Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view

Abstract

Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer.

Point-spread function engineering enhances digital Fourier microscopy

“…While the loss of low- q information upon reducing the observation volume has been noted before, 7,10 we demonstrate how reducing one dimension of an ROI limits the range of wave vectors one can probe. As the ROI is reduced, particles leave the observation volume quicker and, therefore, long-time dynamics are lost.…”

“…Methods to characterize such dynamics include single-particle tracking, 1 utilizing real-space image data, and dynamic light scattering (DLS), 2 using reciprocal space data. Combining elements of both real and reciprocal space methods is, among related digital Fourier microscopy techniques, differential dynamic microscopy (DDM) which, since its initial description in 2008, 3 has been used to characterize the dynamics of bacteria, 4,5 colloids, 6 DNA 7 and protein clusters. 8 By analyzing in the Fourier domain a time series of real-space images one can use DDM to extract the temporal decay rate of density fluctuations within the sample across a range of wave vectors providing data analogous to that of DLS.…”

“…8 By analyzing in the Fourier domain a time series of real-space images one can use DDM to extract the temporal decay rate of density fluctuations within the sample across a range of wave vectors providing data analogous to that of DLS. A notable feature of DDM is that such real-space images can be acquired with a variety of microscopy techniques (e.g., dark-field, 9 confocal, 10 light-sheet 7 ) making this method widely accessible. Here, we show that DDM can be extended with point-spread function (PSF) engineering.…”

Perspective: Differential dynamic microscopy extracts multi-scale activity in complex fluids and biological systems

“…The finite focal depth also sets a maximum timescale over which sample dynamics can be observed (similarly to the fact that particle tracks would have a typical maximum length, also from finite depth of focus). This has been explored systematically in confocal microscopy 18 and in light-sheet 25 …”

Differential dynamic microscopy microrheology of soft materials: A tracking-free determination of the frequency-dependent loss and storage moduli

“…These data are treated via an image processing algorithm [22] or equivalent versions of it [23] that combines image differences and spatial Fourier transformations to obtain as a result the intermediate scattering function f (q,t) that is typically probed in DLS experiments as a function of the scattering wave vector q and time t [24]. Since its introduction, DDM has been profitably used and also extended by several groups [25][26][27][28][29][30][31][32][33][34] for a variety of applications [35]. In particular, DDM has been recently proven to be an effective tool to measure also the rotational dynamics of anisotropic colloidal particles in solution.…”