Light-sheet microscopy enables considerable speed and phototoxicity gains, while quantitative-phase imaging confers label-free recognition of cells and organelles, and quantifies their number-density that, thermodynamically, is more representative of metabolism than size. Here, we report the fusion of these two imaging modalities onto a standard inverted microscope that retains compatibility with microfluidics and open-source software for image acquisition and processing. An accelerating Airy-beam light-sheet critically enabled imaging areas that were greater by more than one order of magnitude than a Gaussian beam illumination and matched exactly those of quantitative-phase imaging. Using this integrative imaging system, we performed a demonstrative multivariate investigation of live-cells in microfluidics that unmasked that cellular noise can affect the compartmental localization of metabolic reactions. We detail the design, assembly, and performance of the integrative imaging system, and discuss potential applications in biotechnology and evolutionary biology.
Light-sheet microscopy has revolutionized bioimaging by enabling approximately an order of magnitude reduction in specimen irradiance compared to confocal imaging. Here, we introduce a light-sheet imaging system that enables an additional order of magnitude reduction in specimen irradiance by operating at the Poisson limit. To operate at this limit, we integrated classical illumination with single-photon detection and wavelet-based image reconstruction. This integration enabled brightness quantification and object recognition from fewer than one detected photon per image pixel, corresponding to more than 10-fold lower irradiance levels than modern systems. We demonstrate how such photon-sparse imaging can eradicate photobleaching and enable both dim and bright object imaging, thus, further enhancing the related gains of light-sheet microscopy.
Light-sheet fluorescence microscopy has greatly improved the speed and overall photostability of optically sectioning cellular and multi-cellular specimens. Similar gains have also been conferred by light-sheet Raman imaging; these schemes, however, rely on diffraction limited Gaussian beams that hinder the uniformity and size of the imaging field-of-view, and, as such, the resulting throughput rates. Here, we demonstrate that a digitally scanned Airy beam increases the Raman imaging throughput rates by more than an order of magnitude than conventional diffraction-limited beams. Overall, this, spectrometer-less, approach enabled 3D imaging of microparticles with high contrast and 1 µm axial resolution at 300 msec integration times per plane and orders of magnitude lower irradiation density than coherent Raman imaging schemes. We detail the apparatus and its performance, as well as its compatibility with fluorescence light-sheet and quantitative-phase imaging towards rapid and low phototoxicity multimodal imaging.
This work presents a new concept to measure the extinction cross section for a single particle in situ. The concept involves recording the hologram produced by the interference of a particle's forward-scattered light with the incident light. This interference pattern is fundamentally connected to the energy flow that gives rise to extinction, and, by integrating this measured pattern, one obtains an approximation for the cross section. Mie theory is used to show that this approximation can be as little as 1% in error of the true value for many cases of practical interest. Moreover, since an image of the particle can be computationally reconstructed from a measured hologram using the Fresnel-Kirchhoff diffraction theory, one can obtain the cross section simultaneously with the particle shape and size.
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