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We have observed whispering gallery modes in the inelastic emission from a 9.8 µm polystyrene bead coated with a monolayer of AlexaFluor 488 dye. Using a separate near-IR trapping laser and an Ar+ excitation laser enables us to isolate and study a single dye-coated bead in a colloidal suspension without causing any bead damage.
©2004 Optical Society of America
1. Introduction
Optical whispering gallery modes (WGMs) were first photographed in millimeter-size CaF2:Sm++ spheres submerged in liquid hydrogen [1]. Subsequently, lasing was observed in 60 µm Rhodamine 6G-doped ethanol microdroplets [2], and a variety of nonlinear optical processes were observed in various liquid microdroplets [3]. Total internal reflection at the liquid-air interface enables the microdroplets to act as very high-Q optical cavities. WGM-based semiconductor lasers have received a lot of recent interest. The efficient coupling of spontaneous emission to the WGMs of a microresonator leads to an enhancement in the Einstein A coefficient at wavelengths commensurate with the WGMs and a reduction in the threshold pump power. Microdisk [4], microcylinder [5] and spiral-shaped micropillar [6] quantum well lasers have been reported. Planar waveguide-coupled microresonators have also been gaining interest for use in channel add-drop filters in wavelength-division multiplexing [7].
In addition, there has been recent interest in using WGMs for biosensing. Proposals have ranged from using the WGMs of a solid dielectric cavity to increase the absorption coupling into a fluorescent analyte layer on the cavity surface [8] to monitoring the transmission of a microresonator to detect trace amount of biological material adsorbed on the surface of the resonator [9]. Vollmer et al. have detected the presence of a layer of bovine serum albumin adsorbed on the surface of a 300 µm silica sphere by observing the shift in the wavelength of a WGM after the monolayer adsorption [10]; however, a tunable laser source is required to detect WGM wavelength-shifts. Since fluorescent-labeling is used extensively in Molecular Biology, we investigate the feasibility of using WGMs to enhance the emission from a dye-coated polystyrene bead, which serves as a model for a layer of fluorescently-labeled adsorbate. WGM-enhanced emission has been observed in dye-doped polystyrene spheres. Benner et al. observed sharp WGM peaks in the fluorescence spectra of single 9.92 µm dye-doped polystyrene beads suspended in water; these spectra were recorded as each sphere floated into and away from the excitation beam [11]. Lasing was observed in single 41 µm polystyrene beads doped with Nile Red dye that were placed on a glass plate [12]. Covalently bonding fluorophores to the surface of a bead enables us to study the WGM-enhanced emission from a surface layer. In this paper, we present the first observation of WGM-enhanced emission from a monolayer of fluorophores covalently-bonded to the surface of a 9.8 µm polystyrene bead in a colloidal suspension.
2. Experimental
The single-beam gradient laser trap, or laser tweezer, has been used extensively as a handle in colloidal systems, e.g., a laser tweezer has been used to trap an emulsion oil microdroplet in order to study its dynamical behavior when it is subjected to cyclodextrin flow [13]. Polystyrene microspheres in a colloidal suspension tend to float into and away from an excitation beam positioned in the suspension. Since visible laser tweezers are known to cause multiphoton damage to polystyrene beads, we use a near infrared laser to trap a dye-coated polystyrene microsphere and a separate Ar+ laser beam to excite the dye emission. This enables us to study the dye emission at various excitation-laser intensities well below the bead damage threshold while the trap-laser intensity is kept constant so that the bead can be held stably in the trap.
A schematic of our experimental setup is shown in Fig. 1. The source of our optical trap is a diode laser assembly (Melles Griot 56ICS115), which uses an anamorphic prism and a microlens to respectively circularize and collimate the output of a near IR laser diode at 832 nm. The laser beam enters an inverted microscope (Leica DMIRB) with a trinocular head. A dichroic mirror external to the microscope is used to combine the trapping beam with an Ar+ laser at 488 nm, which is used to excite the dye fluorescence. A Plan FLOUTAR 100X 1.3 N.A. oil immersion objective focuses the two laser beams into a sample chamber. We place gimbal-mounted mirrors at the respective eyepoints of the two laser beam-paths so that we can independently translate the laser beams in the sample chamber by steering the mirrors without losing any laser power at the sample. The IR beam overfills the back aperture of the microscope objective to ensure the formation of a strong trap.
