. Osten, “Development of functional sub-100nm structures with 3D two-photon polymerisation technique and optical methods for characterization,” J. Laser Appl. 24(4), 042004 (2012). [CrossRef]

7.

W. Haske, V. W. Chen, J. M. Hales, W. T. Dong, S. Barlow, S. R. Marder, and J. W. Perry, “65 nm feature sizes using visible wavelength 3-D multiphoton lithography,” Opt. Express 15(6), 3426–3436 (2007). [CrossRef] [PubMed]

8.

X. Z. Dong, Z. S. Zhao, and X. M. Duan, “Improving spatial resolution and reducing aspect ratio in multiphoton polymerization nanofabrication,” Appl. Phys. Lett. 92(9), 091113 (2008). [CrossRef]

9.

J. Fischer and M. Wegener, “Three-dimensional direct laser writing inspired by stimulated-emission-depletion microscopy,” Opt. Mater. Express 1(4), 614–624 (2011). [CrossRef]

10.

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873). [CrossRef]

11.

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994). [CrossRef] [PubMed]

12.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009). [CrossRef]

13.

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006). [CrossRef] [PubMed]

14.

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006). [CrossRef] [PubMed]

15.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009). [CrossRef] [PubMed]

16.

P. A. Pellett, X. L. Sun, T. J. Gould, J. E. Rothman, M. Q. Xu, I. R. Corrêa Jr, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express 2(8), 2364–2371 (2011). [CrossRef] [PubMed]

17.

K. Friedemann, A. Turshatov, K. Landfester, and D. Crespy, “Characterization via two-color STED microscopy of nanostructured materials synthesized by colloid electrospinning,” Langmuir 27(11), 7132–7139 (2011). [CrossRef] [PubMed]

18.

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999). [CrossRef] [PubMed]

19.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000). [CrossRef] [PubMed]

20.

T. F. Scott, B. A. Kowalski, A. C. Sullivan, C. N. Bowman, and R. R. McLeod, “Two-color single-photon photoinitiation and photoinhibition for subdiffraction photolithography,” Science 324(5929), 913–917 (2009). [CrossRef] [PubMed]

21.

L. Li, R. R. Gattass, E. Gershgoren, H. Hwang, and J. T. Fourkas, “Achieving λ/20 resolution by one-color initiation and deactivation of polymerization,” Science 324(5929), 910–913 (2009). [CrossRef] [PubMed]

22.

J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater. 22(32), 3578–3582 (2010). [CrossRef] [PubMed]

23.

J. Fischer and M. Wegener, “Three-dimensional optical laser lithography beyond the diffraction limit,” Laser Photon. Rev. 7(1), 22–44 (2013). [CrossRef]

24.

Y. S. Cao, Z. S. Gan, B. H. Jia, R. A. Evans, and M. Gu, “High-photosensitive resin for super-resolution direct-laser-writing based on photoinhibited polymerization,” Opt. Express 19(20), 19486–19494 (2011). [CrossRef] [PubMed]

25.

D. Kunik, S. J. Ludueña, S. Costantino, and O. E. Martínez, “Fluorescent two-photon nanolithography,” J. Microsc. 229(3), 540–544 (2008). [CrossRef] [PubMed]

26.

L. Rayleigh, “On the theory of optical images, with special reference to the microscope,” Philosoph. Mag. J. Science 42(255), 167–195 (1896). [CrossRef]

27.

C. M. Sparrow, “On spectroscopic resolving power,” Astrophys. J. 44, 76–86 (1916). [CrossRef]

28.

S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature 412(6848), 697–698 (2001). [CrossRef] [PubMed]

29.

S. Juodkazis, V. Mizeikis, K. K. Seet, M. Miwa, and H. Misawa, “Two-photon lithography of nanorods in SU-8 photoresist,” Nanotechnology 16(6), 846–849 (2005). [CrossRef]

30.

D. F. Tan, Y. Li, F. J. Qi, H. Yang, Q. H. Gong, X. Z. Dong, and X. M. Duan, “Reduction in feature size of two-photon polymerisation using SCR500,” Appl. Phys. Lett. 90(7), 071106 (2007). [CrossRef]

31.

S. H. Park, T. W. Lim, D. Y. Yang, N. C. Cho, and K. S. Lee, “Fabrication of a bunch of sub-30nm nanofibers inside microchannels using photopolymerization via a long exposure technique,” Appl. Phys. Lett. 89(17), 173133 (2006). [CrossRef]

32.

