Transmission and Confocal Fluorescence Microscopy and Time

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J. Phys. Chem. B 1998, 102, 8440-8451

Transmission and Confocal Fluorescence Microscopy and Time-Resolved Fluorescence Spectroscopy Combined with a Laser Trap: Investigation of Optically Trapped Block Copolymer Micelles Thomas Gensch,† Johan Hofkens,† Jan van Stam,† Herman Faes,† Serge Creutz,‡ Kenji Tsuda,† Robert Je´ roˆ me,‡ Hiroshi Masuhara,§ and Frans C. De Schryver*,† Department of Chemistry, Katholieke UniVersiteit LeuVen, Celestijnenlaan 200F, BE-3001 HeVerlee, Belgium, Center for Education and Research on Macromolecules (CERM), UniVersity of Lie` ge, Sart-Tilman B6, BE-4000 Lie` ge, Belgium, and Department of Applied Physics, Osaka UniVersity, Suita, Osaka 565, Japan ReceiVed: May 28, 1998; In Final Form: August 10, 1998

Optical trapping was combined with transmission microscopy (TM), confocal and nonconfocal fluorescence scanning microscopy (CFSM and FSM, respectively), and confocal and nonconfocal time-resolved fluorescence spectroscopy (CTRFS and TRFS, respectively) to study latex particles and block copolymer micelles. Dyelabeled latex particles of various size, in polymer composite films as well as optically trapped in solution, were studied with CFSM to characterize the limits of the setup. CFSM revealed that the resolution in the xand y-directions was near the theoretical limit, i.e., 200-250 nm. CTRFS on the labeled latex particles revealed that the decay time of the label was not influenced by the polymer matrix nor the optical trap. Poly(tert-butylstyrene-block-sodium methacrylate) micelles (diameter approximately 30-40 nm) in deuterated aqueous solutions could be optically trapped, this region of high copolymer micelle concentration being referred to as a trapped cluster. In the transmission images, trapped clusters of 1.5-2 µm diameter were detected. Fluorescence images were obtained using perylene as a fluorophore that is specifically dissolved within the block copolymer micelles. The size of the trapped cluster, estimated from TM and FSM images, increases with increasing irradiation time and power, respectively. In the TM images, the trapped cluster appears as a dark spot (low transmission) with a bright (high transmission) corona-like ring around it. The appearance of the corona is explained as a light deflection phenomenon; i.e., the trapped cluster acts as lens due to a lateral refractive index gradient. When the corona is taken into account when the diameter of the trapped clusters is calculated, a very good agreement is found between TM and FSM. Long irradiation times lead to the formation of large trapped clusters, which are stable for about 10 s, with diameters of several hundred nanometers, while, for short irradiation times, the trapped cluster is smaller and disappears within a time less than 1 s. With CFSM it could be shown that the trapped particle has a spot size of approximately 1.7 µm in the region of the IR laser focus, while the diameter extends up to 5 µm without using the confocal imaging capability. The reason for this is that the conditions for optical trapping are fulfilled not only in but also above and below the focal region. Due to the high numerical aperture, a dumbbell-like shape of the trapped cluster results.

1. Introduction Imaging spectroscopy techniques with spatial resolution have become more important in the study of molecules in complex environments, when inhomogeneities in the local surrounding are present, e.g., biological membranes, Langmuir-Blodgett films, or organic solids. Interactions between the molecules of interest and between them and their environment can cause significant changes of the spectroscopic observables. To make a characterization with higher spatial resolution possible, combining microscopy and modern spectroscopy allows a correlation between spatial, spectroscopic, and time-resolved information. Confocal fluorescence scanning microscopy (CFSM) with fluorescence imaging has, theoretically, a spatial resolution approaching 0.6λ/(x2NA) (NA stands for the numerical aper* To whom correspondence should be addressed. Telephone: +32-1632 74 05. Telefax: +32-16-32 79 89. E-mail: Frans.DeSchryver@Chem. KULeuven.ac.be. † Katholieke Universiteit Leuven. ‡ University of Lie ` ge. § Osaka University.

ture) in the lateral and 1.4nλ/NA2 (n is the object medium refractive index) in the axial direction.1 Optical trapping, a technique pioneered by Ashkin and Dziedzic,2,3 is based on the momentum conservation in interactions of photons with molecules.4 It is established by the radiation pressure exerted by an electronically nonresonant light beam, focused into a solution through a high magnification lens with large numerical aperture. The optically trapped particles can be of a very different nature, such as droplets,5-7 polymer beads,4,8-11 biological cells,12-19 and single macromolecules.20-26 The size of the trapped particle can range from 30 µm down to 20 nm.4 Usually, the refractive index of the particle has to be higher than that of the solvent, although this problem can be circumvented by the use of a scanning beam. The past decade led to an impressive development of complex experimental setups using the optical trapping technique in different fields of biology15-17 and chemistry.22 Recently, a number of studies on the molecular association of Rayleigh particles were published. Rayleigh particles have a diameter much shorter than the wavelength of visible light;

