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Collagen network as the scaffold for spontaneously distributed optical resonators
Organic Electronics 39 (2016) 100e104
Contents lists available at ScienceDirect
Organic Electronics journal homepage: www.elsevier.com/locate/orgel
Collagen network as the scaffold for spontaneously distributed optical resonators Konrad Cyprych a, *, Martyna Janeczko a, Ileana Rau b, Francois Kajzar b, Jaroslaw Mysliwiec a a b
Advanced Materials Engineering and Modelling Group, Wroclaw University of Science and Technology, Wyb, Wyspianskiego 27, 50-370 Wroclaw, Poland Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street No 1, 011061, Bucharest, Romania
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2016 Received in revised form 26 September 2016 Accepted 27 September 2016
The idea behind this work was to acquire one dimensional lasing in the Rhodamine 6G dye doped collagen fibers with the control of random lasing phenomenon properties by changing the dye molecular arrangement. We show that a simple manipulation of the dye concentration in biopolymeric fibril matrix bulk and an additional use of a-cyclodextrin (a-CD) molecules determine formation of specific dye aggregates and as a consequence the shift of the random lasing emission wavelength in the desired direction. The analysis of the light transport mean free path in function of the dye and (a-CD) concentration was done with the use of coherent back scattering technique. © 2016 Elsevier B.V. All rights reserved.
Keywords: Collagen Random lasing a-Cyclodextrin Rhodamine 6G
1. Introduction Bioderived and bioinspired materials are of immense current interest because they can provide novel, attractive physical properties while being able to be produced by “green”, environmentfriendly technologies. One of them is collagen, whose name originates from Greek cola, meaning glue [1]. Collagen is a chiral molecule with finds a growing interest for application in photonics. It is also one of the most spread proteins in human body which plays a role of scaffold for mammalian tissues providing them the elasticity and the durability. Structurally collagen is composed of three polypeptide chains forming a-helix, linked together by hydrogen bonds between hydroxylysine and hydroxyproline and by covalent bonds, that can form different collagen folding forms. Several examples of interesting photonic applications of collagen have been presented in the past. The building unit of collagen is the tropocollagen. It is a noncentrosymmetric molecule, exhibiting second harmonic generation (SHG) as observed by several research groups. The first observation of SHG in collagen was reported by Vasilenko et al. as early as in 1965 [2] what was confirmed later by Fine and Hansen as well as by Roth and Freund [3e5]. Yova et al.
* Corresponding author. E-mail address: [email protected] (K. Cyprych). http://dx.doi.org/10.1016/j.orgel.2016.09.032 1566-1199/© 2016 Elsevier B.V. All rights reserved.
have shown the optical second harmonic generation by collagen Type I. A broad wavelength tuning of harmonic wavelength from 383 to 532 nm was achieved [6]. The same group presented also Rhodamine 6G (Rh6G) and collagen application for energy transfer, in which Rhodamine serves as a donor in the system after the excitation to S2 energy level by 1064 nm via two photon absorption. Collagen, as an acceptor, has shown the fluorescence emission maximum intensity at 390 nm [7]. As a part of the light amplification system, collagen was used as an integral building block of a bone tissue, in which the random lasing phenomenon was observed, however the crucial role in this process was attributed to the hydroxyapatite, responsible for strong light scattering, in order to acquire spontaneously formed optical resonators [8]. Basing on this approach a method of morphology deformations detection, recording the changes in random lasing emission in bone during a stress applied to the tissue was developed [9]. The random lasing, was also observed in the egg membranes, enriched with Rhodamine 6G. Such a natural membrane, among the others, contains the collagen in its structure. Dense network of fibril proteins, positioned in random directions, are acting as scatterers for the optical cavities formation [10]. Cyclodextrins (CD) are circularly formed poly-glucose, playing a crucial role in the drug delivery as its carriers. The structure of cyclodextrin forms geometrical cone with hydrophobic interior and hydrophilic exterior [11]. Application of this material is driven by
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
the formation of specific interactions inside the circular structure, capable to block diffusion of the compound from its interior. The mechanism standing for an efficient CD use, e.g. in drug delivery, is based on the cell membrane permeability for glucose moieties, reaching and penetrating them. The same happens with cyclodextrins, releasing their package of carried molecule inside the targeted cell. It was found that Rh6G and b-cyclodextrin are forming inclusion complexes (IC) having relation to the fluorescence emission wavelength and intensity from them [12,13]. However, the mentioned inclusion complexes are not revealing the detailed structure of Rh6G aggregates as it was done in the work of Martinez, concerning the laponite clay [14,15]. There have been accounted that formation of IC are responsible for the enhancement of fluorescence signal as presented for Rhodamine B and bis(cyclodextrin)s host [16]. In the present work we demonstrate that in the system based on fibril collagen proteins, serving role of a host material for fluorescent dyes and CD penetrating protein structure, lasing action can be achieved and due to the specific aggregates formation the emission wavelength can be easily shifted. 