T. J. A. Wolf, J. Fischer, M. Wegener, and A. N. Unterreiner, “Pump-probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography,” Opt. Lett. 36(16), 3188–3190 (2011). [CrossRef] [PubMed]

33.

J. Fischer and M. Wegener, “Ultrafast polymerization inhibition by stimulated emission depletion for three-dimensional nanolithography,” Adv. Mater. 24(10), OP65–OP69 (2012). [CrossRef] [PubMed]

34.

B. Harke, P. Bianchini, F. Brandi, and A. Diaspro, “Photopolymerization inhibition dynamics for sub-diffraction direct laser writing lithography,” ChemPhysChem 13(6), 1429–1434 (2012). [CrossRef] [PubMed]

35.

I. Sakellari, E. Kabouraki, D. Gray, V. Purlys, C. Fotakis, A. Pikulin, N. Bityurin, M. Vamvakaki, and M. Farsari, “Diffusion-assisted high-resolution direct femtosecond laser writing,” ACS Nano 6(3), 2302–2311 (2012). [CrossRef] [PubMed]

36.

D. Van Steenwinckel, R. Gronheid, F. Van Roey, P. Willems, and J. H. Lammers, “Novel method for characterizing resist performance,” J. Micro-Nanolith. Mem. 7, 023002 (2008).

37.

H. B. Sun, S. Matsuo, and H. Misawa, “Three-dimensional photonic crystal structures achieved with two-photon-absorption photopolymerization of resin,” Appl. Phys. Lett. 74(6), 786–788 (1999). [CrossRef]

38.

J. Fischer, T. Ergin, and M. Wegener, “Three-dimensional polarization-independent visible-frequency carpet invisibility cloak,” Opt. Lett. 36(11), 2059–2061 (2011). [CrossRef] [PubMed]

39.

J. A. Chai, L. S. Wong, L. Giam, and C. A. Mirkin, “Single-molecule protein arrays enabled by scanning probe block copolymer lithography,” Proc. Natl. Acad. Sci. U.S.A. 108(49), 19521–19525 (2011). [CrossRef] [PubMed]

40.

R. Schlapak, J. Danzberger, T. Haselgrübler, P. Hinterdorfer, F. Schäffler, and S. Howorka, “Painting with biomolecules at the nanoscale: biofunctionalization with tunable surface densities,” Nano Lett. 12(4), 1983–1989 (2012). [CrossRef] [PubMed]

41.

R. A. Vega, D. Maspoch, K. Salaita, and C. A. Mirkin, “Nanoarrays of single virus particles,” Angew. Chem. Int. Ed. Engl. 44(37), 6013–6015 (2005). [CrossRef] [PubMed]

42.

C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber, “Geometric control of cell life and death,” Science 276(5317), 1425–1428 (1997). [CrossRef] [PubMed]

43.

F. Klein, B. Richter, T. Striebel, C. M. Franz, G. Freymann, M. Wegener, and M. Bastmeyer, “Two-component polymer scaffolds for controlled three-dimensional cell culture,” Adv. Mater. 23(11), 1341–1345 (2011). [CrossRef] [PubMed]

OCIS Codes
(180.0180) Microscopy : Microscopy
(110.4235) Imaging systems : Nanolithography
(220.4241) Optical design and fabrication : Nanostructure fabrication

ToC Category:
Microscopy

History
Original Manuscript: January 2, 2013
Revised Manuscript: March 29, 2013
Manuscript Accepted: March 31, 2013
Published: April 25, 2013

Virtual Issues
Vol. 8, Iss. 6 Virtual Journal for Biomedical Optics

Citation
Richard Wollhofen, Julia Katzmann, Calin Hrelescu, Jaroslaw Jacak, and Thomas A. Klar, "120 nm resolution and 55 nm structure size in STED-lithography," Opt. Express 21, 10831-10840 (2013)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-21-9-10831


