Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug discovery, clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial resolution.
A major challenge in three-dimensional (3D) microscopy is to obtain accurate spatial information while simultaneously keeping the microscopic samples in their native states. In conventional 3D microscopy, axial resolution is inferior to spatial resolution due to the inaccessibility to side scattering signals. In this study, we demonstrate the isotropic microtomography of free-floating samples by optically rotating a sample. Contrary to previous approaches using optical tweezers with multiple foci which are only applicable to simple shapes, we exploited 3D structured light traps that can stably rotate freestanding complex-shaped microscopic specimens, and side scattering information is measured at various sample orientations to achieve isotropic resolution. The proposed method yields an isotropic resolution of 230 nm and captures structural details of colloidal multimers and live red blood cells, which are inaccessible using conventional tomographic microscopy. We envision that the proposed approach can be deployed for solving diverse imaging problems that are beyond the examples shown here.
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that are intracellularly accumulated as distinct insoluble granules by various microorganisms. PHAs have attracted much attention as sustainable substitutes for petroleum-based plastics. However, the formation of PHA granules and their characteristics, such as localization, volume, weight, and density of granules, in an individual live bacterial cell are not well understood. Here, we report the results of three-dimensional (3D) quantitative label-free analysis of PHA granules in individual live bacterial cells through measuring the refractive index distributions by optical diffraction tomography (ODT). The formation and growth of PHA granules in the cells of Cupriavidus necator, the best-studied native PHA producer, and recombinant Escherichia coli harboring C. necator poly(3-hydroxybutyrate) (PHB) biosynthesis pathway are comparatively examined. Through the statistical ODT analyses of the bacterial cells, the distinctive characteristics for density and localization of PHB granules in vivo could be observed. The PHB granules in recombinant E. coli show higher density and localization polarity compared with those of C. necator, indicating that polymer chains are more densely packed and granules tend to be located at the cell poles, respectively. The cells were investigated in more detail through real-time 3D analyses, showing how differently PHA granules are processed in relation to the cell division process in native and nonnative PHA-producing strains. We also show that PHA granule–associated protein PhaM of C. necator plays a key role in making these differences between C. necator and recombinant E. coli strains. This study provides spatiotemporal insights into PHA accumulation inside the native and recombinant bacterial cells.
The development of optical and computational techniques has enabled imaging without the need for traditional optical imaging systems. Modern lensless imaging techniques overcome several restrictions imposed by lenses, while preserving or even surpassing the capability of lens-based imaging. However, existing lensless methods often rely on a priori information about objects or imaging conditions. Thus, they are not ideal for general imaging purposes. The recent development of the speckle-correlation scattering matrix (SSM) techniques facilitates new opportunities for lensless imaging and sensing. In this review, we present the fundamentals of SSM methods and highlight recent implementations for holographic imaging, microscopy, optical mode demultiplexing, and quantification of the degree of the coherence of light. We conclude with a discussion of the potential of SSM and future research directions.
It has been recently demonstrated that wavefront information can be directly measured using the speckle‐correlation scattering method. The realization of speckle‐correlation scattering requires the use of a diffusive layer; however, it poses technical challenges in characterizing the transmission matrix of the diffusive layer or precisely fabricating an engineered random metasurface. Herein, liquid crystal geometric phase diffusers are exploited for single‐shot reference‐free holographic imaging based on speckle‐correlation scattering. It is demonstrated that the use of a commercial geometric phase diffuser provides precise holographic measurements in a simple imaging setup, with no calibration required. This low‐cost, simple, and high‐resolution wavefront sensing approach inspired by liquid crystal materials may find direct applications in metrology, industrial inspection, and biophotonics.
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