pubs.acs.org/Langmuir © 2010 American Chemical Society
Confocal Laser Imaging and Annealing of Quantum-Dot-Coated Silica Colloidal Crystals Richard E. Beckham† and Michael A. Bevan*,‡ †
Chemical Engineering, Texas A&M University, College Station, Texas 77843, and ‡Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21218 Received October 23, 2009. Revised Manuscript Received February 7, 2010
We demonstrate the ability to image, melt, and anneal repulsive electrostatic and attractive depletion colloidal crystals of quantum-dot-decorated SiO2 microparticles (QDSs). QDS colloidal crystals showed increased thermal motion, melting, and convection only in the presence of CSLM imaging, which we attribute to quantum dot (QD) heating. We exploit this local heating effect to anneal a single defect-rich, polycrystalline domain. Our results demonstrate a method to anneal colloidal crystals locally with focused lasers and the role of heating when using many QD tags per particle in CSLM imaging.
Confocal scanning laser microscopy (CSLM) has become a powerful tool for 3D imaging in colloidal systems.1-3 At the same time, quantum dots (QD) have emerged as tags with superior luminescent properties over conventional fluorophores.4-6 Because QDs do not photobleach, can be excited by a range of wavelengths, and have high emission intensities, they have the potential to be useful tags for CSLM imaging. However, QDs have not been used in CSLM imaging studies of colloidal interactions, dynamics, and structure or as part of broader investigations of colloidal assembly or phase behavior. One issue with single QDs is that their emission is temporally discontinuous, which is commonly referred to as “blinking”.7 This has been overcome in several studies by coating micrometersized colloidal particles with many QDs so that their ensemble emission effectively averages out single QD blinking.8 We have successfully used such quantum-dot-coated microparticles with evanescent wave illumination to perform quantitative total internal reflection microscopy measurements of particle-surface interaction potentials.9 Such composite microparticles appear to have luminescent properties that are well suited to CSLM studies of concentrated 3D colloidal assembly. *To whom correspondence should be addressed. E-mail: mabevan@ jhu.edu. (1) Dinsmore, A. D. Three-dimensional confocal microscopy of colloids. Appl. Opt. 2001, 40, 4152-4159. (2) Prasad, V.; Semwogerere, D.; Weeks, E. R. Confocal microscopy of colloids. J. Phys.: Condens. Matter 2007, 19, 113102. (3) Beckham, R. E.; Bevan, M.A. Interfacial colloidal sedimentation equilibrium I. intensity based confocal microscopy. J. Chem. Phys. 2007, 127, 164708. (4) Chan, W. C.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Luminescent quantum dots for multiplexed biological detection and imaging. Curr. Opin. Biotechnol. 2002, 13, 40-46. (5) Alivisatos, P. The use of nanocrystals in biological detection. Nat. Biotechnol. 2004, 22, 47-52. (6) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005,307, 538-544. (7) Shimizu, K. T.; Neuhauser, R. G.; Leatherdale, C. A.; Empedocles, S. A.; Woo, W. K.; Bawendi, M. G. Blinking statistics in single semiconductor nanocrystal quantum dots. Phys. Rev. B 2001, 63, 205316. (8) Chung, I.; Witkoskie, J. B.; Zimmer, J. P.; Cao, J.; Bawendi, M. G. Extracting the number of quantum dots in a microenvironment from ensemble fluorescence intensity fluctuations. Phys. Rev. B 2007, 75, 045311. (9) Everett, W. N.; Beckham, R. E.; Meissner, K.; Bevan, M. A. Evanescent wave excited luminescence from levitated quantum dot. Langmuir 2007,23, 8950-8956.
