- Email: [email protected]
Fluorescent sensing using biconical tapers
Sensors and Actuators B 96 (2003) 315–320
Fluorescent sensing using biconical tapers Pawel J. Wiejata a , P.M. Shankar b , R. Mutharasan a,∗ b
a Department of Chemical Engineering, Drexel University, Philadelphia, PA 19104, USA Department of Electrical and Computer Engineering, Drexel University, Philadelphia, PA 19104, USA
Received 16 October 2002; accepted 13 June 2003
Abstract Single mode fibers were heat drawn to into biconical tapers with a waist diameter of 3.69 m, and a waist length of 7.1 mm. The taper is shown experimentally to eject 460 nm light and collect the emitted fluorescent light at 516 nm. Flourescein solution at concentration of 10 to 60 M was placed in the waist region, and excitation light of 460 nm was launched at one of the fiber, and 516 nm light intensity was measured at the other end of the fiber. The 516 nm fluorescence signal was found to be proportional to the fluorescein concentration. The results reported in this paper show in practical terms, the feasibility of measuring fluorescence through evanescent coupling, thus demonstrating the use of tapered fibers as fluorescence sensors with potential applications in medical and biomedical areas. © 2003 Elsevier B.V. All rights reserved. Keywords: Fluorescin; Microvolume; Tapered fiber; Evanescent fluorescence
1. Introduction Development of new technologies for sensing biomolecules and properties of cells have become important in recent years, because of their applications in biological and biomedical sciences [1–10]. Sensors are needed that are capable of measuring cell properties at single cell resolution in the areas of clinical, cellular, and pharmaceutical applications. This evolving area of in vivo sensing requires very small size sensors. The current state of technology does not permit high spatially resolved measurements to be performed easily or efficiently, with most of such tests being currently performed in vitro and with large volumes of analytes. Optical fibers offer a simple and rapid means of detecting and sensing biological phenomena, such as metabolic shifts, alteration in cellular morphology, and other metabolic events with ease. There are several physical and optical effects in fibers that can be used in sensing. However, the evanescent field of the fibers has been used often for applications in biochemical [2,3,5,7], biomedical [1,4,6,8–10], and environmental sensing [11–13]. The strength of the evanescent field depends on several factors, the index of the core, index of the surrounding medium, the radius of the fiber and the operating wavelength. It is possible to manipulate the strength of the evanescent field to
sense, characterize, and quantify optical properties of samples in the evanescent region. Tapering the fiber provides an easy access to the evanescent field in fibers, enabling stronger interaction with the sample, thus making the sensors versatile. Tapered fiber tips have been used as nanodelivery devices [4,9] because of the ability of the tips to produce laser beams of extremely small diameters (<100 nm). They have also been used in fluorescence sensing of biomolecules. However, in these applications, the fiber tips were used as light delivery devices, rather than as sensing elements. Thus, these tapered tips do not constitute in situ sensors, where the three features, namely, light delivery, interaction with the sample, and sensing are accomplished by the same fiber [14]. A new kind of biological sensing technique using optical fibers is presented here. Instead of the traditional approach of using a cuvette in fluorescence measurement, a tapered fiber is used to measure the fluorescence emission. Using tapered fibers fabricated from commercially available optical fibers, evanescent field is used to deliver the excitation radiation as well as collect the emission radiation. The fabrication, characterization, and in vitro sensing using tapered fibers is presented. 2. Materials and methods
∗ Corresponding author. Tel.: +1-215-895-2236; fax: +1-215-895-2227. E-mail address: [email protected] (R. Mutharasan).
0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-4005(03)00548-3
The section gives descriptions of the materials and methods used in this work. The details of the fiber, fluorescing
316
P.J. Wiejata et al. / Sensors and Actuators B 96 (2003) 315–320
agent, instrumentation used, characterization and measurement techniques are provided.