10.1021/jp9824104 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/03/1998

Optically Trapped Block Copolymer Micelles examples of such particles are polymer aggregates23-27 and single macromolecules.21,22 The association process was characterized by transmission and fluorescence imaging microscopy, as well as by stationary and time-resolved fluorescence spectroscopy. Formation of a trapped cluster (diameter of the cluster: e25 µm) was observed in aqueous solution due to local heating at the focal spot and subsequent phase transition of the N-isopropylacrylamide polymer.23-25 The radiation pressure can lead to cluster formation in organic solvents,21,22 as well as in aqueous and deuterated (D2O) solutions,27 with no heating in the focal spot in the latter. Polymers in the optical trap can undergo conformational changes, as proven by differences in fluorescence spectra22,26 and fluorescence decays.22 In this article, information obtained by transmission microscopy (TM), confocal and nonconfocal scanning fluorescence microscopy (CFSM and FSM, respectively), confocal and nonconfocal time-resolved fluorescence spectroscopy (CTRFS and TRFS, respectively), and optical trapping is combined. First, the performances of the different parts of the setup are demonstrated on and controlled with test samples, i.e., dyelabeled latex particles embedded in poly(vinyl alcohol) (PVA) films or in solution. In a subsequent step, block copolymer micelles served as optically trapped objects. The enrichment of block copolymer micelles near the focal spot of the trapping laser was investigated. The optical trap was realized by using a high-power continuous wave Nd:YAG laser, of which the laser beam is focused into a diffraction limited spot using a microscope with high numerical aperture (NA ) 1.3) and large magnification (100×). The interaction of the IR light with the nonabsorbing solutesswhich have a higher refractive index than the bulk solventsleads to a spatially symmetric radiation force acting on the micelles. The association process was visualized by the detection of the fluorescence of an added chromophore, perylene. This method allows the estimation of the size and shape of the trapped cluster. The system investigated, i.e., block copolymer micelles, is also described by its micellar aggregation number. By the use of another fluorescent probe, i.e., a ruthenium complex, the aggregation number was determined for these micelles. 2. Experimental Section 2.1. Materials and Samples. Polymer films consisting of submicrometer (500, 220, and 100 ((10) nm) courine-labeled latex particles in PVA were used to estimate the capability of the setup, while coumarine-labeled latex particles with a diameter of 1 and 4.5 µm in H2O were used for the trapping experiments. The latex particles were from PolySciences, and coumarine 485 (Kodak) in methanol (ACROS, Spectrograde) was used as reference in the time-resolved fluorescence experiments. The excitation and emission wavelengths were 420 and 500 nm, respectively, for all time-resolved measurements. For the measurements on block copolymer micelles, the diblock copolymer poly(tert-butylstyrene-block-sodium methacrylate) (tBSM), Figure 1, was used, of which the synthesis and characteristics previously have been described.28 tBSM consists of 26 tert-butylstyrene units in the hydrophobic block and 37 sodium methacrylate ones in the hydrophilic block, calculated from the average molecular weights 4200 and 5300, respectively (Mw/Mn ) 1.1). tBSM forms micelles in aqueous solution, and at room temperature, no exchange of polymer molecules between the micelles occurs within a time frame of at least 3 h, while a slow exchange is observed at 60 °C.28 Two other block copolymers, i.e., a methyl methacrylate-sodium methacrylate diblock copolymer (MMM) and a sodium meth-

J. Phys. Chem. B, Vol. 102, No. 43, 1998 8441

Figure 1. Structure of poly(tert-butylstyrene-block-sodium methacrylate).

acrylate-(tert-butylamino)ethyl methacrylate triblock copolymer with hydrophilic outer blocks (MBAEMM), were used to prove that the trapping of tBSM is not a feature of this specific block copolymer. For the fluorescence imaging, perylene (99+, ACROS) was used. For the determination of the micellar aggregation number, tris(2,2′-bipyridyl)ruthenium(II) chloride (RUBIPY) (Fluka) was used as fluorescent probe and 9-methylanthracene (9-MA) (Eastman Kodak) as quencher. Triton X-100 (TX-100) (scintillation grade, ACROS) was used as micellar reference system. All chemicals were used as received. All solvents for stock solutions were of spectroscopic grade. Stock solutions of perylene in chloroform, RUBIPY in absolute ethanol, and 9-MA in absolute ethanol were used. From the stock solutions, a known volume, to give the desired final concentration, was pipetted into a volumetric flask and the solvent evaporated off. The concentration of the tBSM in the aqueous (water of Milli-Q quality) or D2O (99.9%, ACROS) solutions for microscopy and spectroscopy measurements was between 0.8 and 1.1 g/L. The solutions were allowed to stir overnight at 50-60 °C, to ensure complete dissolution and distribution of the probe. The composition of the samples is given in Table 1. To remove dust and other impurities, the block copolymer solutions for the optical trapping experiments were filtered using a syringe and filters with different pore sizes (Millipore, Millex 0.1, 0.22, and 0.45 µm). The loss of perylene absorbance was less then 10%, which indicates that the block copolymer concentration was not significantly reduced by the filtering process. 2.2. Methods. The use of a high repetition rate, modelocked, solid-state titanium-sapphire laser (Spectra Physics, Model 3960), pumped by a continuous beam-locked argon ion laser (Spectra Physics, Model 2080) as picosecond excitation source, permits a fast measure of fluorescence decays with high time resolution. The frequency doubled light of the titaniumsapphire laser has a pulse width at half-maximum smaller than 2 ps, implying that the time resolution is predominantly determined by the high-speed photodetector and the electronic circuit used for single photon counting measurements. The frequency doubled output of the laser was guided to a Biorad MRC 600 scanning unit (SU), which is connected to a Nikon Diaphot 300 inverted microscope to perform CFSM measurements. The optical image is built up by scanning the excitation laser beam over a fixed sample. Light emitted from the sample passes the objective, mirrors, and a circular variable aperture, before reaching the photodetector. For time-resolved data collection through the microscope, a cooled microchannel plate (MCP, Hamamatsu R3809U-50, cooled by a Hamamatsu C2773 cooler) was used as detection system, placed close to the

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Gensch et al.

TABLE 1: Composition of the Micellar Samples Used for Optical Trapping, Confocal Microscopy, and Time-Resolved Fluorescence Measurements Triton X-100 sample

cTX-100 (mM)

cperylene (µM)

solvent

cNaCl (mM)

tx-1 tx-2 tx-3 tx-4 tx-5 tx-6

69 69 69 69 69 3.5

11 11 11 11 22.5 22.5

H2O H2O H2O H2O H2O H2O

0 100 200 500 0 0

Poly(tert-butylstyrene-block-sodium methacrylate sample

ctBSM (g/L)

cperylene (µM)

solvent

cNaCl (mM)

tBSM-1 tBSM-2 tBSM-3 tBSM-4

0.84 0.96 0.96 1.1

22.5 22.5 11 2.5

D2O H2O H2O H2O

0 0 500 0

sample

ctBSM (g/L)

cRUBIPY (µM)

c9-MA (mM)


0.70 0.70 0.70 0.70

1 1 1 1

0 32 64 80

solvent H2O H2O H2O H2O

a Samples tx-1 to tx-5 were used for time-resolved fluorescence measurements and tx-4 to tx-6 for fluorescence quantum yield estimations. Sample tx-3 was measured both through the microscope and in ordinary SPC mode, while the others were measured in SPC mode. Samples tx-1 to tx-5 contain only micelles with none or one perylene per micelle, while in sample tx-6 40% of the perylene molecules are dissolved in micelles with more than one perylene. Sample tBSM-1 was used for optical trapping and for time-resolved fluorescence measurements through the microscope. Sample tBSM-2 was used for time-resolved fluorescence measurements in SPC mode. Samples tBSM-2 to tBSM-4 were used for fluorescence quantum yield determinations. Samples tBSM-5 to tBSM-8 were used for the determination of the tBSM micellar aggregation number by time-resolved fluorescence quenching measurements. The samples were measured in SPC mode.