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In this work, we describe the fabrication of quantumdot-coated SiO2 microparticles (QDSs) and their assembly into colloidal crystals for imaging with CSLM. We investigate colloidal crystals formed either via the gravitational consolidation of QDSs experiencing electrostatic repulsion in aqueous media or via the condensation of QDSs experiencing depletion attraction due to nonadsorbing SiO2 nanoparticles in nonaqueous media. We demonstrate how CSLM imaging melts these crystals in contrast to video microscopy (VM) imaging and provide evidence that suggests QD heating by the focused scanning laser as the source of melting. We exploit this heating effect to anneal colloidal crystal defects locally by controlling the CSLM imaging region, which has limited precedent.10,11 QDs (TOPO-capped CdSe/ZnS core-shell particles) dispersed in toluene were synthesized and purified using standard protocols.12,13 QDs were functionalized with dihydrolipoic acid (DHLA) via a 24 h reaction in toluene/1-butanol.14 SiO2 particles (1 μm) for use in the assembly of colloidal crystals were prepared in multiple growth steps in 1-butanol,15 coated with 3-aminopropyltrimethoxysilane (APS), and then conjugated with DHLA/ QDs in toluene/1-butanol (Figure 1). Commercial 2 μm SiO2 particles (Bangs) for use as tracer particles were also coated with APS and DHLA/QDs. After conjugation to SiO2 particles, QDs had an emission maximum at ∼535 nm when excited with a (10) Korda, P. T. Grier, D. G. Annealing thin colloidal crystals with optical gradient forces. J. Chem. Phys. 2001, 114, 7570-7573. (11) St. John, A. N.; Lyon, L. A. Local control over phase transitions in microgel assemblies. J. Phys. Chem. B 2008, 112, 11258-11263. (12) Peng, Z. A. Peng, X. G. Formation of high-quality CdTe, CdSe, and CdS nanocrystals using CdO as precursor. J. Am. Chem. Soc. 2001, 123, 183-184. (13) Talapin, D. V.; Rogach, A. L.; Kornowski, A.; Haase, M.; Weller, H. Highly luminescent monodisperse CdSe and CdSe/ZnS nanocrystals synthesized in a hexadecylamine-trioctylphosphine oxide-trioctylphospine mixture. Nano Lett. 2001 1, 207-211. (14) Clapp, A. R.; Goldman, E. R.; Mattoussi, H. Capping of CdSe-ZnS quantum dots with DHLA and subsequent conjugation with proteins. Nat. Protocols 2006, 1, 1258-1266. (15) Bogush, G. H.; Tracy, M. A.; Zukoski, C. F., IV. Preparation of monodisperse silica particles: control of size and mass fraction. J. Non-Cryst. Solids 1988, 104, 95-106. (16) Stober, W.; Fink, A.; Bohn, E. Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 1968, 26, 62-69. (17) van Helden, A. K.; Jansen, J. W.; Vrij, A. Preparation and characterization of spherical monodisperse silica dispersions in non-aqueous solvents. J. Colloid Interface Sci. 1981, 81, 354-368.
Published on Web 02/11/2010
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Figure 1. Initial CSLM image of the first layer (adjacent to a microscope slide) of an electrostatic colloidal crystal of quantumdot-decorated SiO2 microparticles (QDSs). Intensity variations between different particles due to different QD loadings produce intensity nonuniformities within the image. Dashed lines show crystal axes. Further CSLM imaging melts this crystal as shown in a video in Supporting Information and in a series of static images in Figure 2. (Left inset) Schematic depiction of the covalent attachment of dihydrolipoic acid-coated ZnS/CdSe core/shell quantum dots to micrometer-sized 3-aminopropyltrimethoxysilane-coated SiO2 colloids in 1-butanol/toluene solutions. (Right inset) CSLM enlarged equatorial cross-sectional image of an isolated single QD-coated SiO2 colloid excited with a 488 nm laser and emitting at 535 nm.
488 nm argon ion laser. SiO2 particles (70 nm) for mediating depletion forces between micrometer-sized SiO2 particles were synthesized in a single step16 and modified with 1-octadecanol.17 All SiO2 particles were purified by repeated sedimentation and redispersion into deionized water or dimethylformamide (DMF). Samples were then introduced into sedimentation cells for VM and CSLM imaging.3 Sedimented colloidal crystals were imaged at the microscope slide surface using an inverted microscope (Zeiss Axiovert 200M) with an oil-immersion 100 objective (Zeiss, NA = 1.4) and either a charge-coupled device camera (Hamamatsu ORCA-ER) in VM imaging or an 488 nm argon ion laser and photomultiplier tube for CSLM imaging (Zeiss LSM 5 Pascal). Repulsive electrostatic colloidal crystals (Figures 1 and 2) grew from initially dense inhomogeneous fluids at microscope slide surfaces into ∼30-μm-deep sediments that compressed the bottom layers into polycrystalline configurations displaying longrange order.3,18 Attractive depletion colloidal crystals (Figure 3) grew from isolated, randomly oriented crystallites at submonolayer concentrations19 that grew into space-filling polycrystalline configurations in sediments with as few as three or four particle layers but typically much deeper. CSLM imaging melted sedimented colloidal crystals of QDSs experiencing either repulsive electrostatic (Figure 2) or attractive (18) Bevan, M. A.; Lewis J. A.; Braun, P. V. Structural evolution of colloidal crystals with increasing ionic strength. Langmuir 2004, 20, 7045-7052 (19) Fernandes, G. E.; Beltran-Villegas, D. J.; Bevan, M. A. Interfacial colloidal crystallization via tunable hydrogel depletants. Langmuir 2008, 24, 10776-10785.