3.2 mm thick) plates together. A 0.2 mm diameter line was scored lengthwise in the center to accommodate the tapered fiber. A 6.4 mm hole was pre-drilled in the top plate to serve as the chamber (∼150 l) for holding the analyte in contact with the tapered region of the fiber. A third piece of Plexiglas (30 mm2 with a 6.4 mm hole in center) was attached to the base with four screws, after a thin layer of silicone vacuum gel was applied to its underside. The gel eliminated capillary outflow of the analyte from the central chamber. All the vertical surfaces of the fiber holder were painted black to reduce reflection and potential measurement errors. Details of this assembly are also available elsewhere. The experimental arrangement used in the fluorescence studies is shown in Fig. 2. It consists of a spectrofluorimeter (Model QM-1, Photon Technology International Inc., PTI) which has been adapted to accept the fiber. The assembly made to hold the cuevette can be removed and replaced with the fiber holder fabricated mentioned above. This spectrofluorimeter unit is equipped with a 75 W Xenon arc lamp (Ushio Inc., Japan) with a monochromator and a photomultiplier tube (PMT, housing model 710, tube model R1527P, PTI Inc.) coupled through a monochromator. Wavelength settings of monochromator and recording of PMT outputs (counts/s) were done through Felix instrument software. The cuevette holding assembly was removed first. The fiber holder was mounted on a manual X–Y–Z positioning platform in the chamber along the axis of the light source and the PMT. A microscope objective (20× UV capable) was mounted to focus the input light into the fiber. In order to make sure that the light measured by the PMT came only from the fiber, a black Delran cylindrical piece was fitted into the opening leading to the PMT. A 125 m diameter hole was drilled at the center of the Delran block to accommodate the fiber tip with Play-doh (available from toy stores) to seal the center hole so as to block leakage of stray light from the chamber. The slide with the tapered fiber was placed on the platform made to accommodate it. This whole unit was kept in a dark box so that no light from the outside enters the spectrofluorometer. A thick black cloth was also used to cover the entire fluorometer to reduce leakage of ambient light. Afterwards, additional setup was configured through the Felix computer software to set the values for scanning range. The settings for the emission scan were the following: each concentration of the material is run twice, first one at a step size of 1 nm and integration time of 1 s and the second one at a step size of 0.25 nm and integration time 5 s. An excitation wavelength of 460 nm was used and the
2.1. Materials A Corguide fiber (Corning Glass Works, NY, attenuation at 1300 and 1500 nm of 0.36 and 0.26 dB/km, respectively) with a core diameter of 8 m and total diameter of 125 m was used in all experiments reported here. The fluorescing agent used in our studies was fluorescin. A stock solution (100 M) was prepared by dissolving 2 ,7 -difluorofluorescin (Molecular Probes) powder in pH 7.3 phosphate buffered saline (PBS) at room temperature. PBS was also used as the reference. The stock solution was diluted as needed. 2.2. Methods While tapering of fibers can be accomplished using chemical etching [15–18] or heat pulling, our experience with both of these techniques showed that heat-pulled tapers provided reproducible smooth tapers. They retain the optical characteristics by virtue of relative arrangement of core and cladding while chemical etching leads to the preferential elimination of the cladding completely. The presence of both the core and cladding in the heat pulled tapers makes them ideal for sensing. The fabrication methods employed were described elsewhere [19]. A taper consists of a narrow waist with contracting and expanding regions on either side as shown in Fig. 1. The next step in our study was the characterization of the fabricated tapers. The diameter of the taper waist and the total length of the taper from beginning to the end (fiber profile) constitute the taper characteristics. They were measured using an optical microscope at 250× (model IMT-2, Olympus, Japan) equipped with a video camera (Cohu Corp.) linked to a computer. The microscope images were acquired using the Scion Image software (Scion Corp.). A microscopic scale, graduated in 10 m was used in conjunction with the acquired image to measure fiber diameter. The fiber diameter was recorded every millimeter from the beginning of the taper to the end. If and when the fiber taper has the right profile, fiber ends are clipped with a fiber optic cleaver (NO-NIK) ensuring a clean 90◦ angle cut at both ends of the fiber, assuring maximum light collection by the fiber. The fiber was placed in a ‘fiber holder’. The fiber holder was made by gluing two Plexiglas (100 mm × 30 mm × Waist DW I0
Unstretched Fiber
Transition Region I
LW Fig. 1. Schematic of a tapered fiber. Lw was typically 6–7 mm. Diameter of taper obtained was typically 3–4 m.
P.J. Wiejata et al. / Sensors and Actuators B 96 (2003) 315–320
317
Monochromator at λ fiber S1
S2
Cuvette chamber
S3
S4
75W Xenon arc lamp
PMT
details
PC
Fiber mounting apparatus
Originally for the cuvette holder L1
Optical fiber L2 to PMT Fiber mounting apparatus Fluorometer chamber
Fig. 2. Apparatus used for monitoring fiber etching in an analytical fluorometer. Slits S2 and S4 were set at 2 nm, while slits S1 and S3 were opened fully. Convex lens (L1 ) and a microscope objective (L2 ) were used to launch light from monochromator into the fiber.