2. Materials and methods For the light amplification studies we have selected the wellknown water soluble Rhodamine 6G luminescent dye which possesses excellent fluorescence quantum yield. Collagen Type I, extracted from bovine derma, in gel form of concentration equal to 2.2%, was obtained from Collagen Department of Division Leather and Footwear Research Institute, Bucharest, Romania where the purification and the preparation procedure was performed according to the work of Albu [17]. Series of thin film samples were prepared by drop casting on glass slides. They were made using the gelatinized collagen with Rh6G concentration varying from 1% up to 3% (w/w) with respect to dry collagen. For one selected Rh6G:collagen ratio equal to 2% (w/ w) additionally a-cyclodextrin purchased from Sigma Aldrich at molar ratios a-CD:Rh6G equal to 100:1, 10:1 and 1:1, respectively, was added. Quality and morphology of prepared thin layers were evaluated with the inverted optical microscope Olympus IX71 and atomic force microscope Dimension V (Veeco) in tapping mode. The random lasing phenomenon studies were done using the experimental setup with pulsed nanosecond Nd:YAG laser, operating at lex ¼ 532 nm, as excitation source with stripe-like beam geometry of dimensions equal to 3.0 0.5 mm. Lasing spectra were collected by the Shamrock SR-163 fiber spectrometer in function of excitation energy density. Simultaneously to the random lasing emission measurements the optical visualization of the excited area was done using optical system of 0.8 mm resolution, capable to observe and correlate the emission to the position on the sample. Additionally to the light amplification experiments, the coherent backscattering (CBS) studies were performed, in order to get the value of light transport mean free path. It is a crucial parameter in analysis of the statistics of the distance that light travels before it is scattered. 3. Results and discussion 3.1. Collagen layers morphology The microscopic studies show that dye doped collagen deposited on the glass slide is forming fibers arranged in random directions at the 2D surface area. The image analysis done with the ImageJ confirms this situation which results from the isotropic distribution of fibrous structures (Fig. 1a) [18]. The dye doped collagen thin films morphology study was performed by the AFM
101
analysis. The acquired topographies, presented in Fig. 1b and d) indicate that the superhelix collagen fiber of diameter about Dfiber ¼ 5 mm is formed and built from random coil macromolecules with diameter less than dcoil < 50 nm. The formed collagen loose protein network on the glass surface, surrounded with not fibrillated protein film containing Rh6G, and Rh6G aggregates in the fiber structure is shown in Fig. 1. All studied samples exhibit similar morphologic properties. Dye free samples, as well as Rh6G doped collagen fibers casted on the glass slide were investigated using experimental system for CBS measurements. As a source of scattered light cw HeeNe laser of 633 nm emission wavelength was used, which is off the absorption of the samples. Incorporation of Rh6G and a-CD do not show significant effect to the calculated light transport mean free path therefore the designated parameters were evaluated statistically indicating the source of scattering overall the fiber network. The light transport mean free path was calculated for all samples using Equation (1) [19e21]. The obtained average value was equal to lt ¼ 16.8 ± 1.8 mm meaning that this property is one of the whole system and not only for the single collagen fiber. Fig. 2 presents, as an example, the coherent backscattering cone for 3% (w/w) Rh6G doped collagen sample.
lt ¼
0:7,l 2p,W
(1)
where lt is light mean transport path, l ¼ 633 nm, and W stands for the full scattering cone width at its half maximum. 3.2. Multimode fiber lasing Collagen fibril proteins, enriched with Rh6G, become to be the light amplifying medium, able to exhibit the phenomenon of amplified spontaneous emission (ASE) and random lasing (RL). We have focused on the lasing action generation based on the distributed 2D network of dye doped collagen fibers. According to the known phenomenon, that we had described previously, we have used random fibril system in order to acquire tunable light emission relaying on formation of Rh6G aggregates sizing effect responsible for the shift of the light emission wavelength [22]. As was expected, the Rh6G aggregation affects to the RL emission properties. In the measured samples an RL tuning from 598 nm to the 610 nm with about ~10 nm spectral FWHM was observed, as presented in Fig. 3. This effect is related to the formation of J-aggregates and higher order aggregates responsible for the red shift of the emission wavelength [14,15]. The lasing modes were not clearly visible, due to the non-resonant feedback meaning the light intensity increasing in the gain medium upon excitation at the gain length [23e25]. With the microscopy analysis done during the lasing it was revealed that Rh6G forms inclusions, which are colocalized with the fibers positions and are acting as scattering centers embedded in the fiber structure as it is shown in Fig. 4. Although Rh6G aggregates are playing the role of scatterers, the formation of resonant modes has not been observed and light is not localized within the inclusions in collagen fibers. Rather than the densely packed 2D fiber network is responsible for amplification with wave energy dissipation with scattering. Changing the pumping beam energy density a simultaneous “blinking”, coming from inclusions along the fibers, was observed. It is accounted as due to the reaching population inversion in the gain region responsible for the “on” and “off”, turning characteristic for random lasing presented in Fig. 4. Loading collagen fibers with increasing amount of Rh6G resulted in lowering the lasing threshold from r1% ¼ 6.0 mJ/cm2 for lowest Rh6G concentration (1% w/w), down to r3% ¼ 3.4 mJ/cm2 for 3% (w/w). This effect is related to the
102
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
Fig. 1. Transmission mode optical microscope image a), and atomic force microscopy analysis of collagen fibers, b,c) size map and profile of single collagen fiber of a diameter about Dfiber ¼ 5 mm, and d,e) enlargement of single fiber with observed single macromolecules of diameter about dcoil ¼ 50 nm. Presented data come from 2% (w/w) Rh6G:collagen layer.