The sample chamber consists of a #1.5 glass microscope cover slip and a glass microscope slide separated by a layer of paraffin film. It is filled with a colloidal suspension of dye-coated polystyrene beads and rests inverted on the microscope stage, with the cover slip in the bottom. The microscope objective is translated vertically such that the center of a trapped bead is held at ≈6 µm above the cover slip. After passing through a dichroic mirror and a beam-splitting prism internal to the microscope, the scattered light is imaged onto a digital still camera (Nikon Coolpix 995) and a video camera (GBC) mounted on the trinocular head, and an imaging spectrograph (Acton SP-300) mounted on the camera port of the microscope. A small fraction of the scattered laser diode light is transmitted by the dichroic mirror and allows us to monitor the position of our laser trap with the video camera. We bypass the beam-splitting prism and image all the back-scattered light onto the entrance slit of the spectrograph when we are recording the inelastic emission spectra from a polystyrene bead. A bandpass filter (Chroma Technology E720SP) is used to filter out the elastically scattered light from both lasers, and a liquid-nitrogen cooled CCD camera (Roper Scientific Spec-10:100) is mounted at the exit plane of the spectrograph to record the inelastic spectra. The magnified image of the bead at the entrance slit of the spectrograph is 3 mm; however, we have to limit the entrance slit of the spectrograph to 30 µm to maximize the spectral resolution of our system. We therefore use the laser tweezer to position the bead such that we are only collecting scattered radiation from an edge of the bead.
Commercially available dye-coated polystyrene beads are usually coated with the popular biological label FITCI and are very susceptible to photobleaching. We therefore coat our beads with Alexa Fluor 488 (A488), a dye whose fluorescence spectrum is very similar to the laser dye Rhodamine 6G (extends from 500 nm to > 600 nm with a peak at ≈525 nm [14]) but is much more photostable. We add A488 succinimidyl ester (Molecular Probes) to a suspension of 9.8 µm diameter amine(NH2)-functionalized polystyrene beads (Interfacial Dynamics Corp.) and mix them in a vortexer for half an hour. It is interesting to note that the surface of each amine-functionalized bead is a “3-dimensional surface” and each amine group is attached to the polystyrene backbone via a 6-carbon aliphatic chain. A nucleophilic addition-elimination reaction occurs through which A488 fluorophores are covalently bonded to the surface of the beads via amide(C-N) bonds (See Fig. 2). After the beads are coated, they are centrifuged. The supernatant containing the unbonded A488 is aspirated, and the beads are resuspended in water. We repeat this centrifugation – aspiration – resuspension procedure 5 times, which results in a colloid of A488-coated beads in water with no background fluorescence. Since the area of each amine group is 0.2 nm2, each polystyrene bead contains 1.5 billion A488 fluorophores. The amine groups, and thus the fluorophores, are randomly distributed over the surface of each bead [15]. Mercaptoethylamine (MEA) is added at a concentration of 0.2 M to the colloidal suspension as an anti-photobleaching agent.
3. Results and discussion
Figure 3 shows the inelastic emission spectra from an A488-coated bead. These spectra were collected when the excitation Ar+ laser was positioned at the center of the bead and they were integrated for one to three minutes. At excitation laser intensity Iex=0.29 W/cm2, the inelastic spectrum from the A488-coated bead resembles the fluorescence spectrum of A488 in bulk solution. The single-pixel or two-pixel wide irregularly-spaced spikes correspond to cosmic rays incident on the CCD camera. At 0.87 W/cm2≤Iex≤86.7 W/cm2, the inelastic emission spectra consist of two sets of regularly-spaced peaks above a broader background. The broader background corresponds to A488 fluorescence emitted into free space modes. The narrow peaks correspond to A488 emission coupled to the lowest radial order WGMs (TE and TM) of the polystyrene bead. These WGMs are localized to a radial region that is closest to the bead’s surface and thus couple efficiently to the A488 emission. Similar WGM-enhanced emission can be observed when the excitation laser was positioned at the edge of the bead that was furthest away from the edge of the bead where the spectra were collected. The bead was stable within the time frame of our data collection. However, at Iex>100 W/cm2, the A488-coated bead photobleached despite the addition of MEA.