entrance/exit aperture of the SU. The fluorescence emitted by the sample is directed by a 100% reflective mirror to the MCP. Wavelength selection takes place by narrow band-pass filters (full width at half-maximum (fwhm), 10 nm around the central wavelength). For optical trapping, a single beam system is coupled to the other side port of the microscope by a dichroic mirror that reflects infrared light of a diode-pumped continuous wave Nd: YAG laser (λem ) 1064 nm, P ) 660 mW) but transmits visible light shorter than 650 nm. For the trapping experiments, a 100×/1.3NA oil immersion lens was used. To properly fill the aperture of this objective, and in order to make the beam convergent, two planconvex lenses (f1 ) 100 mm) were used with the flat surfaces facing one another. One of the lenses is placed on a x,y,z translation stage, and its position is changed to optimize the focal spot size and depth. Although the output of the Nd:YAG laser is 12 W, the highest trapping power available was 660 mW due to losses in the microscope setup.8 A more detailed description of the setup is given elsewhere.29 Two different modes were used for the time-resolved fluorescence measurements: One where the emission was collected through the microscope and one where the solution was irradiated in an ordinary quartz cuvette and the emission measured at a right angle to the excitation light. The first mode will be referred to as “microscopy mode” and the latter as “SPC mode”. For microscopy mode measurements on the latex particles, sample cells were constructed by placing two cover glasses with a thickness of 100 ( 20 µm on a slide glass (1 cm between the edges of the two cover glasses). A few droplets

of a solution containing labeled latex particles were added, and a third cover glass was put on top of the two other cover glasses. In this way, a solution layer of approximately 100 µm was obtained. For measurements on the block copolymer micelles, the same procedure was followed. For SPC mode measurements, 1 × 1 cm quartz cuvettes were used. For the CFSM imaging measurements of tBSM, perylene was used as chromophore. The solutions were characterized both with respect to their absorption (Perkin-Elmer Lambda-6) and their steady-state emission and excitation spectra (SPEX Fluorolog 1680 combined with a SPEX Spectroscopy Laboratory Coordinator DM1B). For the determination of the aggregation number of the tBSM micelles, RUBIPY was used as fluorescent probe and 9-MA as quencher. Fluorescence decays, differing only in quencher concentration, can be analyzed according to30-34

F(t) ) A1 exp(-A2t - A3[1 - exp(-A4t)])

(1)

where F(t) is the fluorescence intensity at time t and the parameters A1-A4 have the following meanings, assuming stationary probe and quencher molecules: A1 is the fluorescence intensity at time t ) 0. A2 is the decay rate at long times. If there is no exchange of probe or quencher between the micellar phase and the bulk, A2 will equal the unquenched decay rate k0. A3 is the average number of quencher molecules per aggregate, assuming a Poissonian distribution. With a known amount of aggregated block copolymer molecules, the average micellar aggregation number, 〈a〉, can be calculated as

〈a〉 ) A3Sm/Qm

(2)

where Sm is the concentration of bound surfactant (for tBSM, the total concentration due to its extremely low cmc) and Qm the concentration of bound quencher (for 9-MA, the total concentration, due to its very low water solubility). A4 equals the first-order quenching rate constant kq for one quencher in a micelle. For micelles with a large volume, the aggregation number determined from eqs 1 and 2 will be a lower limit value. For a long-lived probe such as RUBIPY, however, large volumes can be probed, and the RUBIPY/9-MA probe/quencher pair has successfully been used for other large micelles.34 As reference system for the time-resolved fluorescence measurements, perylene in TX-100 micelles was used. The TX100 concentration was 69 or 3.5 mM and the perylene concentration 11 or 22 µM, to yield only singly occupied micelles and micelles with more than one perylene molecule, respectively. A series of measurements with added salt was also performed with NaCl concentrations between 100 and 500 mM. Fluorescence decays were analyzed by a nonlinear leastsquares iterative reconvolution method, based on the Marquardt algorithm. The single and global curve analysis methods used have been discussed earlier.35-40 3. Results and Discussion Latex particles and block copolymer micelles (diameter 3040 nm, assuming spherical micelles) in D2O were optically trapped. The latex particles were measured in order to define the instrument resolution and estimate reliability and will be treated in an introductory section. The results of the measurements on block copolymer micelles are subsequently discussed.

Optically Trapped Block Copolymer Micelles


Figure 2. CFSM images of 220 nm labeled latex particles in a PVA film. The spot size is given under each image. Panel a shows “all beads”, b “a few beads”, and c a single bead.

Figure 3. CFSM images of a PVA film with labeled latex particles. The two particles at the center of image asindicated in the imagesare selectively excited at 420 nm and imaged during 15 s. The process is repeated until complete photobleaching is reached, resulting in image b. The relative intensity of the label emission as a function of illumination time is given in c.

3.1. Latex Particles. For CFSM and FSM measurements, the labeled latex particles were excited at 420 nm. A fluorescence image was obtained by scanning the laser beam over the entire view and detecting the emission. A higher magnification is obtained by a zoom facility, i.e., scanning over a smaller area of the sample, without loss of resolution. At higher zoom factors, however, a smaller area of the sample will be illuminated at constant laser intensity, which might result in a pronounced bleaching. Finally, one single latex particle was imaged, using latex particles of different size. A typical example of the confocal images is given for 220 nm particles in Figure 2. These measurements prove that the lateral accuracy is at least 220 ( 20 nm. From Figure 2b, the resolution of the setup can be estimated to be on the order of 200 nm. Besides confirming the resolution accuracy of the setup, polymer films consisting of coumarine-labeled latex particles were investigated for photobleaching effects. This was done by exciting the coumarine derivative at 420 nm and collecting the emission at wavelengths longer than 475 nm. Two latex particles were selectively excited and imaged during 15 s, and this process was repeated until complete photobleaching was achieved, Figure 3a,b. The relative emission intensity decreased exponentially with time with a bleaching constant of 3 × 10-3 s-1, Figure 3c. Comparing the intensities at time t ) 0 and t ) 10 min, the total emission intensity drops to about one-sixth of its initial value. For time-resolved fluorescence experiments this bleaching effect has to be taken into account, and, instead of collecting fluorescence decay curves with 104 peak counts, the accumulation was stopped when the signal-to-noise ratio was one-third of its initial value. For CTRFS measurements, the microscope and single photon counting method were combined, by directing the emission to an MCP, with a single beam optical trapping technique to measure fluorescence decays of dyes incorporated in the latex particles. These particles have a diameter of 1 and 4.5 µm, respectively, but only the 4.5 µm particles will be discussed, as both samples gave similar results.