3. Results and discussion We will now describe the results obtained starting with the characteristics of the fiber taper used in our measurements. Fluorescence measurements with the fiber tapers were compared to those obtained from the traditional cuevette setup. 3.1. Fiber profile Several tapered fibers were fabricated using the technique described in our previous work [19]. All the tapers made by this technique had similar physical attributes. In Fig. 3, a profile of a typical taper is shown. This fiber had a 3.69 m diameter waist and a waist length of 7.1 mm. The transition regions are not normally symmetric with respect to the waist and this is attributed to the drawing technique used. Because of this, it was decided to launch the light at the steeper taper and collect the resulting light response at the more gradually sloped end. This resulted in a better light collection at that end. Most of the tapered fibers fabricated had waist diameters in the range of 3–4 m. 3.2. Fluorescin spectra in a cuvette Experiments were conducted in the traditional cuvette arrangement to establish clearly the excitation and emission
peaks of fluorescin. Fluorescin dye is pH sensitive. The exicitation and emission scans corresponding to pH 7.3 are shown in Fig. 4a and b, respectively. Excitation was varied from 450 to 550 nm and emission 516 nm was measured and is presented Fig. 4a. Note that 460 nm excitation gives higher response than when excited at 502 nm, the second peak in Fig. 4a. In Fig. 4b emission spectra is given for excitation at 460 nm. For the experiments reported in this paper, it was decided to excite at 460 nm because the light intensity was higher at that wavelength. The added benefit is that 460 nm excitation is farther away from the fluorescence peak (516 nm), making it easier to observe fluorescence with less interference from the excitation wavelength. Fluorescence scans were undertaken for different concentrations of fluorescin. The results are shown in Fig. 5. We will now compare these results to those obtained from the tapered fiber.
Taper radius ( m)
emission scan was undertaken in the range of 450–532 nm the first time and 500–532 nm the second time. The second range was used to examine the fluorescence emission more carefully. The full range of the PMT is 3.5 × 106 counts/s. The noise floor in measurement was 30 counts/s.
80 60 40 20 0 -20 -40 -60 -80 0
1
2
3 4 5 Taper Length (mm)
6
7
Fig. 3. Typical taper profile obtained using heat pulling. Initially fiber diameter is 125 m and the waist of this taper was 3.69 m, total taper length was 7.1 mm. Under a microscope (400×) the surfaces appeared smooth without any protrusions.
P.J. Wiejata et al. / Sensors and Actuators B 96 (2003) 315–320
Count (photons/s)
2.0E+06 1.8E+06 1.6E+06 1.4E+06 1.2E+06 1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05 0.0E+00 450 460 470 480 490 500 510 520 530 540 550 (a) Excitation Wavelength (nm)
Count (photons/s)
1.4E+06
0 µΜ 20 µΜ 60 µΜ
350 300 250 200 150 100 50 0 500
510
520
530
540
Wavelength (nm)
1.2E+06
Fig. 6. Evanescent fluorescence spectra using 3.11 m tapered fiber focusing on the fluorescence area. This run was performed at 0.25 nm wavelength steps and 5 s integration time. Only three concentrations are shown, past 60 M the fluorescence intensity starts to decrease.
1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05
0.0E+00 450 460 470 480 490 500 510 520 530 540 550 (b) Emission Wavelength (nm)
Fig. 4. Excitation and emission spectra for 0.1 M fluorescin in PBS, pH 7.3 in a cuvette. Panel (a) represents the absorption spectra. Note two absorption peaks at 460 and 504 nm. Panel (b) represents the emission spectra and we can observe a fluorescence peak at 516 nm. Excitation used was 460 nm.
3.3. Fluorescence characteristics of the tapered fiber The experiments were repeated now with the tapered fiber with the smallest waist (diameter of 3.11 m). Several
1.E+07 0 mM
1.E+06 Count (photons/s)
400
Count (photons/s)
318
20 µΜ 40 mM 60 µΜ
1.E+05 1.E+04 1.E+03 1.E+02 480
490
500
510
520
530
Wavelength (nm)
Fig. 5. Fluorescence spectra in a cuvette. Experiments were performed at 460 nm excitation wavelength and fluorescence in the cuvette was measured at the right angle geometry. Base level of excitation source in the wavelength region of interest is about 1000 counts/s. The emission peak is broad and does change slightly with concentration.
