Three-dimensional fluorescence imaging of DBD-plasma aminated porous polypropylene

J.-E. Ehlers1, A. Hinze2, C.-P. Klages2, K.-H. Gericke1

1Institut für Physikalische und Theoretische Chemie, Technische Universität Braunschweig, Braunschweig, Germany

2Institut für Oberflächentechnik, Technische Universität Braunschweig, Braunschweig, Germany

Abstract: Porous polypropylene substrates (PP membranes) were aminated area-selectively with atmospheric pressure DBD microplasma. The amino groups were labeled with a fluorescent dye and investigated with two-photon microscopy. Three-dimensional distributions of amino group density could be acquired with sub-micrometer resolution. Amino group densities have shown to be significantly higher than for compact substrates. Keywords: PP membrane, DBD-plasma amination, two-photon fluorescence microscopy

1. Micro structured plasma treatment of polymer surfaces

Polypropylene is a saturated hydrocarbon with good mechanical properties as well as hydrophobicity and chemical inertness with respect to biochemical reactions. By means of atmospheric-pressure DBD microplasma treatment using silicone-based plasma stamps, it is possi-ble to introduce an area-selective surface modification [1, 2]. The introduction of amino groups for instance yields area-selective hydrophilicity and nucleophilicity. The reactive amino groups may serve as anchor groups for all kinds of biosynthesis used in bioassays. The concept allows for building up microarrays with spot diameters in the micrometer range. 2. Employment of novel substrate materials

Polyethylene (PE) or polypropylene (PP) foils are often-used substrates for plasma treatment. These foils show a compact morphology, i.e. a dense surface on a macroscopic scale. In contrary, substrates like PP mem-branes (PP-M) exhibit a high specific surface area with voids on the scale of micrometers or even less. This property is of avail for increasing the surface density of plasma generated amino groups on polymers to such a degree that can never be achieved for compact surfaces. 3. Characterization and visualization of amino groups on porous materials with fluorescence microscopy

Due to the porosity of PP membranes, the analysis of the three-dimensional distribution of amino groups is not feasible for most analysis methods. Fluorescence micros-copy is a suitable analysis method for surfaces since it is an absolute measurement method with low detection limits. Fluorescence is a good sensor for characterization of microenvironments and the labeling dyes used are often highly chemically selective. There exist commer-cially available fluorogenic tags for amino groups, e.g. fluorescamine or NBD-F [3]. These substances only yield

fluorescence upon excitation when bound to an amino group, hence showing a high chemical specificity. The fluorescence intensity correlates directly with the amino group density. 4. Two-photon microscopy (TPM)

Providing a sufficiently high photon flux, a molecule can absorb two photons simultaneously if the sum of the photon energies corresponds to the energy of an excited state of the molecule. However, owing to the very low two-photon absorption cross sections compared to the one-photon process, pulsed lasers are required that supply temporary high power. The most often laser system used for two-photon excitation is a titanium doped sapphire laser (Ti:Sa) that provides femtosecond pulses. Since it has a tunable output wavelength, ranging typically from 700 to 1000 nm [4], that would correspond to a one- photon excitation from 350 to 500 nm in first approxima-tion, it is a versatile excitation source in fluorescence microscopy. The excitation with near infrared light leads to an excellent spectral discrimination of fluorescence from the excitation light. As a direct consequence of high photon fluxes required for observable two-photon absorp-tion, light is only absorbed in the focal point region of a focused laser beam. This leads to an intrinsic three-dimensional resolution that is only diffraction limited. The excitation volume exhibits a spheroid shape that is called point spread function whose lateral and axial radii can easily be calculated in good approximation [5]. Additionally, two-photon excitation takes advantage of low light scattering and high penetration depths for most samples due to near infrared excitation wavelengths [6]. The pulsed excitation allows for time-resolved measure-ments of fluorescence decay that provides possibility to fluorescence lifetime imaging (FLIM), which in turn gives rise to information on the local chemical environ-ment [7]. The data can either be acquired by a time-gated light intensifier coupled to a CCD camera or time-corre-lated single photon counting with either a photomultiplier

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7.2. Amino group imaging and 3D rendering

Two-photon fluorescence microscopy allows visual-izing the morphology and topology of complex substrates as it applies to PP membranes. Thereby it is able to re-solve features down to few hundreds of nanometers in a quality comparable to scanning electron microscopy in that dimension as can be seen in Fig. 4 and 5.

Fig. 4 Top side surface of a PP-M – TPM image of primary amino groups (left) and SEM image (right).

Fig. 5 Bottom side surface of a PP-M – TPM image of primary amino groups (left) and SEM image (right).

While the top side of the membrane is governed by large prolate, irregular pores, the bottom side is character-ized by honeycomb-like depressions and smaller pore sizes (0.2 µm according to manufacturer). Both features are well resolved. In contrast to SEM images, TPM images contain chemical information. In this case, the fluorescence specifically comes from labeled primary amino groups in which the fluorescence intensity is pro-portional to the amino group surface density. This can easily be verified when acquiring an image of the edge of an aminated spot as shown in Fig. 6. The amino group density decreases towards the edge of the spot and vanishes almost completely outside the spot.

Fig. 6 Edge of an aminated spot on PP-M. A false color intensity code is used for better visualization as in

subsequent pictures (bar right of the picture). The absolute intensity values differ from image to image.