Fig. 3. Normalized inelastic emission spectra from a single A488-coated polystyrene bead. The spectra at Iex=8. 67 W/cm2 and 0.87 W/cm2 have been multiplied by 10, and the spectrum at Iex=0.29 W/cm2 has been multiplied by 3.
It is very likely that the WGM-enhanced inelastic emission we observe from the A488-coated bead is due to lasing from A488. The lack of a tunable laser source prevents us from measuring the cold-cavity Q of our A488-coated bead. Using a Lorenz-Mie resonance algorithm [16], we calculated that for a 9.8 µm polystyrene bead suspended in water with a refractive index ratio of 1.59/1.33=1.20, the cold-cavity Q of the lowest radial order WGMs for a perfectly spherical polystyrene bead in the 500–600 nm region is between 300 and 2000. The extended nature of the non-uniform surface of our A488-coated bead (see Fig. 2) further suggests that the cold-cavity Q of an A488-coated bead is likely to be less than that of a perfect polystyrene sphere. However, all the WGM peaks in Fig. 3 have a FWHM of 0.2 nm to 0.3 nm, which is less than a typical cold-cavity linewidth of Δλ=λ/Q=524.5 nm/1000=0.5 nm. It is well known that line-narrowing of stimulated emission can occur above the lasing threshold [17]. Therefore, the WGM-enhanced inelastic emission from A488 we observe is probably due to lasing from A488, with a lasing threshold between 0.29 W/cm2 and 0.87 W/cm2. In addition, the image of the bead in Fig. 4b shows two bright spots which have typically been associated with laser spots arising from two counterpropagating laser beams at the rim of a spherical microcavity [18].
Fig. 4. 8-second exposures of an A488-coated bead recorded by the digital still camera. The Ar+ excitation laser was positioned at the center of the bead in Fig. 4(a) and at the edge of the bead in Fig. 4(b).
To ascertain that the narrow peaks we observed is not due to some unknown artifact of A488-coated beads suspended in water, we studied the WGM-enhanced emission from an A488-coated bead suspended in a propanol-water mixture with a 40% propanol mole fraction such that the refractive index of the mixture is 1.38 [19]. Since the refractive index ratio between polystyrene and propanol (1.59/1.38=1.15) is less, the WGMs are less well-confined inside the bead and should thus broaden spectrally. Using the same algorithm as above, we calculated that the cold-cavity Q’s for the WGMs of a 9.8 µm polystyrene sphere in this propanol-water mixture are < 10, which is indeed less than that for the same sphere in water. Fig. 5 shows the inelastic spectrum from an A488-coated bead suspended in a propanol-water mixture with an Ar+ laser intensity Iex=86.7 W/cm2. The FWHM of each WGM is ≈0.6 to 0.7 nm, which is broader than the WGM from an A488-bead suspended in water because of the lower Q’s of the propanol-water WGMS.
4. Conclusion
We have observed the WGM-enhanced inelastic emission from a monolayer of A488 fluorophores on the surface of a 9.8 µm polystyrene bead trapped in an optical trap. It is likely that the WGM-enhanced emission is due to A488 lasing, with a lasing threshold between 0.29W/cm2 and 0.87 W/cm2. The WGMs we observed are surprisingly narrow, especially if we consider the surface morphology of the bead. Further experiments will probe a potential lasing-transition and investigate the effects of surface morphology WGMs.
Acknowledgments
We gratefully acknowledge Dr. Theresa Lynn for a helpful suggestion, Dr. Steven Hill for providing the FORTRAN code of the algorithm described in [16], and colleagues in the Chemistry Department at Pomona College for many Organic Chemistry tutorials and for the use of lab equipment. PGS was supported by research funds from the Pomona College Dean’s Office.
References and links
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