tapered fibers with similar geometric characteristics were tested. We report here results obtained with the 3.11 m waist diameter fiber. The cuevette assembly was removed and replaced with the fiber holder. All alignments were done as described in our previous work [19]. The emission scans are shown in Fig. 6. Increased fluorescence is seen with increased fluorescin concentration. The observed fluorescence could have been excited only from the light transmitted through the fiber interacting with the solution surrounding the taper via the evanescent field [20]. Since the tapered fiber was continuous and all the possible avenues of light leakage into the PMT were eliminated, the fluorescence observed came only from the fluorescence emission light being guided into the fiber through the evanescent coupling mechanism. Thus, these results, we believe, establish that the tapered fiber can act both as a transmitter and a delivery mechanism of the excitation energy to the optically active solution surrounding the fiber and simultaneously pick up the longer wavelength fluorescence light. Even though the emission counts are much lower than those seen in the cuvette, the fluorescence emission is of sufficient magnitude to show the optical functioning of the sensor. Another interesting result is plotted in Fig. 7. Since the cuvette and fiber geometries have their own respective maxima with fluorescin concentration, it is useful to normalize the signal with respect to the maximum for comparison of the two arrangements. The normalized fluorescent light is plotted as a function of the concentration of the fluorescin. One clearly sees that as the fluorescin concentration increases, the fluorescent emission increases, reaching a peak at 60 M for the fiber and 40 M in the cuvette, and then decreases at higher concentration. This suggests that there is a limit of detection for this sensor, indicating potentially inner filter effects quenching the fluorescence. The results in Fig. 7 also seem to suggest that the inner filter effect is slightly
Normalized fluorescent intensity
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319
assist as a sensor, but also a quantitative tool. In comparison to a tapered tip based fluorescence measuring system, the tapered fiber sensor reported here offers greater practical opportunities for in vivo medical applications. This is because the light collection in a tapered tip sensor depends on an open geometry, which is unsuitable for in vivo applications.
1.2 1.0 0.8 0.6 0.4
Acknowledgements
Fiber Cuvette
0.2
This work was supported (in part) by the State of Pennsylvania under The Nanotechnology Institute.
0.0 0
10
20
30
40
50
60
70
80
90
100
Fluorescin Concentration ( M) Fig. 7. Plot of normalized fluorescence light intensity as a function of fluorescin concentration. Normalized value is calculated as a fraction of maximum fluorescence in the respective geometry. The tapered fiber used had a waist diameter of 3.11 m.
less in the fiber geometry compared to cuvette.It should be noted that fluorescence measured using the tapered geometry is along the axis of the fiber, while in cuvette geometry, it is orthogonal. Because the source of fluorescence signal is from the tapered region, one can be certain as to the location of the fluorescent sample, while in open optical path geometry, one can be certain of the direction, but not the location. Thus, sensing fluorescence using tapered geometry offers the added advantage of localization.In tapered fiber sensing, the source of excitation light is the evanescent filed, and thus is of low intensity compared to cuvette. Therefore, the emission signal is correspondingly smaller when compared to open cuvette geometry.
4. Conclusions In this paper, we have shown that tapered fibers have the capability for fluorescence sensing with the potential applications in various biological and medical applications. The preliminary results reported here demonstrate the concept and supports the hypothesis that the tapered region of the fiber can act as a mechanism to deliver the excitation wavelength to the sample (biomolecules) and pickup the longer wavelength fluorescence radiation generated by the sample. This unique ability of the tapered fiber makes it an ideal choice to be part of a probe to examine the presence or absence of specific analytes in solutions. The small size of the waist and small volume of analyte needed also make the taper geometry a ideal choice in cases where a sample volume available may be very small and limited. All these properties make the tapered fiber well suited for in vivo applications because the tapered region incorporates all aspects of sensing. Additional research is necessary to undertake a more careful calibration and analysis so that this probe may not only
References [1] U. Bindig, M. Meinke, I. Gersonde, S. Kravchik, S. Citron, A. Katzir, G. Muller, Detection of malignant tissue by using infrared microscopy and fiberoptic spectroscopy, in: Proceedings of the SPIE 4253 Optical Fibers and Sensors for Medical Applications, 2001, p. 108–117. [2] J.-W. Choi, J.H. Park, S.C. Lee, D.-I. Kim, W.H. Lee, Analysis of culture flouorescence by a fiber-optic sensor in Nicotiana tabacum plant cell culture, Korean J. Chem. Eng. 12 (5) (1995) 528–534. [3] H. Chuang, P. Macuch, M.B. Tabacco, Optical sensors for detection of bacteria. Part 1. General concepts and initial development, Anal. Chem. 73 (2001) 462–466. [4] B.M. Cullum, G.D. Griffin, G.H. Miller, T. Vo-Dinh, Intracellular measurements in mammary carcinoma cells using fiber-optic nanosensors, Anal. Biochem. 277 (2000) 25–32. [5] A.P. Ferreira, M.M. Werneck, R.M. Ribeiro, Development of an evanescent-field fibre optic sensor for Escherichia coli O157:H7, Biosens. Bioelectron. 16 (2001) 399–408. [6] S. McCulloch, D. Uttamchandani, Development of fibre optic micro-optrode for intracellular pH measurements, IEE Proc. Optoelectron. 144 (3) (1997) 162–167. [7] S. Pilevar, C.C. Davis, F. Portugal, Tapered optical fiber sensor using near-infrared fluorophores to assay hybridization, Anal. Chem. 70 (10) (1998) 2031–2037. [8] N. Nath, S.R. Jain, S. Anand, Evenescent wave fibre optic sensor for detection of L. donovani specific antibodies in sera of kala azar patients, Biosens. Bioelectron. 