Fig. 2. Coherent backscattering cone for 3% (w/w) Rh6G doped collagen sample with calculated mean light transport mean free path lt ¼ 16.2 mm.
increasing probability of excitation in the presence of larger amount of Rh6G, but still above the lasing threshold there were not seen any spatial modes testifying the light amplification was not reached on the spatially localized feedback mechanism [26,27]. Basing on the acquired results and the described phenomenon we focused on the direct control of the lasing emission wavelength due to the predictions of aggregates formation, favoring especially the smaller ones. The elaborated solution was found taking advantage of the a-cyclodextrin properties. Samples containing collagen, Rh6G and a-cyclodextrin in different ratios [aCD]:[Rh6G] ranging from 1:1 up to 100:1 (mol/mol) were prepared. In contrary to the lasing emission observed from higher order aggregates, a shift to lower wavelengths in function of the increasing a-CD amount was obtained. As it can be seen in Fig. 5 just taking advantage of the hydrophilic-hydrophobic interactions between aCD and Rh6G and formation of different types of aggregates, we were able to tune the emission wavelength for almost 20 nm. Applied cyclodextrin becomes a carrier for Rhodamine 6G molecules, favoring only the specific aggregates sizes binding. This behavior is observed only in solid-state form of the collagen layers. The solutions prepared for layer fabrication, containing Rh6G, collagen and a-CD exhibit emission at 550 nm, what indicates that
Fig. 3. Normalized random lasing intensities from single collagen fibers doped with increasing amount of Rh6G: from 1% (w/w) (green), 2% (w/w) (yellow) and 3% (w/w) (red) with respect to collagen dry mass. With increasing dopant content the emission wavelength shifts from l1% ¼ 598 nm to l3% ¼ 610 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the fluorescence is originating from Rh6G monomers. The results concerning lasing properties of Rh6G doped collagen with different amounts of a-CD are presented in Table 1. Similarly as for Rh6G doped collagen, the emission from samples enriched with a-CD is colocalized with collagen fibers. This fact indicates that Rhodamine, stabilized by cyclodextrin is adsorbed by protein macrostructures. The mechanism behind the binding is not clear yet. The localization of inclusion complexes in the collagen fibers with and without a-CD has been cross-checked by the analysis of lasing stability in time. The observed material degradation at energy density well above the lasing threshold shows comparable lasing stability for Rh6G doped collagen as well as in the presence of a-CD. Unfortunately the designated stabilities indicate a high oxygen permeability and dye bleaching according to the similar DNA based systems [28,29].
3.3. Gain regions in the fiber structure Characteristic occurrence of inclusion complexes embedded in the collagen were distributed along the single fiber length. The
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
103
Fig. 4. Series of microscopic images showing colocalization of RL emission and dense collagen fiber network. Presented formation of optical resonators in the series of photos taken below threshold, at lasing and collagen network visible after Rh6G bleaching.
Fig. 5. Normalized intensities of lasing emission of a-CD:Rh6G in collagen in selected molar ratios 0:1 (mol/mol) (black), 1:1 (mol/mol) (green), 10:1 (mol/mol) (yellow), 100:1 (mol/mol) (red), Rh6G concentration with respect to dry collagen was equal 2% (w/w). Effect of the a-CD on the RL emission with visible shift to the shorter wavelengths for 100:1 M ratio a-CD:Rh6G in collagen matrices is clearly seen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
acquired microscopic images (Fig. 6) were subjected to the image particle analysis, what allowed to describe statistically the excited objects. Shining objects diameter at lasing emission conditions have shown log-normal distribution with the maximum at dobj ¼ 2.5 ± 0.5 mm. What seems to be interesting is that the Rh6G is not uniformly distributed along fibers forming random arranged 1D system coupled to 2D distributed network. Statistically calculated excitation areas sizes correlate well with the collagen fibers diameter. This relation was observed for all samples regardless the a-CD or Rh6G content. We believe that further modifications of collagen fibers with embedded Rh6G and a-CD might result in the
Fig. 6. Objects diameters acquired from image analysis performer on Rh6G doped collagen.