TABLE 2: Analysis of the Time-Resolved Fluorescence Decay of Latex Particles, Labeled with a Coumarine Derivative, Trapped and in a PVA Film sample 4.5 µm 1.0 µm all beads a few beads single bead

χ2

|Z(χ2)|

0.97 1.08

0.4 1.1

500 nm Latex Particle in PVA Film 2.4 ( 0.1 1.2 ( 0.1 1.18 2.3 ( 0.1 1.3 ( 0.1 1.04 2.3 ( 0.1 1.3 ( 0.1 1.05

2.6 0.5 0.7

τsample (ns)

τref (ns)

Trapped Particles 2.3 ( 0.1 1.3 ( 0.1 2.3 ( 0.1 1.3 ( 0.1

A series of images was collected, separated by a time step, i.e., every 10 s an image was taken, or a z-step, i.e., 2 µm variations in the z-direction. In a time series, 10 consecutive frame scans were collected with a time interval of 10 s, in this way visualizing the Brownian motion of surrounding particles while the trapped latex particle remains fixed. The latex particles cluster easily, and during the 100 s experiment, the cluster regions become larger. The fluorescence decay curves of clusters of labeled latex particles and of one individual particle were analyzed, and the results are given in Table 2. In the z-series, five images were merged. Whereas the central 4.5 µm particle remains trapped and in focus, the other particles come in and out of focus. Latex particles of 4.5 and 1 µm were trapped with highly focused IR light and simultaneously excited at 420 nm. The fluorescence decay was detected at 500 nm and analyzed in the same way as that for the time series, Table 2. From these results, it can be concluded that neither the environment, i.e., solution or film, nor the trapping influences the decay time of the coumarine label. It has been reported in the literature that picosecond timeresolved fluorescence measurements and submicrometer spaceresolved confocal images, obtained from samples in solution or embedded in film, can be combined.41-45 Extending the combination to also include scanning confocal microscopy with laser trapping opens new fields or applications with a higher accuracy.


Gensch et al.

Figure 4. TM (a) and FSM images (b) and their line scans (c and d, respectively) of the cluster formed by tBSM micelles in the optical trap (P1064 ) 220 mW, ttrap ) 120 s).

3.2. Block Copolymer Micelles. The amphiphilic block copolymer poly(tert-butylstyrene-block-sodium methacrylate) was chosen for a more detailed investigation. In the TM and FSM images, trapped clusters of high copolymer micelle concentration were detected. For the FSM images, perylene was used as a fluorophore, specifically dissolved within the block copolymer micelles. From CFSM, the shape of the trapped cluster is revealed, in agreement with the geometry of the optical trap. The tBSM-1 solution showed distinct changes in both TM and FSM images when the trapping laser was switched on. In Figure 4a,b, the TM and FSM images, respectively, after 2 min at a trap power of 220 mW, are given. The circle in the FSM image has a diameter of about 4 µm, and the fluorescence intensity increases monotonically from the rim to the center. The dark spot in the TM imagesindicating less transmissions is smaller (diameter: 1.7 µm). Around this dark spot, a large ring with higher transmission (width: 0.9 µm) appears. These observations are more clearly seen in the line profiles, Figure 4c,d. Furthermore, the good accordance between the TM and FSM results ensures that the trapped objects really are the tBSM micelles. From TM only, it is not possible to discriminate between a micelle and, e.g., a trapped dust particle. The fluorescence measurements, however, measure the emission of the excited perylene probe molecule, which exclusively is dissolved in the micelles. Two other block copolymers, i.e., MMM and MBAEMM, showed similar behavior when optically trapped, results not shown. The trapping of the block copolymer micelles of these two polymers proves that the trapping of tBSM is not a feature specific to this polymer. Moreover, no significant difference

TABLE 3: Cluster Diameters for Different Trap Powers (tTrap ) 1500 S) As Obtained by TM and FSM P (mW)

d (µm)/T (spot)

d (µm)/TM (spot + corona)

660 220 60

2.1 ( 0.2 1.7 ( 0.2 1.3 ( 0.2

4.6 ( 0.2 3.6 ( 0.2 2.7 ( 0.2

d (µm)/FSM 5.2 ( 0.2 4.1 ( 0.2

was found between aqueous solutions and D2O as solvent, besides the obvious heating effect in H2O. The heating-up in the focal spot led to deformation of the cluster after some time of trapping. Because of this effect, all further trapping experiments were performed in D2O. The corona around the dark central spot in the TM images deserves thorough examination. A closer inspection of the TM images in the literature shows similar corona features as well as interference patterns. Neither the nature of these features, nor the definition of the cluster (or “particle”) diameter is given. If the corona in the present study on the block copolymer micelles would be an interference through diffraction based processes, it should be present as well in the studies on the latex spheres. In the TM images of latex particles, however, the corona is not observed. Alternatively, the corona around the cluster in the optical trap can be explained as a Rayleigh particle specific property, i.e., a concentration gradient of the block copolymer micelles reflecting the radial dependence of the gradient force. The polymer concentration gradient leads to a refractive index gradient, i.e., the cluster acts as a lens for the transmission probe light, leading to a bright ring pattern on the image. Explaining the corona as a light refraction phenomenon leads to a different interpretation of the TM images: Not the diameter



Figure 5. Line scans of TM images at three different trap powers, ttrap ) 500 s: (dotted line) P1064 ) 660 mW; (full line) P1064 ) 220 mW; (broken line) P1064 ) 60 mW.