3780 DOI: 10.1021/la904031s
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depletion interactions (Figure 3). Specifically, during the process of CSLM imaging, QDS microparticles in static, equilibrated crystals were observed to: (1) first display increasing rates and magnitudes of Brownian motion on lattice positions, (2) followed by a sudden (first-order) loss of long-range order and onset of isotropic liquid like structure (note nonlinear time scale of image sequence in Figure 2), and (3) then, specifically in the repulsive electrostatic QDS case, display subsequent convective motion toward the center of the CSLM imaging region where small particles eventually disappeared due to vertical convection and new particles arrived from the image periphery. All of these effects can be seen in a movie for the repulsive electrostatic crystal in Supporting Information. For the repulsive electrostatic crystal, Figure 2 shows melting and subsequent convection in (1) static images at different times, (2) Fourier transforms (FTs) of images, (3) a plot of 6-fold order, ψ6,18,19 in the FTs versus time, (4) tracer particle locations in each image, and (5) a plot of tracer particle trajectories. From the onset of CSLM scanning up to the melting point (0-25 s in Figure 2), tracer particles show insignificant motion on length scales greater than the particle radii, and the FTs show gradual broadening and then the disappearance of peaks from each consecutive coordination shell (consistent with Lindemann and Hansen-Verlet melting criteria20,21). After melting (25-100s in Figure 2), all particles display a convective motion toward the center of the imaging region as captured by the trajectories of the larger tracer particles in Figure 2. The rate and degree of melting increased as the CSLM laser intensity was increased. The rate of motion associated with increased thermal motion, melting, and convection was sufficiently slow so that 2D CSLM images of the first layer of particles adjacent to the wall displayed no distortion up to (but not including) conditions that produced the highest rates of convection (Figure2). In repulsive electrostatic crystals, “fast Z-line scans” used to obtain cross-sectional images showed vertical convective “plumes” of QDS particles moving up and away from the bottom microscope slide surface and centered within the CSLM imaging region, although the motion was too rapid to resolve individual particles. In contrast, long periods of VM imaging with conventional transmitted light illumination did not affect sedimented QDS colloidal crystals in any quantifiable way. In particular, VM imaging did not produce melting, convection, or any perceptible increase in particle thermal motion. Simultaneous CSLM and VM imaging revealed melting and convection only during CSLM imaging. Upon cessation of CSLM imaging, QDS convection stopped and all samples recrystallized as monitored nonintrusively with VM. QDSs experiencing depletion attraction recrystallized more slowly than electrostatically stabilized QDSs for kinetic reasons. For the QDSs having depletion attraction, VM showed melting only within the CSLM imaging region and recrystallization from unmelted crystals at the edge of the imaging region when CSLM imaging was stopped (nuclei were also observed in some cases within fluid domains). Colloidal crystals of electrostatically stabilized QDSs melted outside of the CSLM imaging region and immediately recrystallized everywhere when CSLM imaging was stopped. These results are consistent with the localized heating of the QDS particles due to absorbance by the QD coatings of the confocal laser illumination. The increased thermal motion of (20) Lindemann, F. A. The calculation of molecular vibration frequencies. Phys. Z. 1910, 11, 609-612. (21) Hansen, J.-P.; Verlet, L. Phase transitions of the Lennard-Jones system. Phys. Rev. 1969, 184, 151-161.
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Figure 2. Static images extracted from a video in Supporting Information of the CSLM melting of electrostatic colloidal crystal via local heating of QDSs through optical absorbance. In each image, the left inset reports the total imaging duration in seconds, the right inset is an eight-bit grayscale FT of each image, and white arrows indicate the net displacement of 2 μm tracer particles from their initial positions. The lower-left plot is the time dependence of 6-fold order, ψ6, in FTs of the video in Supporting Information as computed for all detectable points (blue, maximum of 19 points including the center and 3 coordination shells) or the first coordination shell (red, maximum of 7 points). The lower-right plot shows 2 μm tracer particle trajectories displaying migration toward the center of the CSLM imaging region with the arrival of new particles at the image periphery, which shows convection associated with laser heating of electrostatic colloidal crystals.