increasing the difference in refractive index of inclusions resulting in stronger light scattering and formation of resonant feedback lasing. 4. Conclusions In this paper we have presented not only a new material for the random lasing applications but also we have demonstrated a possibility of the lasing emission manipulation. By utilizing aggregates formation property of Rh6G it was possible to shift the emission wavelength to longer ones, whereas the presence of a-CD forced formation of specific Rh6G aggregates resulting in blue shift of the emission. The observed random lasing emission testify that the emission is based on the non-resonant mechanism but with the emission localized in inclusions distributed network, what was confirmed by microscopic studies and colocalization with collagen fibers.
Table 1 Summary of lasing properties of Rh6G:collagen samples and of a-CD containing system in different molar ratios to Rh6G. Rh6G c [%] (w/ Emission wavelength lmax w) [nm]
Lasing threshold rth[mJ/ cm2]
a-CD:Rh6G (mol/mol) 2% (w/w) Rh6G
Emission wavelength lmax [nm]
Lasing threshold rth[mJ/ cm2]
1.0 2.0 3.0
6.0 4.0 3.4
0:1 1:1 10:1 100:1
606 608 606 594
4.0 9.8 7.2 3.5
598 606 610
104
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
Acknowledgements This work was financially supported by the Polish National Science Centre (UMO-2013/11/N/ST4/01488), and Wroclaw University of Science and Technology. The authors acknowledge also the financial support of Romanian Ministry of Education, Research, Youth and Sports, through the UEFISCDI organism, under Contract Number 3/2012, Code Project PN-II-PT-PCCA-2011-3.1-0316. The authors would like to acknowledge Dr. M. Albu Kaya for supplying the collagen samples. References [1] J. Grote, F. Kajzar, K.-S. Lee, I. Rau, Disp. Imaging (2015) 239. [2] L.S. Vasilenk, V.P. Chebotae, Y.V. Troitski, Sov. Phys. JETP U. S. S. R. 21 (1965) 513. [3] S. Fine, W.P. Hansen, Appl. Opt. 10 (1971) 2350. [4] S. Roth, I. Freund, J. Chem. Phys. 70 (1979) 1637. [5] S. Roth, I. Freund, J. Appl. Crystallogr. 15 (1982) 72. [6] E. Georgiou, T. Theodossiou, V. Hovhannisyan, K. Politopoulos, G.S. Rapti, D. Yova, Opt. Commun. 176 (2000) 253. [7] T. Theodossiou, E. Georgiou, V. Hovhannisyan, K. Politopoulos, D. Yova, J. Opt. A Pure Appl. Opt. 3 (2001) L1. [8] Q. Song, S. Xiao, Z. Xu, J. Liu, X. Sun, V. Drachev, V.M. Shalaev, O. Akkus, Y.L. Kim, Opt. Lett. 35 (2010) 1425. [9] Q. Song, Z. Xu, S.H. Choi, X. Sun, S. Xiao, O. Akkus, Y.L. Kim, Biomed. Opt. Express 1 (2010) 1401. [10] H. Dengfeng, X. Min, L. Xiyun, Y. Ming, Y. Tao, W. Chuanke, L. Tingshuai,
L. Shenye, Laser Phys. Lett. 13 (2016) 065603. [11] P. Semeraro, V. Rizzi, P. Fini, S. Matera, P. Cosma, E. Franco, R. García, ~ ez, J.A. Gabaldo n, I. Fortea, E. Pe rez, M. Ferra ndiz, Dyes M. Ferr andiz, E. Nún Pigments 119 (2015) 84. [12] M.M. Asimov, B.A. Bushuk, M.A. Senyuk, A.P. Stupak, A.N. Rubinov, J. Appl. Spectrosc. 66 (1999) 697. [13] S. Hamai, K. Sasaki, J. Incl. Phenom. Macro 45 (2003) 19. [14] V.M. Martinez, F.L. Arbeloa, J.B. Prieto, T.A. Lopez, I.L. Arbeloa, J. Phys. Chem. B 108 (2004) 20030. [15] V.M. Martinez, F.L. Arbeloa, J.B. Prieto, I.L. Arbeloa, J. Phys. Chem. B 109 (2005) 7443. [16] Y. Liu, Y. Chen, S.-X. Liu, X.-D. Guan, T. Wada, Y. Inoue, Org. Lett. 3 (2001) 1657. [17] M. Albu, Lap. LAMBERT Acad. Publ. Saarbr. Ger. (2011) 23. [18] J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, Nat. methods 9 (2012) 676. [19] M.B. van der Mark, M.P. van Albada, A. Lagendijk, Phys. Rev. B 37 (1988) 3575. [20] E. Akkermans, P.E. Wolf, R. Maynard, Phys. Rev. Lett. 56 (1986) 1471. [21] E. Akkermans, P.E. Wolf, R. Maynard, G. Maret, J. Phys. Paris 49 (1988) 77. ska, F. Kajzar, I. Rau, J. Mysliwiec, Adv. Device Mater. 1 [22] K. Cyprych, Z. Kopczyn (2015) 69. [23] H. Cao, J.Y. Xu, Y. Ling, A.L. Burin, E.W. Seeling, X. Liu, R.P.H. Chang, IEEE J. Sel. Top. Quantum Electron 9 (2003) 111e119. pez, Sci. Rep. (2015) 16848. [24] A. Consoli, C. Lo [25] H.E. Türeci, L. Ge, S. Rotter, A.D. Stone, Science 320 (2008) 643. [26] M. Leonetti, C. Conti, C. Lopez, Nat. Phot. 5 (2011) 615e617. [27] A. Consoli, C. Lopez, Opt. Express 24 (2016) 10. [28] L. Sznitko, A. Szukalski, K. Cyprych, P. Karpinski, A. Miniewicz, J. Mysliwiec, Chem. Phys. Lett. 576 (2013) 31. [29] B.A. Weissman, N.A. Brennan, D.A. Lee, I. Fatt, Investig. Ophthalmol. Vis. Sci. 31 (1990) 334.