of the dark spot, but the sum of the dark spot diameter and twice the corona width yields the diameter of the trapped cluster. If the corona is counted as a part of the cluster in the TM image, then a good agreement between cluster diameters based on TM and FSM experiments is found, Table 3. The significant larger cluster diameters from FSM, as compared to the TM images (the dark spot plus corona is approximately 15% smaller), can partially be understood in terms of the higher sensitivity of the fluorescence microscopy. The properties of the clusters as a function of laser power were investigated. Trapping could be observed for P1064 > 50 mW, and an increase of P1064 leads to an increase of the spot width in TM and FSM and the bright ring (corona) in TM. Figure 5 shows the TM line profiles at three different trap powers. The corona is more pronounced at the left side of the spot, which can be explained by a slightly nonparallel alignment of the trap beam to the optical axis of the microscope. The cluster height increases distinctively between 60 and 220 mW trap power, but does not change very much between 220 and 660 mW. The diameter of the dark spot in the TM images is more than a factor of 2 smaller than the diameter obtained from FSM. Borowicz et al.22 and Hofkens et al.24 report a high-power breakdown of the cluster formation in the optical trap for poly(N-vinylcarbazole) and poly(N-isopropylacrylamide), respectively. Although similar trap powers and block copolymer concentrations were used in this study, no indication for a smaller size of the cluster or unstable cluster formation were found at high trap powers. The reported studies on cluster formation of Rayleigh particles in an optical trap use TM images only for the characterization of the cluster size.21-27 To corroborate that the trapped object is indeed a polymer micelle, independent spectral information orsas in this studysa fluorescence image and a fluorescence decay, is needed. Borowicz et al. used a polymer that itself is fluorescent,21,22 while Hofkens et al. modified their polymer by a covalent attachment of the chromophore to the polymer chain.26 In the present study, noncovalent labeling was chosen, i.e., perylene, localized within the polymeric micelles, as a fluorophore. The time dependence of the appearance of the trapped cluster was observed at different powers of the trapping laser with TM and FSM. Figure 6 shows the time dependence of the spot growth in TM and FSM at P1064 ) 220 mW. The growth kinetics are identical within the error of the measurements, which was also observed at other trap powers. At a trapping power of 220 mW and after 150 s of trapping, the size of the trapped cluster does not change. Faster formation of the trapped cluster was observed with increasing trap powers. This result suggests that the cluster formation is a dynamic process: when

Figure 6. Cluster size as a function of trap time from TM (circles) and FSM (squares). The size is calculated from changes in intensity and normalized in order to allow an accurate comparison between the two techniques.

the maximum cluster size is reached, the number of micelles migrating in the cluster and diffusing from the cluster are simular. From this moment on, an increase in laser power results only in faster establishing of the equilibrium. Simular results were reported before.24 CFSM images of the trapped clusters were recorded to investigate the shape of the cluster in the axial direction in more detail. Parts a and d of Figure 7 show a confocal and a nonconfocal image, respectively, of the trapped cluster after 120 s trapping with P1064 ) 660 mW. The lateral size of the trapped cluster, as determined by fluorescence photon counting at constant PM gain, is largely reduced in the confocal image compared to the nonconfocal one. Parts b and c of Figure 7 show CFSM images at intermediate apertures of the variable iris in front of the detector of the confocal fluorescence unit, and give further support for this observation. These pictures are between the confocal and nonconfocal images and shows as expectedsintermediate trapped cluster sizes. The micelles forming the trapped cluster are assumed to remain single entities in the trap, due to some specific features of their characteristics. First, the hydrophilic shell is negatively charged, so there will be a substantial electrostatic repulsion between the micelles. Second, it is known that tBSM forms very stable, “frozen”, micelles at room temperature,28 leading to a reduced probability of merging the micelles in the trapped cluster into a “super structure”. The 3-fold reduction in cluster diameter upon decreasing the pinhole iris, as seen in Figure 8, can be explained with the confocal imaging properties and the spatial distribution of the attractive gradient force in the optical trap. The gradient force is linearly proportional to the gradient of the electric field vector. The IR laser used for trapping has a TEM00 mode and, owing to that, a spatial Gaussian distribution. The use of a microscopy lens with high numerical aperture leads to a rotationally symmetric cone with large base angle and a diffraction limited laser focal spot, which is defined as 1/e2 of the Gaussian intensity distribution,46 with a diameter of ca. 1 µm. After passing through the spot, the beam widens again. The depth of the focus, i.e., approximately 3 µm for 1064 nm laser light, is a measure of the distance between the focus and the beginning of the perfect cone region. The electric field gradients perpendicular and parallel to the light directions increase up to the beam focus and subsequently decrease again. The gradient force is point-symmetric to the beam focus, resulting in a trap potential with a dumbbell shape, Figure 9. Due to the nonsymmetric scatter force, this dumbbell is slightly distorted; i.e., the trapping is less efficient in the region behind


Gensch et al.

Figure 7. Image of the trapped cluster with P1064 ) 660 mW and ttrap ) 30 s: (a) CFSM; (b, c) fluorescence microscopy images with partial confocality; (d) FSM.

Figure 8. Cluster diameter as a function of pinhole size in FSM imaging, P1064 ) 660 mW and ttrap ) 30 s. Pinhole diameters 700 µm and 4 mm correspond to confocal and fully nonconfocal imaging, respectively.

Figure 9. Schematic cross-section of the cluster formed in the optical trap.

the focal point, where scatter and gradient forces act in opposite directions. Hence, a dumbbell shape distribution of the block copolymer micelles in the optical trap is expected. This would easily be observed if the trap beam and the optical axis of the TM or FSM would be perpendicular. In the present case, however, they are parallel and identical with the z-direction of the microscope. Theoretically, TM and FSM have no resolution in the z-direction and only aberrations of optical components will lead to a contrast between in-focus and out-of-focus information. The confocal imaging in CFSM leads to a high weighting of the in-focus and a low weighting of the out-offocus fluorescence photons, resulting in a z-axis resolution, which can be calculated as 750 and 850 nm for the used λex and λem, respectively. The diameter decrease seen in the fluorescence images, Figures 7 and 8, can for this reason be explained with an increasing z-resolution and the dumbbell shape

of the cluster formed of the block copolymer micelles. Borowicz et al. refer to attractive chemical interactions between the trapped objects together with the forces due to the optical trap to explain the formation of a trapped cluster.22 In the present system attractive chemical interactions are unlikely to play a role, and the optical gradient forces alone lead to the trapped cluster. Some representative fluorescence decays, measured in microscopy mode, of perylene in TX-100 and tBSM micelles are shown in Figure 10. The fluorescence decay of perylene in TX-100 micelles is monoexponential with a decay time of 6.15 ns. Adding NaCl to the solution conserves the monoexponential decay but shortens the decay time; see Table 4. The decay of perylene in tBSM micelles, on the other hand, can, with the exception of very low perylene loading (vide infra), be described by a biexponential decay function. The resulting decay times


J. Phys. Chem. B, Vol. 102, No. 43, 1998 8447 TABLE 4: Results of the Analysis of the Fluorescence Decaysa Triton X-100 sample

τ (ns)

tx-1 tx-2 tx-3 tx-4

6.15 6.10 6.08 6.03

Poly(tert-butylstyrene-block-sodium methacrylate) sample

R1

τ1 (ns)

R2

τ2 (ns)

tBSM-1 untrapped tBSM-1 trapped tBSM-2 tBSM-4

0.70 0.74 0.91 1

5.6 5.2 5.9 5.6

0.30 0.26 0.09

1.6 1.8 3.0

sample

τ (ns)


893.6

A3

A4 ()kq) (ns-1)

〈a〉

0.076 0.156 0.183

0.009

178 182 171

a For the biexponential analysis, R and R are the weight factors 1 2 connected to the exponentials with decay times τ1 and τ2, respectively. See also Table 1 for sample composition and eqs 1 and 2 for the meanings of A3, A4, and 〈a〉.