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References

  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science248(4951), 73–76 (1990). [CrossRef] [PubMed]
  2. J. H. Strickler and W. W. Webb, “Three-dimensional optical data storage in refractive media by two-photon point excitation,” Opt. Lett.16(22), 1780–1782 (1991). [CrossRef] [PubMed]
  3. S. Maruo, O. Nakamura, and S. Kawata, “Three-dimensional microfabrication with two-photon-absorbed photopolymerization,” Opt. Lett.22(2), 132–134 (1997). [CrossRef] [PubMed]
  4. F. Burmeister, S. Steenhusen, R. Houbertz, U. D. Zeitner, S. Nolte, and A. Tünnermann, “Materials and technologies for fabrication of three-dimensional microstructures with sub-100 nm feature sizes by two-photon polymerization,” J. Laser Appl.24(4), 042014 (2012). [CrossRef]
  5. J. F. Xing, X. Z. Dong, W. Q. Chen, X. M. Duan, N. Takeyasu, T. Tanaka, and S. Kawata, “Improving spatial resolution of two-photon microfabrication by using photoinitiator with high initiating efficiency,” Appl. Phys. Lett.90(13), 131106 (2007). [CrossRef]
  6. V. F. Paz, M. Emons, K. Obata, A. Ovsianikov, S. Peterhänsel, K. Frenner, C. Reinhardt, B. Chichkov, U. Morgner, and W. Osten, “Development of functional sub-100nm structures with 3D two-photon polymerisation technique and optical methods for characterization,” J. Laser Appl.24(4), 042004 (2012). [CrossRef]
  7. W. Haske, V. W. Chen, J. M. Hales, W. T. Dong, S. Barlow, S. R. Marder, and J. W. Perry, “65 nm feature sizes using visible wavelength 3-D multiphoton lithography,” Opt. Express15(6), 3426–3436 (2007). [CrossRef] [PubMed]
  8. X. Z. Dong, Z. S. Zhao, and X. M. Duan, “Improving spatial resolution and reducing aspect ratio in multiphoton polymerization nanofabrication,” Appl. Phys. Lett.92(9), 091113 (2008). [CrossRef]
  9. J. Fischer and M. Wegener, “Three-dimensional direct laser writing inspired by stimulated-emission-depletion microscopy,” Opt. Mater. Express1(4), 614–624 (2011). [CrossRef]
  10. E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie9(1), 413–418 (1873). [CrossRef]
  11. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett.19(11), 780–782 (1994). [CrossRef] [PubMed]
  12. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics3(3), 144–147 (2009). [CrossRef]
  13. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006). [CrossRef] [PubMed]
  14. K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods3(9), 721–723 (2006). [CrossRef] [PubMed]
  15. C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature457(7233), 1159–1162 (2009). [CrossRef] [PubMed]
  16. P. A. Pellett, X. L. Sun, T. J. Gould, J. E. Rothman, M. Q. Xu, I. R. Corrêa, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express2(8), 2364–2371 (2011). [CrossRef] [PubMed]
  17. K. Friedemann, A. Turshatov, K. Landfester, and D. Crespy, “Characterization via two-color STED microscopy of nanostructured materials synthesized by colloid electrospinning,” Langmuir27(11), 7132–7139 (2011). [CrossRef] [PubMed]
  18. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett.24(14), 954–956 (1999). [CrossRef] [PubMed]
  19. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A.97(15), 8206–8210 (2000). [CrossRef] [PubMed]
  20. T. F. Scott, B. A. Kowalski, A. C. Sullivan, C. N. Bowman, and R. R. McLeod, “Two-color single-photon photoinitiation and photoinhibition for subdiffraction photolithography,” Science324(5929), 913–917 (2009). [CrossRef] [PubMed]
  21. L. Li, R. R. Gattass, E. Gershgoren, H. Hwang, and J. T. Fourkas, “Achieving λ/20 resolution by one-color initiation and deactivation of polymerization,” Science324(5929), 910–913 (2009). [CrossRef] [PubMed]
  22. J. Fischer, G. von Freymann, and M. Wegener, “The materials challenge in diffraction-unlimited direct-laser-writing optical lithography,” Adv. Mater.22(32), 3578–3582 (2010). [CrossRef] [PubMed]
  23. J. Fischer and M. Wegener, “Three-dimensional optical laser lithography beyond the diffraction limit,” Laser Photon. Rev.7(1), 22–44 (2013). [CrossRef]