12 (6) (1997) 491–498. [9] T. Vo-Dinh, J.-P. Alarie, B.M. Cullum, G.D. Griffin, Antibody-based nanoprobe for measurement of a fluorescent analyte in a single cell, Nature Biotechnol. 18 (2000) 764–767. [10] C. Zhou, P. Pivarnik, S. Auger, A. Rand, S. Letcher, A compact fiber-optic immunosensor for Salmonella based on evanescent wave excitation, Sens. Actuators B 42 (1997) 169–175. [11] C. Bariain, I.R. Matias, F.J. Arregui, M. Lopez-Amo, Tapered optical-fiber-based pressure sensor, Opt. Eng. 39 (8) (2000) 2241– 2247. [12] C. Bariain, I.R. Matias, F.J. Arregui, M. Lopez-Amo, Optical fiber humidity sensor based on a tapered fiber coated with agarose gel, Sens. Actuators B 69 (2000) 127–131. [13] Y. Zaatar, D. Zaouk, J. Bechara, A. Khoury, C. Llinaress, J.-P. Charles, Fabrication and characterization of an evanescent wave fiber optic sensor for air pollution control, Mater. Sci. Eng. 74 (2000) 296–298. [14] F.P. Payne, H.S. Mackenzie, Novel applications of monomode fiber tapers, Soc. Photo Opt. Instrum. Eng. 1504 (1991) 165–175. [15] T.J. Brophy, P.M. Shankar, Formation and measurement of tapers in optical fibers, Rev. Sci. Instrum. 64 (9) (1993) 2650–2654. [16] C.E. Chryssouo, Theoretical analysis of tapering fused silica optical fibers using a carbon dioxide laser, Opt. Eng. 38 (10) (1999) 1645– 1649.
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[17] G.A. Valaskovic, M. Holton, G.H. Morrison, Parameter, control, characterization and optimization in the fabrication of optical fiber near-field probes, Appl. Opt. 34 (7) (1995) 1215–1228. [18] H.S. Haddock, P.M. Shankar, R. Mutharasan, Fabrication of biconical tapered optical fibers using hydrofluoric acid, Mater. Sci. Eng. B 97 (2003) 87–93.
[19] H.S. Haddock, P.M. Shankar, R. Mutharasan, Evanescent sensing of biomolecules and cells, Biosens. Actuators Chem. 88 (2003) 67– 74. [20] W. Henry, Evanescent field devices: a comparison between tapered optical fibers and polished or D fibers, Opt. Quan. Electronics 26 (1994) S261–S272.
Fluorescent sensing using biconical tapers Pawel J. Wiejata a , P.M. Shankar b , R. Mutharasan a,∗ b
a Department of Chemical Engineering, Drexel University, Philadelphia, PA 19104, USA Department of Electrical and Computer Engineering, Drexel University, Philadelphia, PA 19104, USA
Received 16 October 2002; accepted 13 June 2003
Abstract Single mode fibers were heat drawn to into biconical tapers with a waist diameter of 3.69 m, and a waist length of 7.1 mm. The taper is shown experimentally to eject 460 nm light and collect the emitted fluorescent light at 516 nm. Flourescein solution at concentration of 10 to 60 M was placed in the waist region, and excitation light of 460 nm was launched at one of the fiber, and 516 nm light intensity was measured at the other end of the fiber. The 516 nm fluorescence signal was found to be proportional to the fluorescein concentration. The results reported in this paper show in practical terms, the feasibility of measuring fluorescence through evanescent coupling, thus demonstrating the use of tapered fibers as fluorescence sensors with potential applications in medical and biomedical areas. © 2003 Elsevier B.V. All rights reserved. Keywords: Fluorescin; Microvolume; Tapered fiber; Evanescent fluorescence
1. Introduction Development of new technologies for sensing biomolecules and properties of cells have become important in recent years, because of their applications in biological and biomedical sciences [1–10]. Sensors are needed that are capable of measuring cell properties at single cell resolution in the areas of clinical, cellular, and pharmaceutical applications. This evolving area of in vivo sensing requires very small size sensors. The current state of technology does not permit high spatially resolved measurements to be performed easily or efficiently, with most of such tests being currently performed in vitro and with large volumes of analytes. Optical fibers offer a simple and rapid means of detecting and sensing biological phenomena, such as metabolic shifts, alteration in cellular morphology, and other metabolic events with ease. There are several physical and optical effects in fibers that can be used in sensing. However, the evanescent field of the fibers has been used often for applications in biochemical [2,3,5,7], biomedical [1,4,6,8–10], and environmental sensing [11–13]. The strength of the evanescent field depends on several factors, the index of the core, index of the surrounding medium, the radius of the fiber and the operating wavelength. It is possible to manipulate the strength of the evanescent field to
sense, characterize, and quantify optical properties of samples in the evanescent region. Tapering the fiber provides an easy access to the evanescent field in fibers, enabling stronger interaction with the sample, thus making the sensors versatile. Tapered fiber tips have been used as nanodelivery devices [4,9] because of the ability of the tips to produce laser beams of extremely small diameters (<100 nm). They have also been used in fluorescence sensing of biomolecules. However, in these applications, the fiber tips were used as light delivery devices, rather than as sensing elements. Thus, these tapered tips do not constitute in situ sensors, where the three features, namely, light delivery, interaction with the sample, and sensing are accomplished by the same fiber [14]. A new kind of biological sensing technique using optical fibers is presented here. Instead of the traditional approach of using a cuvette in fluorescence measurement, a tapered fiber is used to measure the fluorescence emission. Using tapered fibers fabricated from commercially available optical fibers, evanescent field is used to deliver the excitation radiation as well as collect the emission radiation. The fabrication, characterization, and in vitro sensing using tapered fibers is presented. 2. Materials and methods
∗ Corresponding author. Tel.: +1-215-895-2236; fax: +1-215-895-2227. E-mail address: [email protected] (R. Mutharasan).