Figure 10. Representative perylene fluorescence decays: (a) Triton X-100 micelles without added salt (sample tx-1); (b) Triton X-100 micelles in the presence of 500 mM NaCl (sample tx-4); (c) tBSM micelles, untrapped and measured through the microscope (sample tBSM-1); (d) tBSM micelles optically trapped and measured through the microscope (sample tBSM-1).

are given in Table 4, and some general trends can be found: First, the decay is faster in the tBSM micelles as compared to the TX-100 micelles, and, second, the major decay component (the longer one) becomes shorter upon trapping. The biexponential decay of perylene in the tBSM micelles indicates that new deactivation paths are opened for excited perylene, which should be accompanied by a lowered fluorescence quantum yield. Using perylene in cyclohexane as reference, with a fluorescence quantum yield47 Φf ) 0.94, the quantum yields of perylene in tBSM and TX-100 micelles were determined. For the TX-100 samples, i.e., samples tx-4, tx-5, and tx-6, the quantum yield is slightly lower, Φf ) 0.8, than in cyclohexane, but no significant difference is seen between the samples, despite the difference in the number of probe molecules per micelle and the ionic strength. For the block copolymer micelles, however, the probe loading clearly influences the quantum yield. The sample tBSM-4, with very few perylene molecules per micelle, has a quantum yield close to the reference’s, i.e., Φf ) 0.9, while the other two, with a difference only in the ionic strength, both have a Φf about a factor of 2 lower. Furthermore, the fluorescence decay of tBSM-4 is monoexponential, as was the decay in the TX-100 samples and that of perylene in cyclohexane, with a decay time of 5.6 ns. From this it can be concluded that the biexponential decay of perylene in the block copolymer micelles is an effect of the high probe loading in these aggregates, i.e., up to 30 perylene molecules per micelle (vide infra). Similar effects on the perylene excited-state photophysics have been reported elsewhere.48,49 From time-resolved fluorescence quenching measurements, using RUBIPY as fluorescent probe and 9-MA as quencher, the tBSM aggregation number was determined to be approximately 177 ( 5, Table 4. As mentioned in Methods section 2.2), the original model leading to eq 1 is not yielding the average aggregation number for such large aggregates as the tBSM micelles,50 but the determined number will be a lower limit aggregation number. Even though there is an uncertainty coupled to the present estimation of the tBSM aggregation number, there is additional support for about 200 tBSM molecules per micelle. First, taking the length and the volume

8448 J. Phys. Chem. B, Vol. 102, No. 43, 1998 of the hydrophobic block into account, i.e., approximately 6575 and 7850 Å3, respectively, it is possible to calculate the aggregation number as the micellar hydrophobic volume divided by the volume of one hydrophobic block. The volume will, taking 65-75 Å as the radius of the hydrophobic core, be between 1.15 × 106 and 1.75 × 106 Å3, and the corresponding aggregation number equals 145-220. Second, a SAXS study on a block copolymer with a similar length of the hydrophobic block, but with styrene instead of a tert-butylstyrene as the repeating unit, yielded51 an aggregation number of approximately 200. Even if the repeating unit is different, this difference should not induce a too extreme change in the aggregation number. This allows us to accept 175 as a good approximation of the aggregation number of the tBSM micelles. It should also be mentioned that no significant difference was observed between measuring through the microscope and measuring in SPC mode, Table 4. The cluster deformation of the block copolymer solution after trapping was investigated by recording the TM image sequences after switching off the trap laser. A remarkable difference for short and long trap times is found using P1064 ) 660 mW. In Figure 11, a sequence after 120 s of trapping is shown. Small changes in the shape of the cluster are visible 0.5 s after switching off the trap laser, inferring mobility within the trapped cluster. After 1 s, the cluster is very faint and undetectable after 3 s (not shown). Similar cluster formation has been reported for poly(N-isopropylacrylamide) in aqueous and in D2O solutions.23-27 In aqueous solution, the absorption of IR light by H2O leads to local heating within the laser focal point. It was shown that this heating is sufficient for the macromolecules near the focus to undergo a phase transition and subsequently form large particles. Hence, the particle formation is dominated by a phase transition, and an influence of the radiation pressure could not be proven. D2O solutions, however, do not significantly absorb at 1064 nm, and the cluster formation in this solvent, which was slower and led to smaller clusters, was explained as a radiation pressure effect. A prolonged trapping time led to the formation of a metastable cluster with an elliptic shape.27 This was attributed to polymer chain entanglement and formation of new polymer aggregate structures, different from those in solution. The formation of metastable clusters on the micrometer scale, together with the difference in the fluorescence decays of the perylene probe, led us to the conclusion that a similar formation of metastable clusters occurs when trapping the block copolymer micelles. In several studies, the minimum size of a particle for optical trapping has been investigated.4,9,22,52,53 Ashkin et al.4 calculated the smallest particle diameter for stable trapping as 15 nm, and, in the same study, they experimentally performed optical trapping for particles with diameters down to 25 nm. Later, other research groups could trap particles with diameters down to 15 nm.9,22 At a diameter of 10-15 nm, the attractive gradient force, with a cubic dependence on the particle radius, becomes smaller than the forces resulting from Brownian motion and convection in the solution. While more interest has been put on trapping a single object, the cluster formation, i.e., the accumulation of many objects within the optical trap, as observed in this study and for example by Borowicz et al.,21,22 is a different phenomenon. Although the optical trapping is not stable for particles with diameters below 10 nm, i.e., an individual particle will leave the trap volume after a certain time due to Brownian motion, the radiation pressure still leads to a concentration of these particles in the optical trap, forming a cluster as observed in TM, FSM,

Gensch et al.