7.3. 3D rendering and axial amino group density profiles

Additionally, TPM is capable of 3D sectioning taking advantage of the intrinsic three-dimensional resolution. Several 2D images at different sampling depths forming a so-called z-stack can be used for 3D rendering. Thus, additional information can be extracted from the data and be visualized. An example is shown in Fig. 7 and 8; Fig. 7 showing the 2D data and the left image of Fig. 8 depicting a selected view on the 3D rendered data.

Fig. 7 Z-stack of a PP-M top side (80×80 µm2). Distance between two images is 2.5 µm.

Fig. 8 3D rendering of amino group density of a PP-M top side spot in the middle (left) and the edge (right).

The images shown have been rendered with Volume Viewer, a free plugin for ImageJ (author: K.U. Barthel, Berlin, Germany). With this tool, the axial distribution of amino groups on a PP membrane can impressively be illustrated. Note that the highest amino group intensity is found not directly on the surface but a few micrometers inside the membrane when averaging over a certain area. This is due to the fact of an undulated surface. Plotting the depth intensity profile of a z-stack with averaging over a sufficiently large area yields a monoexponential decay of amino group density with respect to depth as depicted in Fig. 9.

Fig. 9 Axial amino group density profiles of single pixels and a 30×30 µm2 area (green line) with a

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7.4. Filamentary discharge – sliding discharge When imaging amino group spots on PP membranes a

remarkable feature of porous materials for plasma treat-ment can be observed. Amino groups not only form on the upper few micrometer of the membrane, but there are some “tubes” going further down into the membrane (cf. Fig. 10, left). Their diameter ranges from about two up to ten micrometer. It is believed that this phenomenon can be assigned to filamentary discharges through the porous substrate. Interestingly, looking only at the surface these filaments cannot be detected. It is assumed that the filaments penetrate the whole membrane thickness.

Fig. 10 3D image of a filamentary discharge (left) and a sliding discharge on a PP-M surface (right).

Another characteristic of area-selectively plasma-treated porous substrates is the appearance of sliding dis-charges on the surface as shown on the right side of Fig. 10. These discharges occur between the plasma stamp cavities and are low in fluorescence intensity, respectively amino group density compared to the spots. Although a 10 kg equivalent compression force is applied to the DBD setup (cf. Fig. 1) and the PDMS is flexible enough to even out the substrate’s imperfection of flatness, it appears as if microscopic cavities remain which give rise to these sliding discharges.

7.5. Application of an additional dielectric barrier and treatment depth comparison

As mentioned in the “materials and methods” section an additional barrier was used for the plasma treatment of PP-M because of its porosity. A 75 µm PP foil is sufficient to guarantee a uniform treatment of the membrane, how-ever, filamentary discharges can still be observed. When omitted, an electrical breakdown is observed in the center of the plasma spots where electric field strength is highest. As consequence, a hole in the center of the spots is gener-ated, having a diameter of several tens of micrometers as shown in Fig. 11.

Fig. 11 3D-rendered image of a plasma-generated hole in the membrane (left) and a fluorescence lifetime section

(right). The structure of the hole indicates that the polymer has

melted during the breakdown. In addition, a fluorescence lifetime image highlights a change in the chemical struc-ture of the polymer. The inner ring (cf. Fig. 11, right) exhibits a lower fluorescence lifetime than the outer part where normal plasma treatment has occurred. Thus, the fluorescence lifetime yields additional information on the plasma treatment of the membrane and can be used as control constant for the analysis of plasma treated poly-mers.

The treatment depth of both top and bottom side, i.e. the 1/e- or z-value has been investigated for plasma treat-ment with and without additional dielectric barrier. It was found that the treatment depth varies significantly from measured area to area. Nevertheless, the trend shows that top and bottom side have a comparable treatment depth of circa 4 µm which is increased to circa 5 µm when omit-ting the additional dielectric barrier. It has to be pointed out that the treatment depth is the same at the edge of the plasma spots; only the amino group density is lower (cf. Fig. 8). Acknowledgement

The support of the VolkswagenStiftung for the project “Microstructured Surface Treatment by Atmospheric-Pressure Microplasmas” (reference number I/81252 and I/81255) under the initiative “Innovative Methods for Manufacturing of Multifunctional Surfaces” is greatly acknowledged. The authors thank Nina Lucas for providing the PDMS plasma stamps. References [1] N. Lucas, V. Ermel, M. Kurrat, S. Büttgenbach, J.

Phys. D: Appl. Phys. 41, 215202, (2008). [2] N. Lucas, A. Hinze, C.-P. Klages, S. Büttgenbach, J.

Phys. D: Appl. Phys. 41, 194012, (2008). [3] K. Imai, T. Toyo’oka, H. Miyano, Analyst, 109, 1365,

(1984). [4] B.R. Masters, P.T.C. So, Microscopy Research and

Technique 63, 3, (2004). [5] W.R. Zipfel, R.M. Williams, W.W. Webb, Nature

Biotechn. 21, 1369, (2003). [6] P.T.C. So et al., Annu. Rev. Biomed. Eng. 02, 399,

(2000). [7] J.R. Lakowicz, Principles of Fluorescence Spectros-

copy 2nd ed., Kluwer Academic, New York, (1999). [8] W. Becker, A. Bergmann, Lifetime Imaging

Techniques for Optical Microscopy, Becker&Hickl GmbH, (2003).

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