8 OPTICS LETTERS / Vol. 32, No. 1 / January 1, 2007

Reshaping a sample fluid droplet: towardcombined performance enhancement of an

evanescent-wave fiber-optic fluorometer

Jianjun Ma and Wojtek J. BockCentre de Recherche en Photonique, Département d’Informatique et d’Ingénierie, Université du Québec en

Outaouais, P.O. Box 1250, Station B, Gatineau, Québec J8X 3X7, Canada

Received August 15, 2006; revised October 3, 2006; accepted October 7, 2006;posted October 11, 2006 (Doc. ID 74119); published December 13, 2006

A modified evanescent-wave fiber-optic fluorometer capable of simultaneous signal enhancement and sup-pression of stray excitation light is examined. Such a capability is achieved by adjusting the shape of a fluo-rophore sample droplet and regulating the distance separating the exit of the illuminating fiber and an un-cladded segment of the receiving fiber, set perpendicular to each other. The effects of sample attenuation andinhomogeneity are also analyzed and evidenced by associated experiments. © 2006 Optical Society ofAmerica

OCIS codes: 060.2270, 060.2370, 300.6280.

Fluorescent spectroscopy is a powerful tool forsample analysis in the biological sciences. For theseapplications, evanescent-wave (EW) excitation ofsamples immobilized on the surface of decladded op-tical fibers is often preferred1 because of its excellentperformance in the measurement of surface-specificevents. However, excitation power levels present achallenge for this technique. Theoretical analysisshows that nearly all the power present in the EWarises from the higher-order modes.1 A large-coremultimode fiber with a high NA is often chosen foreasy and highly efficient light coupling, dramaticallyreducing manufacturing cost. Its mode number is de-termined by N=0.5k2a2�NA�2, where k=2� /� is thewave number in free space and a is the radius of thefiber core. The percentage of the power contributed toEW excitation is limited by a factor of approximately1.33N−1/2, where N is the number of modes propa-gated by the fiber.2 This limitation reveals that amultimode fiber suffers lower EW excitation powerdue to the large number of propagating modes. Al-though the larger cladding distributed mode field of asingle-mode or a few-mode fiber enhances the EW ex-citation power and has been investigated,3,4 the smallcore size of such a fiber complicates device fabrica-tion, light coupling, and alignment of additional com-ponents. The total fluorescent signal, of course, couldbe increased by using a longer sensing length of thefiber,5 a solution widely adopted by scientists. Unfor-tunately, this requires an increase of the sample vol-ume. Moreover, two typical EW-based fiber probes,which feature access to one or two fiber ends for ex-citation light delivery and signal collection, showother drawbacks. Access to one end requires a beamsplitter and lens system or a fiber coupler; access totwo ends involves the use of a high-performancefilter-detector system to remove the strong stray lightfrom the light source. To address all these existingproblems, we propose a modified EW-based fiber fluo-rometer capable of simultaneous signal enhance-

ment, stray excitation light suppression, and mea-

0146-9592/07/010008-3/$15.00 ©

surement of reduced sample volume, featuring asimplified architecture and low cost.

As illustrated in Fig. 1, a 1.8 m multimode fiberwith a core/cladding size of 400/425 �m serves as areceiving fiber, or r-fiber. A 5 cm length of cladding atone end is removed. Its tip is positioned in a chamberto block the ambient stray light. The other end of thefiber is connected to a USB2000 spectrometer fromOcean Optics that is interrogated by a computer. Theilluminating fiber, or i-fiber, having a core/claddingsize of 300/330 �m and positioned at 90° to sym-metrical axis z of the r-fiber, launches the excitationlight from a 633 nm He–Ne laser source to thesample. By use of two precision translation stage sys-

Fig. 1. Experiments for the proposed sensor. (a) Experi-mental setup. The fiber tip is positioned in a micro darkchamber. The inset indicates the three positions of thei-fiber for sample excitation. The photographs in (b) and (c)show the sample, the i-fiber, and the r-fiber associated with

positions 1 and 2 of (a), respectively.