Figure 11. TM images of a cluster formed in the optical trap after a short trap time at several times after blocking the trap laser, P1064 ) 660 mW and ttrap ) 120 s.

and CFSM. The lower limit of particle sizes for this accumulation process has still to be investigated. Borowicz et al. showed the accumulation of 10 nm long polymer chains.22 The block copolymer micelles used in this study have a hydrophobic radius of about 7 nm, vide supra. The whole micellar size, however, will be larger, as the hydrophilic blocks form a layer around the hydrophobic core. In a SAXS study on a similar block copolymer, this layer was found to be approximately 10 nm thick.51 So, very likely the micelles are of a size where stable trapping of a single micelle is rather difficult but accumulation of a cluster still possible. The observation of the fast disappearance of the cluster after blocking the trap beam within 1 s, Figure 11, supports this idea. After prolonged optical trapping the block copolymer micelles have started to interact and form larger subclusters which are stable



Figure 12. TM images of a cluster formed in the optical trap after a long trap time at several times after blocking the trap laser, P1064 ) 660 mW and ttrap ) 1500 s.

on a 10 s time scale, Figure 12. After 1 s, the trapped cluster has split in at least four smaller clusters of 0.5-1.5 µm size. These clusters move out of the visible part of the solution within 15 s. On this time scale these smaller clusters are stable and do not dissemble. The difference in size, compare Figure 11c,d, is attributed to movements out of focus in the z-direction of these clusters. 4. Conclusions Using different microscopy techniques to characterize latex particles and the photokinetics of an attached coumarine label demonstrates that the setup used in this investigation allows the determination of fluorescence decay times with a high precision from large as well as from small regions of the sample,

while the microscopy resolution is near the theoretical limit. The excellent agreement of the fluorescence decay times estimated from single optically trapped latex particles with that estimated from many particles in solution rules out any influence of the infrared laser trap beam on the photophysics of the dye label. The formation of an optically trapped cluster of tBSM block copolymer micelles demonstrates, for the first time, the accumulation due to radiation pressure of small, dynamic, noncovalently bound polymer aggregates. The small size of the micelles as well as their structure directs the future research toward trapping of even smaller polymer aggregates, more complicated structures such as liposomes and proteins, and large monomolecular objects, e.g., dendrimers. The size and the shape of the trapped tBSM micelle cluster is estimated to be a

8450 J. Phys. Chem. B, Vol. 102, No. 43, 1998 dumbbell of approximately 10 µm length, with maximum and minimum diameters of 5 and 2 µm, respectively. From TM and CTRFS measurements, the increase of tBSM micelles in the dumbbell shaped trapped cluster could be followed. Near the focal spot, the micelle concentration is about 50 times higher than the bulk concentration. The number of micelles in the trapped cluster can be calculated to be between 104 at the lowest trap power and up to 105 for the highest power. The trapped cluster does not contain micelles fixed in space, however, as there is an equilibrium of micelles leaving and entering the cluster. The TM images show a pronounced structure, i.e., a bright corona around a dark central spot. This phenomenon is attributed to the structure of the trapped cluster: The concentration of micelles in the cluster reflects the attractive potential produced by the radiation force within the optical trap. This leads to a lateral and axial concentration gradient, increasing toward the focal point, of the tBSM micelles. The corona appears due to a lens effect of the micelles in the cluster, which has rotational symmetry, on the light used for TM imaging. A very good agreement between the diameters determined from TM and FSM is obtained if considering the corona in the TM images as a part of the trapped cluster. The trapped cluster is a structure stable for several seconds if irradiated for long times, e.g., 1500 s. From TRFS measurements, using RUBIPY as probe and 9-MA as quencher, the dimensions of the tBSM micelles could be determined. The micellar aggregation number, approximately 175, and the concentration the probe used for FSM, CFSM, and CTRFS, i.e., perylene, shows that the number of perylene molecules per micelle is as high as 30. This leads to a biexponential perylene fluorescence decay in the tBSM micelles, which is accompanied with a lower quantum yield of fluorescence. Compared with the perylene fluorescence decay kinetics in untrapped micelles, the decay times upon optical trapping of the micelles are significantly shorter. From reference measurements on perylene dissolved in neutral micelles in the presence of a salt, the decay time shortening upon trapping can be explained by the high concentration of micelles within the trapped cluster, leading to changes in the local environment, e.g., the ionic strength. Acknowledgment. T.G. thanks the EU for a postdoctoral fellowship within the framework of the TMR-program. J.H. thanks FWO for a postdoctoral fellowship. J.v.S. is a postdoctoral fellow at the K.U. Leuven. H.F. acknowledges the IWT for a scholarship. K.T. thanks Mitsubishi Paper Mills, Japan, for financial support. S.C. and R.J. thank Akzo Nobel N.V. for financial support. H.M. and F.C.D.S thank the Monbusho International Scientific Research Program, Japan (Joint Research Grant 09044085), for travel support. The continuing support of the Belgian Fund for Scientific Research and the Ministry of Scientific Programming (DWTC) through IUAP/PAI-IV/11 is gratefully acknowledged. The authors thank Anton De Gezelle for performing the major part of the time-resolved fluorescence measurements on the block copolymer micelles. References and Notes (1) Brakenhoff, G. J.; Blom, P.; Barends, P. J. Microsc. 1979, 117, 219. (2) Ashkin, A. Phys. ReV. Lett. 1970, 24, 156. (3) Ashkin, A.; Dziedzic, J. M. Appl. Phys. Lett. 1971, 19, 283. (4) Ashkin, A.; Dziedzic, J. M.; Bjo¨rkholm, J. E.; Chu, S. Opt. Lett. 1986, 11, 288. (5) Nakatani, K.; Uchida, T.; Misawa, H.; Kitamura, N.; Masuhara, H. J. Phys. Chem. 1993, 97, 5197.

