Characterizing Concentrated, Multiply Scattering, and Actively Driven Fluorescent Systems with Confocal Differential Dynamic Microscopy

Abstract

We introduce confocal differential dynamic microscopy (ConDDM), a new technique yielding information comparable to that given by light scattering but in dense, opaque, fluorescent samples of micron-sized objects that cannot be probed easily with other existing techniques. We measure the correct wave vector q-dependent structure and hydrodynamic factors of concentrated hard-sphere-like colloids. We characterize concentrated swimming bacteria, observing ballistic motion in the bulk and a new compressed-exponential scaling of dynamics, and determine the velocity distribution; by contrast, near the coverslip, dynamics scale differently, suggesting that bacterial motion near surfaces fundamentally differs from that of freely swimming organisms. DOI: 10.1103/PhysRevLett.108.218103 PACS numbers: 87.64.mk, 47.57.EÀ, 78.35.+c, 82.70.Dd Fluorescence imaging is an important and versatile form of optical microscopy. Fluorescent tags can selectively identify specific features within an image, thereby enhancing contrast; this is particularly powerful in biology and soft-matter physics. A major difficulty, however, is that all fluorescent objects within the illumination beam emit light, even if outside the microscope's focal plane, hindering collection of high-quality images. Using a confocal pinhole, which limits detected light to only that originating from the focal plane, confocal microscopy allows true 3D imaging. By its very nature, however, confocal microscopy is relatively slow; collecting a 3D stack of images usually requires several seconds, limiting the study of dynamics to relatively slow phenomena, characterized by time scales well in excess of a second [1,2].Even traditional bright field microscopy is limited in its ability to follow rapid dynamics; by contrast, another optical method, dynamic light scattering (DLS), is wellsuited to characterize dynamics at high speeds, specifically ensemble averages as a function of scattering wave vector q, albeit at the cost of losing real-space information [3]. One way to combine DLS with the advantages of real-space imaging in the wide field is differential dynamic microscopy (DDM), which extends to lower-q information analogous to that given by DLS [4-6]. However, DDM has thus far been restricted to wide field imaging; consequently, like DLS, DDM probes only dilute suspensions [4][5][6]. No equivalent method exists for fluorescence, particularly in high-concentration samples where imaging is obscured. This severely limits the use of fluorescence microscopy for studies of dynamics in dense samples.In this Letter, we introduce a new technique using confocal fluorescence microscopy that provides a powerful probe not only of rapid dynamics but also of the static structure of dense, fluorescent samples that multiply scatter light, precluding their study with other techniques. Motivated by DDM analysis, we examine the Fourier spectra of the differences between pairs of images within a sequence; the short-time differences confirm diffusive motion of hard-sphere-like colloidal ...

Mass transport properties of the tetrahydronaphthalene/n-dodecane mixture measured by investigating non-equilibrium fluctuations

“…It is also worth pointing out that in contrast to a conventional static NFS experiment, this approach enables us to obtain the background noise B(q) without performing a separate measurement. The double-frame differential dynamics approach has proven to be very useful and has already been applied in many other realizations of the NFS technique [51,52,[55][56][57][58].…”

Structural Transition in a Fluid of Spheroids: A Low-Density Vestige of Jamming

“…The thermodynamics of a fluid of simple spheres is wellknown and almost completely understood [1,2]. However, the constituents of real matter are typically nonspherical.…”

“…This observation suggests a deep connection to exist between the physics of jamming and the thermodynamics of simple fluids. DOI: 10.1103/PhysRevLett.116.098001 The thermodynamics of a fluid of simple spheres is wellknown and almost completely understood [1,2]. However, the constituents of real matter are typically nonspherical.…”

“…Therefore, the effective volume occupied by an oblate spheroid is a sphere of diameter b, which is the corresponding solid of revolution. In a fluid of such effective spheres, the first peak position [1,2] is r p ¼ b, so that r p =L ¼ t −1=3 . Once again, this prediction [solid line in Fig.…”

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

“…Furthermore, for laser diffraction and dynamic light scattering, only diluted samples that are free from multiple scattering and light absorption can be measured. To overcome this difficulty, several new techniques have been proposed for the measurement of dynamic light scattering from undiluted, concentrated solutions, such as cross-correlation spectroscopy 5,6 , low-coherence dynamic light scattering 7,8 , diffusingwave spectroscopy 9,10 , and differential dynamic microscopy 11,12 .…”