0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-4005(03)00548-3
The section gives descriptions of the materials and methods used in this work. The details of the fiber, fluorescing
316
P.J. Wiejata et al. / Sensors and Actuators B 96 (2003) 315–320
agent, instrumentation used, characterization and measurement techniques are provided.
3.2 mm thick) plates together. A 0.2 mm diameter line was scored lengthwise in the center to accommodate the tapered fiber. A 6.4 mm hole was pre-drilled in the top plate to serve as the chamber (∼150 l) for holding the analyte in contact with the tapered region of the fiber. A third piece of Plexiglas (30 mm2 with a 6.4 mm hole in center) was attached to the base with four screws, after a thin layer of silicone vacuum gel was applied to its underside. The gel eliminated capillary outflow of the analyte from the central chamber. All the vertical surfaces of the fiber holder were painted black to reduce reflection and potential measurement errors. Details of this assembly are also available elsewhere. The experimental arrangement used in the fluorescence studies is shown in Fig. 2. It consists of a spectrofluorimeter (Model QM-1, Photon Technology International Inc., PTI) which has been adapted to accept the fiber. The assembly made to hold the cuevette can be removed and replaced with the fiber holder fabricated mentioned above. This spectrofluorimeter unit is equipped with a 75 W Xenon arc lamp (Ushio Inc., Japan) with a monochromator and a photomultiplier tube (PMT, housing model 710, tube model R1527P, PTI Inc.) coupled through a monochromator. Wavelength settings of monochromator and recording of PMT outputs (counts/s) were done through Felix instrument software. The cuevette holding assembly was removed first. The fiber holder was mounted on a manual X–Y–Z positioning platform in the chamber along the axis of the light source and the PMT. A microscope objective (20× UV capable) was mounted to focus the input light into the fiber. In order to make sure that the light measured by the PMT came only from the fiber, a black Delran cylindrical piece was fitted into the opening leading to the PMT. A 125 m diameter hole was drilled at the center of the Delran block to accommodate the fiber tip with Play-doh (available from toy stores) to seal the center hole so as to block leakage of stray light from the chamber. The slide with the tapered fiber was placed on the platform made to accommodate it. This whole unit was kept in a dark box so that no light from the outside enters the spectrofluorometer. A thick black cloth was also used to cover the entire fluorometer to reduce leakage of ambient light. Afterwards, additional setup was configured through the Felix computer software to set the values for scanning range. The settings for the emission scan were the following: each concentration of the material is run twice, first one at a step size of 1 nm and integration time of 1 s and the second one at a step size of 0.25 nm and integration time 5 s. An excitation wavelength of 460 nm was used and the
2.1. Materials A Corguide fiber (Corning Glass Works, NY, attenuation at 1300 and 1500 nm of 0.36 and 0.26 dB/km, respectively) with a core diameter of 8 m and total diameter of 125 m was used in all experiments reported here. The fluorescing agent used in our studies was fluorescin. A stock solution (100 M) was prepared by dissolving 2 ,7 -difluorofluorescin (Molecular Probes) powder in pH 7.3 phosphate buffered saline (PBS) at room temperature. PBS was also used as the reference. The stock solution was diluted as needed. 2.2. Methods While tapering of fibers can be accomplished using chemical etching [15–18] or heat pulling, our experience with both of these techniques showed that heat-pulled tapers provided reproducible smooth tapers. They retain the optical characteristics by virtue of relative arrangement of core and cladding while chemical etching leads to the preferential elimination of the cladding completely. The presence of both the core and cladding in the heat pulled tapers makes them ideal for sensing. The fabrication methods employed were described elsewhere [19]. A taper consists of a narrow waist with contracting and expanding regions on either side as shown in Fig. 1. The next step in our study was the characterization of the fabricated tapers. The diameter of the taper waist and the total length of the taper from beginning to the end (fiber profile) constitute the taper characteristics. They were measured using an optical microscope at 250× (model IMT-2, Olympus, Japan) equipped with a video camera (Cohu Corp.) linked to a computer. The microscope images were acquired using the Scion Image software (Scion Corp.). A microscopic scale, graduated in 10 m was used in conjunction with the acquired image to measure fiber diameter. The fiber diameter was recorded every millimeter from the beginning of the taper to the end. If and when the fiber taper has the right profile, fiber ends are clipped with a fiber optic cleaver (NO-NIK) ensuring a clean 90◦ angle cut at both ends of the fiber, assuring maximum light collection by the fiber. The fiber was placed in a ‘fiber holder’. The fiber holder was made by gluing two Plexiglas (100 mm × 30 mm × Waist DW I0
Unstretched Fiber
Transition Region I
LW Fig. 1. Schematic of a tapered fiber. Lw was typically 6–7 mm. Diameter of taper obtained was typically 3–4 m.