Gensch et al. (6) Tamai, N.; Ito, T.; Masuhara, H. Jpn. J. Appl. Phys. 1996, 35, L547. (7) Yao, H.; Inoue, Y.; Ikeda, H.; Nakatani, K.; Kim, H.-B.; Kitamura, N. J. Phys. Chem. 1996, 100, 1494. (8) Misawa, H.; Koshioka, M.; Sasaki, K.; Kitamura, N.; Masuhara, H. J. Appl. Phys. 1991, 70, 3829. (9) D′Helon, C.; Dearden, E. W.; Rubinztein-Dunlop, H.; Heckenberg, N. R. J. Mod. Opt. 1994, 41, 595. (10) Kitamura, N.; Sasaki, K.; Misawa, H.; Masuhara, H. In Microchemistry: Spectroscopy and Chemistry in Small Domains; Masuhara, H., De Schryver, F. C., Kitamura, N., Tamai, N., Eds.; North-Holland: Amsterdam, 1994; p 35. (11) Chestnut, M. H. Curr. Opin. Colloid Interface Sci. 1997, 2, 158. (12) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Nature 1987, 330, 769. (13) Block, S. M.; Blair, D. F.; Berg, H. C. Nature 1989, 338, 514. (14) Svoboda, K.; Block, S. M. Annu. ReV. Biophys. Biomol. Struct. 1994, 23, 247. (15) Ashkin, A.; Schuetze, K.; Dziedzic, J. M.; Euteneuer, U.; Schliwa, M. Nature 1990, 348, 346. (16) Svoboda, K.; Schmidt, C. F.; Schnapp, B. J.; Block, S. M. Nature 1993, 365, 721. (17) Yin, H.; Wang, M. D.; Svoboda, K.; Landlick, R.; Block, S. M.; Gelles, J. Science 1995, 270, 1653. (18) Masters, B. R. Opt. Eng. 1995, 34, 684. (19) Spizzirri, P. G.; Hill, J. S.; Kahl, S. B.; Ghiggino, K. P. Lasers Med. Sci. 1996, 11, 237. (20) Chiu, D. T.; Zare, R. N. Chem. Eur. J. 1997, 3, 335. (21) Borowicz, P.; Hotta, J.-I.; Sasaki, K.; Masuhara, H. J. Phys. Chem. B 1997, 101, 5900. (22) Borowicz, P.; Hotta, J.-I.; Sasaki, K.; Masuhara, H. J. Phys. Chem. B 1998, 102, 1896. (23) Ishikawa, M.; Misawa, H.; Kitamura, N.; Fujisawa, R.; Masuhara, H. Chem. Lett. 1993, 481. (24) Hofkens, J.; Hotta, J.-I.; Sasaki, K.; Masuhara, H.; Faes, H.; De Schryver, F. C. Mol. Cryst. Liq. Cryst. 1996, 283, 165. (25) Ishikawa, M.; Misawa, H.; Kitamura, N.; Fujisawa, R.; Masuhara, H. Bull. Chem. Soc. Jpn. 1996, 69, 59. (26) Hofkens, J.; Hotta, J.-I.; Sasaki, K.; Masuhara, H.; Taniguchi, T.; Miyashita, T. J. Am. Chem. Soc. 1997, 119, 2741. (27) Hofkens, J.; Hotta, J.; Sasaki, K.; Masuhara, H.; Iwai, K. Langmuir 1997, 13, 414. (28) van Stam, J.; Creutz, S.; Je´roˆme, R.; De Schryver, F. C. Submitted for publication. (29) Vanoppen, P.; Hofkens, J.; Latterini, L.; Jeuris, K.; Faes, H.; De Schryver, F. C.; Kerimo, J.; Barbara, P. F.; Rowan, A. E.; Nolte, R. J. M. In Applied Fluorescence in Chemistry, Medicine, and Biology; Rettig, W., Strehmel, B., Schrader, S., Eds.; Springer: Berlin, accepted for publication. (30) Infelta, P. P.; Gra¨tzel, M.; Thomas, J. K. J. Phys. Chem. 1974, 78, 190. (31) Roelants, E.; De Schryver, F. C. Langmuir 1987, 3, 209. (32) van Stam, J.; Lindblad, C.; Almgren, M. Prog. Colloid Polym. Sci. 1991, 84, 8. (33) Reekmans, S.; Bernik, D.; Gehlen, M.; van Stam, J.; Van der Auweraer, M.; De Schryver, F. C. Langmuir 1993, 9, 2289. (34) Dutt, G. B.; van Stam, J.; De Schryver, F. C. Langmuir 1997, 13, 1957. (35) Knutson, J. R.; Beechem, J. M.; Brand, L. Chem. Phys. Lett. 1983, 102, 501. (36) Lo¨froth, J.-E. Eur. Biophys. J. 1985, 13, 45. (37) Ameloot, M.; Beechem, J. M.; Brand, L. Biophys. Chem. 1986, 23, 155. (38) Boens, N.; Malliaris, M.; Van der Auweraer, M.; Luo, H.; De Schryver, F. C. Chem. Phys. 1988, 121, 199. (39) Boens, N.; Ameloot, M.; Yamazaki, I.; De Schryver, F. C. Chem. Phys. 1988, 121, 73. (40) Gehlen, M. H.; De Schryver, F. C. Chem. ReV. 1993, 93, 199. (41) Sasaki, K.; Koshioka, M.; Masuhara, H. Appl. Spectrosc. 1991, 45, 1041. (42) Masuhara, H.; Kitamura, N.; Misawa, H.; Sasaki, K.; Koshioka, M. J. Photochem. Photobiol. A: Chem. 1992, 65, 235. (43) Tamai, N.; Asahi, T.; Masuhara, H. ReV. Sci. Instrum. 1993, 64, 2496. (44) Ghiggino, K. P.; Spizzirri, P. G.; Smith, T. A. In Microchemistry. Spectroscopy and Chemistry in Small Domains; Masuhara, H., De Schryver, F. C., Kitamura, N., Tamai, N., Eds.; North-Holland: Amsterdam, 1994; p 197. (45) Ghauharali, R. I.; van Driel, R.; Brakenhoff, G. J. J. Microsc. 1997, 185, 375. (46) Webb, R. H. Rep. Prog. Phys. 1996, 59, 427. (47) Berlman, I. B. Handbook of fluorescence spectra of aromatic molecules; Academic Press: New York, London, 1971.

Optically Trapped Block Copolymer Micelles (48) Liu, Y. S.; Ware, W. R. J. Phys. Chem. 1993, 97, 5987. (49) Van der Auweraer, M. Personal communication. (50) Almgren, M.; Alsins, J.; Mukhtar, E.; van Stam, J. J. Phys. Chem. 1988, 92, 4479.

J. Phys. Chem. B, Vol. 102, No. 43, 1998 8451 (51) Creutz, S.; Lombardo, D.; Lesieur, P.; Gaspard, J. P.; Je´roˆme, R.; Williams, C. Unpublished results. (52) Harada, Y.; Asakura, T. Opt. Commun. 1996, 124, 529. (53) Ren, K. F.; Grehan, G.; Gouesbet, G. Appl. Opt. 1996, 35, 2702.

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