Contents lists available at ScienceDirect
Organic Electronics journal homepage: www.elsevier.com/locate/orgel
Collagen network as the scaffold for spontaneously distributed optical resonators Konrad Cyprych a, *, Martyna Janeczko a, Ileana Rau b, Francois Kajzar b, Jaroslaw Mysliwiec a a b
Advanced Materials Engineering and Modelling Group, Wroclaw University of Science and Technology, Wyb, Wyspianskiego 27, 50-370 Wroclaw, Poland Faculty of Applied Chemistry and Materials Science, University Politehnica of Bucharest, Polizu Street No 1, 011061, Bucharest, Romania
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 July 2016 Received in revised form 26 September 2016 Accepted 27 September 2016
The idea behind this work was to acquire one dimensional lasing in the Rhodamine 6G dye doped collagen fibers with the control of random lasing phenomenon properties by changing the dye molecular arrangement. We show that a simple manipulation of the dye concentration in biopolymeric fibril matrix bulk and an additional use of a-cyclodextrin (a-CD) molecules determine formation of specific dye aggregates and as a consequence the shift of the random lasing emission wavelength in the desired direction. The analysis of the light transport mean free path in function of the dye and (a-CD) concentration was done with the use of coherent back scattering technique. © 2016 Elsevier B.V. All rights reserved.
Keywords: Collagen Random lasing a-Cyclodextrin Rhodamine 6G
1. Introduction Bioderived and bioinspired materials are of immense current interest because they can provide novel, attractive physical properties while being able to be produced by “green”, environmentfriendly technologies. One of them is collagen, whose name originates from Greek cola, meaning glue [1]. Collagen is a chiral molecule with finds a growing interest for application in photonics. It is also one of the most spread proteins in human body which plays a role of scaffold for mammalian tissues providing them the elasticity and the durability. Structurally collagen is composed of three polypeptide chains forming a-helix, linked together by hydrogen bonds between hydroxylysine and hydroxyproline and by covalent bonds, that can form different collagen folding forms. Several examples of interesting photonic applications of collagen have been presented in the past. The building unit of collagen is the tropocollagen. It is a noncentrosymmetric molecule, exhibiting second harmonic generation (SHG) as observed by several research groups. The first observation of SHG in collagen was reported by Vasilenko et al. as early as in 1965 [2] what was confirmed later by Fine and Hansen as well as by Roth and Freund [3e5]. Yova et al.
* Corresponding author. E-mail address: [email protected] (K. Cyprych). http://dx.doi.org/10.1016/j.orgel.2016.09.032 1566-1199/© 2016 Elsevier B.V. All rights reserved.