P.J. Wiejata et al. / Sensors and Actuators B 96 (2003) 315–320
317
Monochromator at λ fiber S1
S2
Cuvette chamber
S3
S4
75W Xenon arc lamp
PMT
details
PC
Fiber mounting apparatus
Originally for the cuvette holder L1
Optical fiber L2 to PMT Fiber mounting apparatus Fluorometer chamber
Fig. 2. Apparatus used for monitoring fiber etching in an analytical fluorometer. Slits S2 and S4 were set at 2 nm, while slits S1 and S3 were opened fully. Convex lens (L1 ) and a microscope objective (L2 ) were used to launch light from monochromator into the fiber.
3. Results and discussion We will now describe the results obtained starting with the characteristics of the fiber taper used in our measurements. Fluorescence measurements with the fiber tapers were compared to those obtained from the traditional cuevette setup. 3.1. Fiber profile Several tapered fibers were fabricated using the technique described in our previous work [19]. All the tapers made by this technique had similar physical attributes. In Fig. 3, a profile of a typical taper is shown. This fiber had a 3.69 m diameter waist and a waist length of 7.1 mm. The transition regions are not normally symmetric with respect to the waist and this is attributed to the drawing technique used. Because of this, it was decided to launch the light at the steeper taper and collect the resulting light response at the more gradually sloped end. This resulted in a better light collection at that end. Most of the tapered fibers fabricated had waist diameters in the range of 3–4 m. 3.2. Fluorescin spectra in a cuvette Experiments were conducted in the traditional cuvette arrangement to establish clearly the excitation and emission
peaks of fluorescin. Fluorescin dye is pH sensitive. The exicitation and emission scans corresponding to pH 7.3 are shown in Fig. 4a and b, respectively. Excitation was varied from 450 to 550 nm and emission 516 nm was measured and is presented Fig. 4a. Note that 460 nm excitation gives higher response than when excited at 502 nm, the second peak in Fig. 4a. In Fig. 4b emission spectra is given for excitation at 460 nm. For the experiments reported in this paper, it was decided to excite at 460 nm because the light intensity was higher at that wavelength. The added benefit is that 460 nm excitation is farther away from the fluorescence peak (516 nm), making it easier to observe fluorescence with less interference from the excitation wavelength. Fluorescence scans were undertaken for different concentrations of fluorescin. The results are shown in Fig. 5. We will now compare these results to those obtained from the tapered fiber.
Taper radius ( m)
emission scan was undertaken in the range of 450–532 nm the first time and 500–532 nm the second time. The second range was used to examine the fluorescence emission more carefully. The full range of the PMT is 3.5 × 106 counts/s. The noise floor in measurement was 30 counts/s.
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Fig. 3. Typical taper profile obtained using heat pulling. Initially fiber diameter is 125 m and the waist of this taper was 3.69 m, total taper length was 7.1 mm. Under a microscope (400×) the surfaces appeared smooth without any protrusions.
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Count (photons/s)
2.0E+06 1.8E+06 1.6E+06 1.4E+06 1.2E+06 1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05 0.0E+00 450 460 470 480 490 500 510 520 530 540 550 (a) Excitation Wavelength (nm)
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Fig. 6. Evanescent fluorescence spectra using 3.11 m tapered fiber focusing on the fluorescence area. This run was performed at 0.25 nm wavelength steps and 5 s integration time. Only three concentrations are shown, past 60 M the fluorescence intensity starts to decrease.