  24. Y. S. Cao, Z. S. Gan, B. H. Jia, R. A. Evans, and M. Gu, “High-photosensitive resin for super-resolution direct-laser-writing based on photoinhibited polymerization,” Opt. Express19(20), 19486–19494 (2011). [CrossRef] [PubMed]
  25. D. Kunik, S. J. Ludueña, S. Costantino, and O. E. Martínez, “Fluorescent two-photon nanolithography,” J. Microsc.229(3), 540–544 (2008). [CrossRef] [PubMed]
  26. L. Rayleigh, “On the theory of optical images, with special reference to the microscope,” Philosoph. Mag. J. Science42(255), 167–195 (1896). [CrossRef]
  27. C. M. Sparrow, “On spectroscopic resolving power,” Astrophys. J.44, 76–86 (1916). [CrossRef]
  28. S. Kawata, H. B. Sun, T. Tanaka, and K. Takada, “Finer features for functional microdevices,” Nature412(6848), 697–698 (2001). [CrossRef] [PubMed]
  29. S. Juodkazis, V. Mizeikis, K. K. Seet, M. Miwa, and H. Misawa, “Two-photon lithography of nanorods in SU-8 photoresist,” Nanotechnology16(6), 846–849 (2005). [CrossRef]
  30. D. F. Tan, Y. Li, F. J. Qi, H. Yang, Q. H. Gong, X. Z. Dong, and X. M. Duan, “Reduction in feature size of two-photon polymerisation using SCR500,” Appl. Phys. Lett.90(7), 071106 (2007). [CrossRef]
  31. S. H. Park, T. W. Lim, D. Y. Yang, N. C. Cho, and K. S. Lee, “Fabrication of a bunch of sub-30nm nanofibers inside microchannels using photopolymerization via a long exposure technique,” Appl. Phys. Lett.89(17), 173133 (2006). [CrossRef]
  32. T. J. A. Wolf, J. Fischer, M. Wegener, and A. N. Unterreiner, “Pump-probe spectroscopy on photoinitiators for stimulated-emission-depletion optical lithography,” Opt. Lett.36(16), 3188–3190 (2011). [CrossRef] [PubMed]
  33. J. Fischer and M. Wegener, “Ultrafast polymerization inhibition by stimulated emission depletion for three-dimensional nanolithography,” Adv. Mater.24(10), OP65–OP69 (2012). [CrossRef] [PubMed]
  34. B. Harke, P. Bianchini, F. Brandi, and A. Diaspro, “Photopolymerization inhibition dynamics for sub-diffraction direct laser writing lithography,” ChemPhysChem13(6), 1429–1434 (2012). [CrossRef] [PubMed]
  35. I. Sakellari, E. Kabouraki, D. Gray, V. Purlys, C. Fotakis, A. Pikulin, N. Bityurin, M. Vamvakaki, and M. Farsari, “Diffusion-assisted high-resolution direct femtosecond laser writing,” ACS Nano6(3), 2302–2311 (2012). [CrossRef] [PubMed]
  36. D. Van Steenwinckel, R. Gronheid, F. Van Roey, P. Willems, and J. H. Lammers, “Novel method for characterizing resist performance,” J. Micro-Nanolith. Mem.7, 023002 (2008).
  37. H. B. Sun, S. Matsuo, and H. Misawa, “Three-dimensional photonic crystal structures achieved with two-photon-absorption photopolymerization of resin,” Appl. Phys. Lett.74(6), 786–788 (1999). [CrossRef]
  38. J. Fischer, T. Ergin, and M. Wegener, “Three-dimensional polarization-independent visible-frequency carpet invisibility cloak,” Opt. Lett.36(11), 2059–2061 (2011). [CrossRef] [PubMed]
  39. J. A. Chai, L. S. Wong, L. Giam, and C. A. Mirkin, “Single-molecule protein arrays enabled by scanning probe block copolymer lithography,” Proc. Natl. Acad. Sci. U.S.A.108(49), 19521–19525 (2011). [CrossRef] [PubMed]
  40. R. Schlapak, J. Danzberger, T. Haselgrübler, P. Hinterdorfer, F. Schäffler, and S. Howorka, “Painting with biomolecules at the nanoscale: biofunctionalization with tunable surface densities,” Nano Lett.12(4), 1983–1989 (2012). [CrossRef] [PubMed]
  41. R. A. Vega, D. Maspoch, K. Salaita, and C. A. Mirkin, “Nanoarrays of single virus particles,” Angew. Chem. Int. Ed. Engl.44(37), 6013–6015 (2005). [CrossRef] [PubMed]
  42. C. S. Chen, M. Mrksich, S. Huang, G. M. Whitesides, and D. E. Ingber, “Geometric control of cell life and death,” Science276(5317), 1425–1428 (1997). [CrossRef] [PubMed]
  43. F. Klein, B. Richter, T. Striebel, C. M. Franz, G. Freymann, M. Wegener, and M. Bastmeyer, “Two-component polymer scaffolds for controlled three-dimensional cell culture,” Adv. Mater.23(11), 1341–1345 (2011). [CrossRef] [PubMed]

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