have shown the optical second harmonic generation by collagen Type I. A broad wavelength tuning of harmonic wavelength from 383 to 532 nm was achieved [6]. The same group presented also Rhodamine 6G (Rh6G) and collagen application for energy transfer, in which Rhodamine serves as a donor in the system after the excitation to S2 energy level by 1064 nm via two photon absorption. Collagen, as an acceptor, has shown the fluorescence emission maximum intensity at 390 nm [7]. As a part of the light amplification system, collagen was used as an integral building block of a bone tissue, in which the random lasing phenomenon was observed, however the crucial role in this process was attributed to the hydroxyapatite, responsible for strong light scattering, in order to acquire spontaneously formed optical resonators [8]. Basing on this approach a method of morphology deformations detection, recording the changes in random lasing emission in bone during a stress applied to the tissue was developed [9]. The random lasing, was also observed in the egg membranes, enriched with Rhodamine 6G. Such a natural membrane, among the others, contains the collagen in its structure. Dense network of fibril proteins, positioned in random directions, are acting as scatterers for the optical cavities formation [10]. Cyclodextrins (CD) are circularly formed poly-glucose, playing a crucial role in the drug delivery as its carriers. The structure of cyclodextrin forms geometrical cone with hydrophobic interior and hydrophilic exterior [11]. Application of this material is driven by
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
the formation of specific interactions inside the circular structure, capable to block diffusion of the compound from its interior. The mechanism standing for an efficient CD use, e.g. in drug delivery, is based on the cell membrane permeability for glucose moieties, reaching and penetrating them. The same happens with cyclodextrins, releasing their package of carried molecule inside the targeted cell. It was found that Rh6G and b-cyclodextrin are forming inclusion complexes (IC) having relation to the fluorescence emission wavelength and intensity from them [12,13]. However, the mentioned inclusion complexes are not revealing the detailed structure of Rh6G aggregates as it was done in the work of Martinez, concerning the laponite clay [14,15]. There have been accounted that formation of IC are responsible for the enhancement of fluorescence signal as presented for Rhodamine B and bis(cyclodextrin)s host [16]. In the present work we demonstrate that in the system based on fibril collagen proteins, serving role of a host material for fluorescent dyes and CD penetrating protein structure, lasing action can be achieved and due to the specific aggregates formation the emission wavelength can be easily shifted. 2. Materials and methods For the light amplification studies we have selected the wellknown water soluble Rhodamine 6G luminescent dye which possesses excellent fluorescence quantum yield. Collagen Type I, extracted from bovine derma, in gel form of concentration equal to 2.2%, was obtained from Collagen Department of Division Leather and Footwear Research Institute, Bucharest, Romania where the purification and the preparation procedure was performed according to the work of Albu [17]. Series of thin film samples were prepared by drop casting on glass slides. They were made using the gelatinized collagen with Rh6G concentration varying from 1% up to 3% (w/w) with respect to dry collagen. For one selected Rh6G:collagen ratio equal to 2% (w/ w) additionally a-cyclodextrin purchased from Sigma Aldrich at molar ratios a-CD:Rh6G equal to 100:1, 10:1 and 1:1, respectively, was added. Quality and morphology of prepared thin layers were evaluated with the inverted optical microscope Olympus IX71 and atomic force microscope Dimension V (Veeco) in tapping mode. The random lasing phenomenon studies were done using the experimental setup with pulsed nanosecond Nd:YAG laser, operating at lex ¼ 532 nm, as excitation source with stripe-like beam geometry of dimensions equal to 3.0 0.5 mm. Lasing spectra were collected by the Shamrock SR-163 fiber spectrometer in function of excitation energy density. Simultaneously to the random lasing emission measurements the optical visualization of the excited area was done using optical system of 0.8 mm resolution, capable to observe and correlate the emission to the position on the sample. Additionally to the light amplification experiments, the coherent backscattering (CBS) studies were performed, in order to get the value of light transport mean free path. It is a crucial parameter in analysis of the statistics of the distance that light travels before it is scattered. 3. Results and discussion 3.1. Collagen layers morphology The microscopic studies show that dye doped collagen deposited on the glass slide is forming fibers arranged in random directions at the 2D surface area. The image analysis done with the ImageJ confirms this situation which results from the isotropic distribution of fibrous structures (Fig. 1a) [18]. The dye doped collagen thin films morphology study was performed by the AFM
101
analysis. The acquired topographies, presented in Fig. 1b and d) indicate that the superhelix collagen fiber of diameter about Dfiber ¼ 5 mm is formed and built from random coil macromolecules with diameter less than dcoil < 50 nm. The formed collagen loose protein network on the glass surface, surrounded with not fibrillated protein film containing Rh6G, and Rh6G aggregates in the fiber structure is shown in Fig. 1. All studied samples exhibit similar morphologic properties. Dye free samples, as well as Rh6G doped collagen fibers casted on the glass slide were investigated using experimental system for CBS measurements. As a source of scattered light cw HeeNe laser of 633 nm emission wavelength was used, which is off the absorption of the samples. Incorporation of Rh6G and a-CD do not show significant effect to the calculated light transport mean free path therefore the designated parameters were evaluated statistically indicating the source of scattering overall the fiber network. The light transport mean free path was calculated for all samples using Equation (1) [19e21]. The obtained average value was equal to lt ¼ 16.8 ± 1.8 mm meaning that this property is one of the whole system and not only for the single collagen fiber. Fig. 2 presents, as an example, the coherent backscattering cone for 3% (w/w) Rh6G doped collagen sample.