2006 Optical Society of America

January 1, 2007 / Vol. 32, No. 1 / OPTICS LETTERS 9

tems, the r-fiber can be moved along the +z and −zdirections as well as the +y and −y directions and thei-fiber can move along the +x and −x directions. AlexaFluor 635 dye conjugate diluted in 0.1 M phosphate-buffered saline6 is the fluid sample used for investi-gation. A tiny drop of the sample is gently injectedinto the point where the i-fiber and the r-fiber meet toform a small droplet for measurements. In the ex-periment, the i-fiber is first adjusted to a positionclose to that of the uncladded segment of the r-fiber.The sample droplet, with a concentration of80 �g/ml, is then dispensed. The fluorescent emis-sion spectrum with the best fluorescent signal level isrecorded by moving the i-fiber along the x axis only,as represented by position 2 in the inset of Fig. 1.Second, by separately adjusting two fibers along the xand y axes, the i-fiber is altered to position 1, wherethe optimum spectrum with maximum fluorescentsignal and minimum background excitation light isgenerated and recorded. Third, another optimumspectrum is recorded with the i-fiber adjusted to po-sition 3.

Figures 2(a) and 2(b) show the optimized spectra atthe three positions for sample concentrations of 80and 10 �g/ml, respectively. Surprisingly, position 2,which is thought to possess the strongest fluorescentemission intensity, indicates a very low signal leveland strong stray excitation light. In contrast, at posi-tion 1 the first maximum fluorescent emission hasthreefold and fourfold intensity increases for the 80and 10 �g/ml concentrations, respectively. For bothconcentrations, the second maxima are seen at posi-tion 3. Noticeably, at both positions 1 and 3, the exci-tation light background is completely eliminated forthe higher-concentration sample. For the lower-concentration sample, only a very weak trace of theHe–Ne laser line is observed at position 1. When thefiber separation is increased from position 1 to 2 or 3,the experiment also shows a gradual rise and fall ofthe fluorescent signal and the stray excitation lighttrace levels, respectively.

The observed results can be explained throughRefs. 5, 7, and 8, which indicate that most of the fluo-rescent power coupled to the guiding modes comesfrom a thin layer of molecules surrounding the sur-face area of the fiber (about a wavelength thick). It istherefore essential to distribute the strong excitationlight power uniformly in this layer, or EW layer, to

Fig. 2. (Color online) Optimized fluorescent emission spec-trum for three i-fiber positions. The units of intensity arebased on the sensitivity of the USB2000 spectrometer at 86photons/count (estimate). (a) and (b) Optimized spectra forsample concentrations of 80 and 10 �g/ml, respectively.

efficiently excite these molecules. Ray tracing of Fig.

1(b), illustrated in Fig. 3(a), reveals that this goal canbe accomplished by reshaping the droplet to create aliquid–air surfaced micro concave mirror and a nar-row sample layer on one side of the target fiber.Based on Snell’s law, all light rays from the i-fiberwill be reflected back toward the EW layer by thismirror through a total internal reflection (TIR) or apartial internal reflection (PIR). Critically, three raygroups will contribute to the EW-layer excitation.The first ray group, rays − and ® in Fig. 3(a), willskim over the target surface. This is further high-lighted in Fig. 3(b). The second ray group, associatedwith ray ¯ in Fig. 1(a) and further shown in Fig. 3(c),has a grazing incident angle at the core surface. Alarge portion of its power will penetrate into the core.With their incident angles close to the critical angle,the rays in the core will experience multiple PIRs atthe inner core surface until all their power is uni-formly transferred to the entire EW layer surround-ing the core. The third ray group, ray ¬ in Fig. 3(a),has a smaller incident angle to the target surface.Most of its energy will pass through the fiber-coresurface twice with very small reflection, as indicatedin Fig. 3(d). The final result is a bright and uniformexcitation light distribution in the EW layer, en-abling the generation of strong fluorescent light andits highly efficient coupling to the guiding modes ofthe fiber. The intensity of fluorescence, IF, can be ex-pressed as IF=KI0d, where I0 is the intensity of theexcitation light; K is a factor associated with the con-centration of the solution, the molecular extinctioncoefficient, and the quantum efficiency of fluores-cence; and d is defined as the optical path length. Forthe first ray group, we use a ray in Fig. 3(b) as a rep-

Fig. 3. (Color online) Ray tracing for the proposed sensorarchitecture, (a) Ray tracing associated with Fig. 1(b). �,thickness of the EW layer. (b) Result for a ray skimmingover the core surface. (c) Result for a ray with a grazing in-cident angle. (d) Result for a ray with a smaller incident

angle. (e) Ray tracing associated with Fig. 1(c).