Figure 3. Analyzed VM images before, during, and after CSLM imaging within the central square region in a polycrystalline, attractive depletion QDS colloidal crystal adjacent to the underlying microscope slide surface. Triangulation based on particle centers is used to visualize the microstructure, with the color scheme indicating bonds between crystalline neighbors from local bond orientational order parameters with gray lines indicating fluid structures and yellow and blue indicating crystalline structures.19 (Left-to-right, top-to-bottom) Polycrystalline structure prior to CSLM imaging, complete melting of all polycrystalline domains within the CSLM imaging region, recrystallization of the central crystal and regrowth of surrounding crystal domains immediately after ceasing CSLM imaging, and re-equilibrated polycrystalline structure showing a decreased central crystal domain size and increases surrounding domain sizes.
particles initially on lattice positions with CSLM imaging appears to be consistent with local particle heating rather than heating (22) Eichmann, S. L.; Anekal, S. G.; Bevan, M. A. Electrostatically confined nanoparticle interactions and dynamics. Langmuir 2008, 24, 714-721.
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of the surrounding fluid or substrate. We note that previous experiments in our laboratory displayed no evidence of heating for either (1) single QD-coated latex microparticles (and gold nanoparticles22) illuminated by an unfocused 1-mm-diameter 488 nm evanescent wave9 or (2) sedimented colloidal crystals of 720 nm SiO2 core-shell particles containing molecular fluorophores23 when imaged for extended periods using the same 488 nm laser and confocal microscope as in this work. As a result, the generation of local power densities sufficient to heat and melt QDS colloidal crystals in Figure 2 appears to arise from increased local absorbance due to (1) CSLM imaging through a high numerical aperture objective that focuses the laser to micrometer scales and (2) high average QD concentrations that arise from monolayer coverages of QDs on SiO2 colloids that are at high concentrations (φ ≈ 0.74) within colloidal crystals. A mechanism based on local heating is also consistent with the dynamic behavior observed as part of the melting process for crystals with different interparticle forces. For the same laser power, colloidal crystals of QDSs experiencing electrostatic repulsion displayed faster and more extensive melting and convection than QDSs experiencing depletion attraction. Because effective hard sphere interactions operate in both the repulsive electrostatic (one component) and attractive depletion (two components) colloidal crystals, melting and freezing are athermal and depend only on the effective particle volume fractions.24 As a result, melting should not depend on T but can arise from local T gradients. From the results in Figures 2 and 3, it appears that T gradients drive convection and that shear melts the repulsive electrostatic colloidal crystals25 more easily and to a greater extent than the attractive depletion crystals, presumably because of a higher shear modulus for the attractive depletion crystal. The apparent T-gradient-driven shear melting of the colloidal crystals (23) van Blaaderen, A.; Vrij, A. Synthesis and characterization of colloidal dispersions of fluorescent, monodisperse silica spheres. Langmuir 1992, 8, 2921-2931. (24) Cheng, Z.; Chaikin, P. M.; Russel, W. B.; Meyer, W. V.; Zhu, J.; Rogers, R. B.; Ottewill, R. H. Phase diagram of hard spheres. Mater. Des. 2001, 22, 529-534. (25) Imhof, A.; van Blaaderen, A.; Dhont, J. K. G. Shear melting of colloidal crystals of charged spheres studied with rheology and polarizing microscopy. Langmuir 1994, 10, 3477-3484.
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in this work is reminiscent of previous studies of electrolyte gradient-driven shear melting of repulsive electrostatic colloidal crystals.18 Modeling the spatiotemporal heating of composite media under transient, nonuniform, 3D illumination is not trivial. A simple estimate of ΔT with laser absorbance can be made for adiabatic heating at a diffraction limited spot using ΔT = [(P/ A)ΘΔt]/[C(V/A)] with (1) power, P = 10-1mW, for a 102 mW laser with 10-1 attenuation by confocal optics and an additional 10-2 software-controlled attenuation; (2) area, A=1 μm2, for a diffraction-limited spot; (3) percent incident power dissipated as heat, Θ = 10-2, for inefficient absorbance and efficient luminescence; (4) illumination time, Δt=1 μs, for a 106 pixels/s scan rate; (5) an average heat capacity, C=106 J/m3K; and (6) a focal point volume/area, V/A = 1-10 μm3/μm2, for complete absorbance over a finite depth/spot area. This calculation suggests ΔT = 0.1-1 K/image, which could produce Τ gradients and the observed convective melting of