lt ¼
0:7,l 2p,W
(1)
where lt is light mean transport path, l ¼ 633 nm, and W stands for the full scattering cone width at its half maximum. 3.2. Multimode fiber lasing Collagen fibril proteins, enriched with Rh6G, become to be the light amplifying medium, able to exhibit the phenomenon of amplified spontaneous emission (ASE) and random lasing (RL). We have focused on the lasing action generation based on the distributed 2D network of dye doped collagen fibers. According to the known phenomenon, that we had described previously, we have used random fibril system in order to acquire tunable light emission relaying on formation of Rh6G aggregates sizing effect responsible for the shift of the light emission wavelength [22]. As was expected, the Rh6G aggregation affects to the RL emission properties. In the measured samples an RL tuning from 598 nm to the 610 nm with about ~10 nm spectral FWHM was observed, as presented in Fig. 3. This effect is related to the formation of J-aggregates and higher order aggregates responsible for the red shift of the emission wavelength [14,15]. The lasing modes were not clearly visible, due to the non-resonant feedback meaning the light intensity increasing in the gain medium upon excitation at the gain length [23e25]. With the microscopy analysis done during the lasing it was revealed that Rh6G forms inclusions, which are colocalized with the fibers positions and are acting as scattering centers embedded in the fiber structure as it is shown in Fig. 4. Although Rh6G aggregates are playing the role of scatterers, the formation of resonant modes has not been observed and light is not localized within the inclusions in collagen fibers. Rather than the densely packed 2D fiber network is responsible for amplification with wave energy dissipation with scattering. Changing the pumping beam energy density a simultaneous “blinking”, coming from inclusions along the fibers, was observed. It is accounted as due to the reaching population inversion in the gain region responsible for the “on” and “off”, turning characteristic for random lasing presented in Fig. 4. Loading collagen fibers with increasing amount of Rh6G resulted in lowering the lasing threshold from r1% ¼ 6.0 mJ/cm2 for lowest Rh6G concentration (1% w/w), down to r3% ¼ 3.4 mJ/cm2 for 3% (w/w). This effect is related to the
102
K. Cyprych et al. / Organic Electronics 39 (2016) 100e104
Fig. 1. Transmission mode optical microscope image a), and atomic force microscopy analysis of collagen fibers, b,c) size map and profile of single collagen fiber of a diameter about Dfiber ¼ 5 mm, and d,e) enlargement of single fiber with observed single macromolecules of diameter about dcoil ¼ 50 nm. Presented data come from 2% (w/w) Rh6G:collagen layer.
Fig. 2. Coherent backscattering cone for 3% (w/w) Rh6G doped collagen sample with calculated mean light transport mean free path lt ¼ 16.2 mm.
increasing probability of excitation in the presence of larger amount of Rh6G, but still above the lasing threshold there were not seen any spatial modes testifying the light amplification was not reached on the spatially localized feedback mechanism [26,27]. Basing on the acquired results and the described phenomenon we focused on the direct control of the lasing emission wavelength due to the predictions of aggregates formation, favoring especially the smaller ones. The elaborated solution was found taking advantage of the a-cyclodextrin properties. Samples containing collagen, Rh6G and a-cyclodextrin in different ratios [aCD]:[Rh6G] ranging from 1:1 up to 100:1 (mol/mol) were prepared. In contrary to the lasing emission observed from higher order aggregates, a shift to lower wavelengths in function of the increasing a-CD amount was obtained. As it can be seen in Fig. 5 just taking advantage of the hydrophilic-hydrophobic interactions between aCD and Rh6G and formation of different types of aggregates, we were able to tune the emission wavelength for almost 20 nm. Applied cyclodextrin becomes a carrier for Rhodamine 6G molecules, favoring only the specific aggregates sizes binding. This behavior is observed only in solid-state form of the collagen layers. The solutions prepared for layer fabrication, containing Rh6G, collagen and a-CD exhibit emission at 550 nm, what indicates that
Fig. 3. Normalized random lasing intensities from single collagen fibers doped with increasing amount of Rh6G: from 1% (w/w) (green), 2% (w/w) (yellow) and 3% (w/w) (red) with respect to collagen dry mass. With increasing dopant content the emission wavelength shifts from l1% ¼ 598 nm to l3% ¼ 610 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
the fluorescence is originating from Rh6G monomers. The results concerning lasing properties of Rh6G doped collagen with different amounts of a-CD are presented in Table 1. Similarly as for Rh6G doped collagen, the emission from samples enriched with a-CD is colocalized with collagen fibers. This fact indicates that Rhodamine, stabilized by cyclodextrin is adsorbed by protein macrostructures. The mechanism behind the binding is not clear yet. The localization of inclusion complexes in the collagen fibers with and without a-CD has been cross-checked by the analysis of lasing stability in time. The observed material degradation at energy density well above the lasing threshold shows comparable lasing stability for Rh6G doped collagen as well as in the presence of a-CD. Unfortunately the designated stabilities indicate a high oxygen permeability and dye bleaching according to the similar DNA based systems [28,29].