10 OPTICS LETTERS / Vol. 32, No. 1 / January 1, 2007

resentative and find that d=2d1�2��R, where � isthe thickness of the EW layer and R is the fiber-coreradius. As a comparison, ray tracing of Fig. 1(c) isplotted in Fig. 3(e), showing that most of the rays car-rying high power have a very short path length d�2�. Therefore, the fluorescent signal enhancementfactor for the first ray group, defined as �1, will be�1=�R /�. For R=200 �m and �=0.6 �m (thickness ofabout one He–Ne laser wavelength), �1�18. Thisclearly explains why launching the rays directly tothe target core surface, as is the case in Figs. 3(d) and3(e), will result in a much lower fluorescent signallevel. Moreover, direct exposure of the target fibercore to strong excitation light will easily introducescattered excitation light into the fiber core as a re-sult of even minor defects of the fiber or inhomogene-ity of the sample, as evidenced by the laser traces inFigs. 2(a) and 2(b) associated with position 2. To es-timate the enhancement factor �2 of the second raygroup, as shown in Fig. 3(c) and from its geometryand Snell’s law, it is easy to prove that all pathlengths in the EW layer are equal to d2. Assumingthat the ray inside the core will experience n reflec-tions, their reflectances and transmittances are Riand Ti �i=1, . . . ,n� at the respective points labeled inFig. 3(c). The total fluorescence generated in the EWlayer will be

IF = �i=1

n

IFi = KI0d2�1 + R1 + T1T2 + T1R2T3 + ¯

+ T1�j=2

i

RjTi� . �1�

Replacing all Ti terms with Ti=1−Ri and expandingEq. (1) lead to the cancellation of most terms. The fi-nal result is

IF = limn→� with R�1

KI0�2d2� + KI0d2�j=1

n

Rj = KI0�2d2�.

�2�

In light of the fact that d2�d1 for a grazing angle,Eq. (2) leads to �2��1. Replacing d2 with d3, we findthat Eq. (2) is also valid for the third ray groupshown in Fig. 3(d), but with d3��. On the otherhand, our experimental results in Figs. 2(a) and 2(b)indicate that the observed total signal enhancementis much lower than the estimated value of �1�18.This may stem from several reasons: first, the excita-tion light experiences loss in the reflection surface ofthe mirror. In contrast, the rays in Fig. 3(e) will carrymuch more excitation power to the EW layer. Second,the path length of the second ray group, d2, tends tobe shorter when the grazing angle increases. Third, alarge portion of excitation light power is carried bythe third ray group with much less excitation effi-ciency.

Other critical factors for the proposed sensor are

also highlighted here. First, the weight of the droplet

affects the shape of the mirror and hence the fluores-cent signal level, as evidenced in Figs. 2(a) and 2(b).Second, for a highly attenuated sample, its perfor-mance deteriorates due to a required long pathlength from the i-fiber to the r-fiber. In contrast, if wereduce the fiber separation in Fig. 1(a), the light ismuch less attenuated, allowing part of the EW layerto be efficiently excited. In the experiment, by dilut-ing Rhodamine 6G to a dark color and following theaforementioned experimental steps, we visibly veri-fied the huge loss of the sample by observing the dis-appearance of the excitation power within a veryshort distance from the i-fiber exit. Then we observeda reduction of the fluorescent signal level when mov-ing the i-fiber from position 1 to 3, proving the analy-sis above. Third, there is a strong association be-tween sample homogeneities and the stray excitationlight. In our experiment, this type of sample is pre-pared by evaporating part of the buffer solvent toform some dye particles in the sample. We observedthe appearance of stray excitation light in the fluo-rescent spectrum, suggesting that these particles dif-fuse the excitation light toward different directions.Some rays are thus able to interact with the EW tailand transfer their power to the guiding mode. Fi-nally, the proposed orthogonal and symmetrical ar-chitecture also leads to a backreflection-free feature,allowing the high-quality signal level to be furtherdoubled by simply connecting both ends of the fiber tothe spectrometer.

In conclusion, we have proposed a modified EW-based fluorometer capable of simultaneous signal en-hancement, stray excitation light suppression, andmeasurement of reduced sample volume. These fea-tures, combined with its simple architecture and lowcost, point to its bright future in chemical, biological,biomedical, and clinical applications as well as envi-ronmental monitoring. The highly efficient couplingof excitation light to the EW layer also makes it agood tool for surface-enhanced Raman scatteringspectroscopy.

The authors gratefully acknowledge support forthis project from the Natural Sciences and Engineer-ing Research Council of Canada and from theCanada Research Chairs Program. J. Ma’s e-mail ad-dress is jianjun@uqo.ca; W. J. Bock’s iswojtek.bock@uqu.ca.

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