1.0E+06 8.0E+05 6.0E+05 4.0E+05 2.0E+05
0.0E+00 450 460 470 480 490 500 510 520 530 540 550 (b) Emission Wavelength (nm)
Fig. 4. Excitation and emission spectra for 0.1 M fluorescin in PBS, pH 7.3 in a cuvette. Panel (a) represents the absorption spectra. Note two absorption peaks at 460 and 504 nm. Panel (b) represents the emission spectra and we can observe a fluorescence peak at 516 nm. Excitation used was 460 nm.
3.3. Fluorescence characteristics of the tapered fiber The experiments were repeated now with the tapered fiber with the smallest waist (diameter of 3.11 m). Several
1.E+07 0 mM
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Fig. 5. Fluorescence spectra in a cuvette. Experiments were performed at 460 nm excitation wavelength and fluorescence in the cuvette was measured at the right angle geometry. Base level of excitation source in the wavelength region of interest is about 1000 counts/s. The emission peak is broad and does change slightly with concentration.
tapered fibers with similar geometric characteristics were tested. We report here results obtained with the 3.11 m waist diameter fiber. The cuevette assembly was removed and replaced with the fiber holder. All alignments were done as described in our previous work [19]. The emission scans are shown in Fig. 6. Increased fluorescence is seen with increased fluorescin concentration. The observed fluorescence could have been excited only from the light transmitted through the fiber interacting with the solution surrounding the taper via the evanescent field [20]. Since the tapered fiber was continuous and all the possible avenues of light leakage into the PMT were eliminated, the fluorescence observed came only from the fluorescence emission light being guided into the fiber through the evanescent coupling mechanism. Thus, these results, we believe, establish that the tapered fiber can act both as a transmitter and a delivery mechanism of the excitation energy to the optically active solution surrounding the fiber and simultaneously pick up the longer wavelength fluorescence light. Even though the emission counts are much lower than those seen in the cuvette, the fluorescence emission is of sufficient magnitude to show the optical functioning of the sensor. Another interesting result is plotted in Fig. 7. Since the cuvette and fiber geometries have their own respective maxima with fluorescin concentration, it is useful to normalize the signal with respect to the maximum for comparison of the two arrangements. The normalized fluorescent light is plotted as a function of the concentration of the fluorescin. One clearly sees that as the fluorescin concentration increases, the fluorescent emission increases, reaching a peak at 60 M for the fiber and 40 M in the cuvette, and then decreases at higher concentration. This suggests that there is a limit of detection for this sensor, indicating potentially inner filter effects quenching the fluorescence. The results in Fig. 7 also seem to suggest that the inner filter effect is slightly
Normalized fluorescent intensity
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assist as a sensor, but also a quantitative tool. In comparison to a tapered tip based fluorescence measuring system, the tapered fiber sensor reported here offers greater practical opportunities for in vivo medical applications. This is because the light collection in a tapered tip sensor depends on an open geometry, which is unsuitable for in vivo applications.
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Acknowledgements
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This work was supported (in part) by the State of Pennsylvania under The Nanotechnology Institute.
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Fluorescin Concentration ( M) Fig. 7. Plot of normalized fluorescence light intensity as a function of fluorescin concentration. Normalized value is calculated as a fraction of maximum fluorescence in the respective geometry. The tapered fiber used had a waist diameter of 3.11 m.
less in the fiber geometry compared to cuvette.It should be noted that fluorescence measured using the tapered geometry is along the axis of the fiber, while in cuvette geometry, it is orthogonal. Because the source of fluorescence signal is from the tapered region, one can be certain as to the location of the fluorescent sample, while in open optical path geometry, one can be certain of the direction, but not the location. Thus, sensing fluorescence using tapered geometry offers the added advantage of localization.In tapered fiber sensing, the source of excitation light is the evanescent filed, and thus is of low intensity compared to cuvette. Therefore, the emission signal is correspondingly smaller when compared to open cuvette geometry.
4. Conclusions In this paper, we have shown that tapered fibers have the capability for fluorescence sensing with the potential applications in various biological and medical applications. The preliminary results reported here demonstrate the concept and supports the hypothesis that the tapered region of the fiber can act as a mechanism to deliver the excitation wavelength to the sample (biomolecules) and pickup the longer wavelength fluorescence radiation generated by the sample. This unique ability of the tapered fiber makes it an ideal choice to be part of a probe to examine the presence or absence of specific analytes in solutions. The small size of the waist and small volume of analyte needed also make the taper geometry a ideal choice in cases where a sample volume available may be very small and limited. All these properties make the tapered fiber well suited for in vivo applications because the tapered region incorporates all aspects of sensing. Additional research is necessary to undertake a more careful calibration and analysis so that this probe may not only
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