3.3. Gain regions in the fiber structure Characteristic occurrence of inclusion complexes embedded in the collagen were distributed along the single fiber length. The
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Fig. 4. Series of microscopic images showing colocalization of RL emission and dense collagen fiber network. Presented formation of optical resonators in the series of photos taken below threshold, at lasing and collagen network visible after Rh6G bleaching.
Fig. 5. Normalized intensities of lasing emission of a-CD:Rh6G in collagen in selected molar ratios 0:1 (mol/mol) (black), 1:1 (mol/mol) (green), 10:1 (mol/mol) (yellow), 100:1 (mol/mol) (red), Rh6G concentration with respect to dry collagen was equal 2% (w/w). Effect of the a-CD on the RL emission with visible shift to the shorter wavelengths for 100:1 M ratio a-CD:Rh6G in collagen matrices is clearly seen. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
acquired microscopic images (Fig. 6) were subjected to the image particle analysis, what allowed to describe statistically the excited objects. Shining objects diameter at lasing emission conditions have shown log-normal distribution with the maximum at dobj ¼ 2.5 ± 0.5 mm. What seems to be interesting is that the Rh6G is not uniformly distributed along fibers forming random arranged 1D system coupled to 2D distributed network. Statistically calculated excitation areas sizes correlate well with the collagen fibers diameter. This relation was observed for all samples regardless the a-CD or Rh6G content. We believe that further modifications of collagen fibers with embedded Rh6G and a-CD might result in the
Fig. 6. Objects diameters acquired from image analysis performer on Rh6G doped collagen.
increasing the difference in refractive index of inclusions resulting in stronger light scattering and formation of resonant feedback lasing. 4. Conclusions In this paper we have presented not only a new material for the random lasing applications but also we have demonstrated a possibility of the lasing emission manipulation. By utilizing aggregates formation property of Rh6G it was possible to shift the emission wavelength to longer ones, whereas the presence of a-CD forced formation of specific Rh6G aggregates resulting in blue shift of the emission. The observed random lasing emission testify that the emission is based on the non-resonant mechanism but with the emission localized in inclusions distributed network, what was confirmed by microscopic studies and colocalization with collagen fibers.
Table 1 Summary of lasing properties of Rh6G:collagen samples and of a-CD containing system in different molar ratios to Rh6G. Rh6G c [%] (w/ Emission wavelength lmax w) [nm]
Lasing threshold rth[mJ/ cm2]
a-CD:Rh6G (mol/mol) 2% (w/w) Rh6G
Emission wavelength lmax [nm]
Lasing threshold rth[mJ/ cm2]
1.0 2.0 3.0
6.0 4.0 3.4
0:1 1:1 10:1 100:1
606 608 606 594
4.0 9.8 7.2 3.5
598 606 610
104
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Acknowledgements This work was financially supported by the Polish National Science Centre (UMO-2013/11/N/ST4/01488), and Wroclaw University of Science and Technology. The authors acknowledge also the financial support of Romanian Ministry of Education, Research, Youth and Sports, through the UEFISCDI organism, under Contract Number 3/2012, Code Project PN-II-PT-PCCA-2011-3.1-0316. The authors would like to acknowledge Dr. M. Albu Kaya for supplying the collagen samples. References [1] J. Grote, F. Kajzar, K.-S. Lee, I. Rau, Disp. Imaging (2015) 239. [2] L.S. Vasilenk, V.P. Chebotae, Y.V. Troitski, Sov. Phys. JETP U. S. S. R. 21 (1965) 513. [3] S. Fine, W.P. Hansen, Appl. Opt. 10 (1971) 2350. [4] S. Roth, I. Freund, J. Chem. Phys. 70 (1979) 1637. [5] S. Roth, I. Freund, J. Appl. Crystallogr. 15 (1982) 72. [6] E. Georgiou, T. Theodossiou, V. Hovhannisyan, K. Politopoulos, G.S. Rapti, D. Yova, Opt. Commun. 176 (2000) 253. [7] T. Theodossiou, E. Georgiou, V. Hovhannisyan, K. Politopoulos, D. Yova, J. Opt. A Pure Appl. Opt. 3 (2001) L1. [8] Q. Song, S. Xiao, Z. Xu, J. Liu, X. Sun, V. Drachev, V.M. Shalaev, O. Akkus, Y.L. Kim, Opt. Lett. 35 (2010) 1425. [9] Q. Song, Z. Xu, S.H. Choi, X. Sun, S. Xiao, O. Akkus, Y.L. Kim, Biomed. Opt. Express 1 (2010) 1401. [10] H. Dengfeng, X. Min, L. Xiyun, Y. Ming, Y. Tao, W. Chuanke, L. Tingshuai,
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